LncRNA SNHG16 facilitates the liver metastasis of colorectal cancer by modulating circulating tumor cell epithelial-mesenchymal transition via a positive feedback loop with the YAP1/TEAD1 complex

doi:10.21203/rs.3.rs-1122369/v1

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LncRNA SNHG16 facilitates the liver metastasis of colorectal cancer by modulating circulating tumor cell epithelial-mesenchymal transition via a positive feedback loop with the YAP1/TEAD1 complex

https://doi.org/10.21203/rs.3.rs-1122369/v1

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Background: Circulating tumor cells (CTCs) are important precursors of colorectal cancer (CRC) metastasis. The epithelial-mesenchymal transition (EMT) process facilitates CTC invasion by allowing these cells to evade antimetastatic checkpoints to mediate distant metastasis. However, the molecular mechanism of CTC EMT remains largely unknown.

Results: Here, we studied the oncogenic long noncoding RNA (lncRNA) SNHG16, whose expression is correlated with advanced TNM stage, distant metastasis, and a poor prognosis in CRC patients. Furthermore, loss- and gain-of function revealed that SNHG16 promoted CRC proliferation, migration, invasion, EMT, mesenchymal-type CTC (MCTC) generation, and liver metastasis through YAP1. Mechanistically, SNHG16 could act as a miRNA sponge to sequester miR-195-5p on Ago2, thereby protecting YAP1 from repression. Moreover, YAP1 could combine with TEA domain transcription factor 1 (TEAD1) to form a YAP1/TEAD1 complex, which could in turn bind to the promoter of SNHG16 and regulate its transcription. Finally, we demonstrated that YAP1 significantly promoted tumor progression, and that SNHG16 rescued the effect of YAP1 on tumor progression.

Conclusion: Herein, we clarified a hitherto unexplored SNHG16-YAP1/TEAD1 positive feedback loop, which may be a candidate target for CRC treatment.

SNHG16

miR-195-5p

YAP1

TEAD1

CTCs

cancer

Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related mortality worldwide¹. To date, metastasis remains the leading cause of cancer-related mortality worldwide². The liver is the most common site of CRC distant metastasis^3-5, and liver metastasis is the main cause of CRC-related death⁶. Metastasis is a complicated process involving multiple steps and driven by multiple mechanisms^7-9. Mounting evidence has demonstrated that circulating tumor cells (CTCs) are the precursors of metastases¹⁰. The epithelial-to-mesenchymal transition (EMT) process, which provides CTCs with several prometastatic traits^11,12, is also implicated in the metastatic process¹³. Generally, epithelial-type CTCs can gain more mesenchymal traits to increase their invasive ability via EMT¹⁴, thereby overcoming antimetastatic bottlenecks^15,16 and achieving great potential for metastasis^17-20. Accumulating evidence revealed that the counts of CTCs, especially mesenchymal-type CTCs (^MCTCs) ²¹, are correlated with tumor metastasis^22,23. Therefore, further illumination of the molecular mechanism of CTC EMT will allow us to understand the mechanisms of CRC liver metastasis better.

The activation of EMT stems from the interplay of many different factors, one of which is the Hippo signaling pathway^24-27. As a downstream transcriptional coactivator of the Hippo pathway, the abnormal expression of yes-associated protein 1 (YAP1) promotes malignant tumor proliferation and metastasis, induces EMT, and may induce drug resistance^28-30. On the other hand, YAP1 in its active form could function as a transcriptional coactivator predominantly mediated by interaction with TEAD transcription factors³¹. MiR-195-5p has been demonstrated to be a tumor suppressor in various cancers³². In our previous study, we also demonstrated that miR-195-5p could potently inhibit the process of EMT via YAP1 in CRC³³. However, we did not find a significant change in miR-195-5p expression when we upregulated or downregulated YAP1. Therefore, further exploration of the upstream miR-195-5p/YAP1 axis has great significance for further clarifying the mechanism of CRC EMT.

Long noncoding RNAs (lncRNAs), transcripts longer than 200 nucleotides with no or limited protein-coding potential³⁴, are also implicated in EMT activation^35,36. The complicated secondary structure of lncRNAs contains many binding sites, which allows lncRNAs to drive many important tumor cellular phenotype changes through their interactions with RNA, chromatin, and protein^34,37. Consequently, lncRNAs could regulate the process of EMT in a variety of ways^38,39. For instance, lncRNAs can function as microRNA (miRNA) sponges to sequester miRNAs from endogenous target mRNAs, thus affecting tumor progression⁴⁰. Among many cancer-related lncRNAs, small nucleolar RNA host gene 16 (SNHG16) is mainly located in the cytoplasm and was initially identified as an oncogene in neuroblastoma⁴¹. It has been demonstrated that lncRNA SNHG16 can regulate cancer cellular proliferation, invasion, EMT, and chemoresistance^42,43; meanwhile SNHG16 can also regulate the migration, invasion, and lipid metabolism of CRC cells, thereby playing pivotal roles in CRC progression⁴⁴. Bioinformatics analysis also found that lncRNA SNHG16 was one of the most upregulated lncRNAs in CRC. Intriguingly, using bioinformatics, we also found the same miR-195-5p response elements between SNHG16 and YAP1. Moreover, YAP1 was the most significantly changed EMT-related transcription factor when we altered the expression of SNHG16 in CRC. Therefore, whether lncRNA SNHG16 could act upstream of miR-195-5p, thus regulating YAP1 expression by posttranscriptional modification, deserves to be further explored. More importantly, as a critical transcriptional coactivator, it remains largely unknown whether YAP1 can activate the transcription of SNHG16.

The present study found that SNHG16 was upregulated in CRC and significantly associated with a poor prognosis in CRC patients. Cox-regression analysis revealed that SNHG16 represented an independent prognostic biomarker in CRC. According to loss- and gain-of function analysis, SNHG16 could regulate CRC proliferation, migration, invasion, EMT, ^MCTC generation, and liver metastasis. Next, we demonstrated that YAP1 was the functional mediator of SNHG16 and that SNHG16 fulfilled its oncogenic role in a YAP1 dependent manner. Mechanistically, SNHG16 could act as a miRNA sponge to sequester miR-195-5p on Ago2, thereby protecting YAP1 from repression. Moreover, YAP1 could combine with TEA domain transcription factor 1 (TEAD1) to form a complex, which in turn binds with the promoter of SNHG16 to regulate its transcription. Herein, we clarified a previous unexplored positive feedback loop between SNHG16 and YAP1/TEAD1. Through this positive feedback loop, SNHG16 promotes CTC EMT and CRC liver metastasis. These findings provide a novel mechanism for CRC liver metastasis and indicate that it may be a potential candidate target in CRC treatment.

SNHG16 is upregulated in CRC tissues and indicates a poor prognosis in CRC patients

To identify the lncRNAs involved in CRC progression, we analyzed the lncRNA expression profiles of CRC in the GSE84984 dataset. A series of abnormally expressed lncRNAs in CRC were identified, and SNHG16 was among the most upregulated lncRNAs (Figure 1A). The top 10 upregulated and downregulated lncRNAs are shown in the heatmap (Figure 1A). The volcano plot shows the upregulated and downregulated lncRNAs in CRC (Figure 1B). As aforementioned, lncRNA SNHG16 plays an important role in tumor progression. Thus, lncRNA SNHG16 was selected for further research.

Initially, we sought to determine the levels of SNHG16 expression from The Cancer Genome Atlas (TCGA) database. As shown in Figure 1C, the expression of SNHG16 was significantly upregulated in CRC tissues (p < 0.001). We further measured the expression of lncRNA SNHG16 in 45 matched normal colorectal and cancerous colorectal tissues via qRT-PCR. LncRNA SNHG16 was significantly upregulated in CRC tissues compared with paired adjacent normal tissues (PANTs) (Fig 1D) and correlated with the advanced tumor-node-metastasis (TNM) stage (Fig 1B).

We then separated 111 CRC tissue samples into two groups based on the median value of SNHG16 expression level. Subsequently, we investigated the correlation between lncRNA SNHG16 expression and clinicopathological significance (Table 1). Evidently, the high expression of lncRNA SNHG16 was positively correlated with lymphovascular invasion (LVI), perineural invasion (PNI), TNM stage (p < 0.001), lymph node metastasis (p < 0.05), and distant metastasis (p < 0.001). Further Kaplan-Meier analysis revealed that SNHG16 high expression was associated with poor overall survival (OS) (p < 0.05) and progression-free survival (PFS) (p < 0.05) in the TCGA cohort (Figure 1F) and our cohort (Fig 1G and Figure 1H).

Cox regression analysis (Table 2) revealed that SNHG16 expression was an independent prognostic factor correlated with poor OS (HR = 3.125, 95%CI = 1.145-8.47). Similarly, we found that lncRNA SNHG16 was significantly upregulated in seven CRC cell lines (Fig 1I); moreover, we detected the highest expression level of SNHG16 in HCT116 and the lowest expression of SNHG16 in DLD1, and thus, these two cell lines were selected for further research. Additionally, in situ hybridization (ISH) showed that SNHG16 was mainly located in the cytoplasm of CRC tumor cells but not in PANT (Fig 1J). Taken together, these results strongly indicated that SNHG16 is upregulated in CRC and is an independent prognostic factor.

The ectopic expression of lncRNA SNHG16 affects the proliferation, migration, invasion, and EMT of CRC cells.

To investigate the role of SNHG16 in tumor progression, loss- and gain-of-function approaches were applied. First, three siRNA sequences of SNHG16 were designed, and qRT-PCR was used to verify the most efficient sequence (Fig S1A). Based on the results, the sequence of si-SNHG16 (si-3) was used to perform a lentiviral-based SNHG16 knockdown stable cell line (Fig 2A), termed Lv-anti-SNHG16. Then, we use lentivirus to establish a DLD1 cell line that stably overexpressed SNHG16, termed Lv-Oe-SNHG16 (Fig 2B). Finally, the knockdown and overexpression effects were validated by PCR (Fig 2A and Fig 2B).

As shown in Fig 2C, SNHG16 knockdown HCT116 cells formed fewer colonies than the control group, indicating that cell proliferation was inhibited by SNHG16 knockdown. In contrast, the SNHG16-overexpressing DLD1 cells formed many more colonies than their negative control cells (Fig 2D). Hence, the overexpression of SNHG16 promoted the proliferation of CRC cells. According to the Transwell migration and invasion assay, the knockdown of SNHG16 significantly reduced the number of cells crossing the membrane (Fig 2E), whereas overexpression of SNHG16 increased the number of migrating and invading cells (Fig 2F). Wound healing assays further revealed the positive effect of lncRNA SNHG16 on CRC migration capacity. Evidently, the knockdown of SNHG16 inhibited the migration speed (Fig 2G), yet the migration speed was faster in the SNHG16 overexpressing DLD1 cells than in their control group (Fig 2H).

Given the critical role of EMT in CRC cellular migration and invasion⁴⁵, we next investigated whether SNHG16 could induce EMT in CRC cells. Western blot and immunofluorescence assay revealed that knockdown of SNHG16 significantly reduced the expression levels of vimentin and N-cadherin but increased E-cadherin level in HCT116 (Fig 2I, Fig 2J, and Fig S1B). In contrast, SNHG16 overexpression promoted the expression of mesenchymal markers but inhibited the expression of epithelial markers in DLD1 cells (Fig 2I, Fig 2J, and Fig S1B).

Altogether, these results demonstrated that SNHG16 could regulate CRC cell migration and invasion by affecting the process of EMT.

LncRNA SNHG16 facilitates CRC cell proliferation, migration, invasion and EMT in a YAP1-dependent manner.

To explore the mechanism by which SNHG16 regulates EMT in CRC cells, we focused on identifying an EMT-related transcription factor that could regulate the progression of CRC⁴⁶. As shown in Fig 3A and 3B, a panel of transcription factors was screened, and YAP1 showed the most significant change among them. Western blot also revealed that SNHG16 could positively regulate the protein level of YAP1 (Fig 3C and Fig S2A), indicating a potential interaction between SNHG16 and YAP1. In addition, the positive association between the expression levels of SNHG16 and YAP1 in CRC tissues further demonstrated that YAP1 was a potential target of SNHG16 (Fig 3D). Furthermore, we have previously demonstrated that the miR-195-5p/ YAP1 axis plays a vital role in CRC progression³³. Thus, YAP1 was selected for further research.

To investigate whether the function of SNHG16 in tumor progression was dependent on YAP1, we performed a rescue experiment. The results showed that SNHG16 knockdown could inhibit the proliferation, migration, and invasion of CRC cells. Evidently, YAP1 overexpression rescued the SNHG16-downregulation-mediated effect on proliferation, migration, and invasion (Fig 3E, Fig S2B). Moreover, SNHG16-overexpression facilitated the proliferation, migration, and invasion of DLD1 cells, and the effect of SNHG16-overexpression on tumor progression was abrogated by YAP1 knockdown (Fig 3F, Fig S2C). As expected, YAP1 could rescue SNHG16’s effect on tumor cellular morphology (Fig S 2D and Fig S 2E). Immunofluorescence and western blotting assays revealed that the overexpression of SNHG16 could promote the EMT of CRC cells, and the promotive effect on EMT could be rescued by YAP1 knockdown; moreover, the SNHG16-knockdown-mediated inhibition of EMT could be attenuated by YAP1 overexpression (Fig 3G, Fig 3H, and Fig S 2F). These results revealed that YAP1 was the functional mediator of SNHG16 in CRC.

MiR-195-5p potently abrogated the effect of the SNHG16/YAP1 axis on tumor progression.

To further investigate how the EMT of CRC cells might be controlled dynamically by the SNHG16/YAP1 axis during tumor metastasis, we focused on the involvement of miRNAs^47,48. According to the hypothesis of competing endogenous RNA (ceRNA), certain lncRNAs have many miRNA response elements (MREs) and can function as miRNA sponges to posttranscriptionally regulate target genes in the cytoplasm, thus playing an important role in the progression of CRC^40,49. In our previous research, we demonstrated that miR-195-5p potently inhibited the EMT of CRC through YAP1³³. Subcellular distribution assays showed that SNHG16 was mainly located in the cytoplasm of HCT116 and DLD1 cells, indicating potential posttranscriptional regulation (Fig 4A and Fig 4B). Through starBase⁵⁰ and LncBase Predicted⁵¹, we identified several miRNAs which have potential SNHG16 binding sites, one of which was miR-195-5p. Furthermore, we found the same miR-195-5p response elements between SNHG16 and YAP1 (Fig 4C), which indicates potential interplay among these three factors. Thus, we selected miR-195-5p as potential downstream target of SNHG16 for further research.

We have previously demonstrated that miR-195-5p was markedly downregulated in CRC tissues and cell lines³³; thus, we further investigated the relationship between the expression of SNHG16 and miR-195-5p. The correlation between lncRNA SNHG16 expression and miR-195-5p expression was assessed in fifty CRC tissues. SNHG16 expression was significantly negatively correlated with miR-195-5p expression in CRC tissues (two-sided Pearson’s correlation, r = -0.6820, p < 0.001) (Fig 4D). Additionally, miR-195-5p was also significantly decreased after SNHG16 overexpression in DLD1 cells (Fig 4E). In contrast, miR-195-5p was drastically increased after knockdown of SNHG16 in HCT116 cells (Fig 4F). However, we did not observe significant changes in SNHG16 when we upregulated or downregulated miR-195-5p (Fig 4G and Fig 4H).

Next, we investigated whether the SNHG16/YAP1 axis promotes the migration, invasion, and EMT progression of CRC cells through miR-195-5p. As illustrated in Figure 4I, SNHG16 knockdown inhibited the proliferation, migration and invasion of CRC cells. Evidently, miR-195-5p inhibitor clearly rescued SNHG16 downregulation-mediated inhibitory effect on the proliferation, migration and invasion of HCT116 cells (Fig 4I). In contrast, miR-195-5p mimics abrogated the enhanced proliferation, migration and invasion ability of SNHG16 overexpressing DLD1 cells (Fig 4J). Then, a wound healing assay further revealed that miR-195-5p could rescue the effect of SNHG16 on migration in CRC cell lines (Fig 4k-L). Furthermore, the inhibition of EMT in HCT116 cells with SNHG16 knockdown could be rescued by miR-195-5p inhibitors (Fig 4M and Fig S 3A). Moreover, miR-195-5p mimics significantly reversed the enhancement of EMT in DLD1 cells with SNHG16 overexpression (Fig 4N and Fig S 3B). In our previous research, we demonstrated that miR-195-5p could also regulate the progression of CRC through YAP1³³. Based on current and previous research, we successfully demonstrated that miR-195-5p could abrogate the effect of the SNHG16/YAP1 axis on tumor progression.

lncRNA SNHG16 functions as a ceRNA and sponges miR-195-5p, further regulating YAP1 expression and facilitating tumor progression.

Next, we sought to examine whether SNHG16-mediated miR-195-5p regulation occurs through direct binding with miR-195-5p. Therefore, RNA-binding protein immunoprecipitation (RIP), biotin-avidin pulldown, and luciferase assays were applied to conduct further research.

MiRNAs are present in the form of miRNA ribonucleoprotein complexes (miRNPs) that contain argounaute 2 (Ago2), the core component of RNA-induced silencing complexes (RISCs)⁵². To validate whether SNHG16 associates with miR-195-5p on Ago2, we performed RIP assays with Ago2 on HCT116 cells transfected with miR-195-5p inhibitor or transfected with negative control (Fig 5A). As shown in Fig 5B, Ago2 protein was successfully immunoprecipitated from HCT116 extracts with the Ago2 antibody. SNHG16 expression was significantly decreased in Ago2 immunoprecipitates purified from miR-195-5p inhibitor-transfected cells, compared with Ago2 immunoprecipitates purified from their negative control cells (Fig 5C). Thus, the RIP assay revealed that SNHG16 was likely in the miR-195-5p-RISC and interacts with miR-195-5p. Additionally, the sequence-specific binding between miR-195-5p and SNHG16 was further validated by a biotin-avidin pulldown system. As Fig 5D shows, the expression of SNHG16 in the miR-195-5p-Bio group was approximately 30 times higher than that in the miR-195-5p-Mut-Bio group or control group. Consistent with our results, the luciferase reporter assays revealed that miR-195-5p significantly inhibited the luciferase activity of the SNHG16-wild type (WT) construct but not of the SNHG16-mutant (mut) construct (Fig 5E-F). Collectively, these results revealed that SNHG16 could act as a miRNA sponge to directly bind with miR-195-5p and exert an inhibitory effect on miR-195-5p expression.

As we previously demonstrated³³, upregulation of miR-195-5p suppresses the proliferation, migration, invasion, and EMT of colon cancer cells through targeting the YAP1 mRNA 3’- untranslated region (UTR). Therefore, we wondered whether ectopically expressed SNHG16 prevents miR-195-5p from inhibiting YAP1, thereby affecting the progression of tumors. Through starBase, we found that there were many identical miR-195-5p response elements between SNHG16 and miR-195-5p (Fig 4A). Thus, we speculated that SNHG16 could regulate the mRNA stability of YAP1 through miR-195-5p. Then, a luciferase assay among SNHG16 (WT and mut), miR-195-5p, and YAP1(Luc-YAP1-wt and Luc-YAP1-mut) was performed. Compared with SNHG16-mut, SNHG16-WT enhanced the luciferase activity of YAP1-WT, while miR-195-5p inhibited the luciferase activity of YAP1-WT. Furthermore, the inhibited luciferase activity of miR-195-5p group on YAP1-WT was rescued by SNHG16 (Fig 5G). We did not find significant changes when we conducted the same experiment on YAP1-mut (Fig 5G). The luciferase assay among these three factors revealed that the overexpression of SNHG16 could prevent miR-195-5p from targeting YAP1. Through western blotting and qRT-PCR in a CRC cell line, we also found that SNHG16 could positively regulate the mRNA and protein expression of YAP1. Furthermore, miR-195-5p rescued SNHG16-mediated regulation on YAP1 (Fig 5H-K, Fig S 4A-4B).

Altogether, these findings indicate that SNHG16 could function as a miRNA sponge to directly bind and sequester endogenous miR-195-5p, thereby preventing it from inhibiting YAP1 expression and affecting tumor progression.

YAP1 combines with TEAD1 to form a complex that binds to the promoter region of SNHG16 and activates its transcription.

Since YAP1 functions as an important transcriptional coactivator, the positive correlation between the expression of SNHG16 and YAP1 drew us to investigate the regulatory role of YAP1 in SNHG16 transcription. Then, we constructed a lentivirus-mediated HCT116 cell line that stably overexpressed or knocked down YAP1, termed Lv-Oe-YAP1 or Lv-anti-YAP1, respectively. Overexpression of YAP1 significantly clearly promoted SNHG16 expression (Fig 6A), whereas knockdown of YAP1 dramatically suppressed the expression level of SNHG16 (Fig 6B). Furthermore, knockdown of YAP1 inhibited the proliferation (Fig S5A), migration (Fig S5C and Fig S5E), invasion (Fig S5C), and EMT (Fig 6C and Fig S 5G) of CRC cell lines. In addition, we observed a promotive effect on tumor progression in the Lv-Oe-YAP1 group (Fig S5B, Fig S5D, Fig S5F, Fig 6C and Fig S5H).

Although we have demonstrated the important role of YAP1 in SNHG16 expression and tumor progression, the mechanism of the YAP1-lncRNA interaction remains largely unknown. Based on existing research, YAP1 cannot bind directly with DNA alone. However, YAP1 can combine with transcription factors to regulate the transcription of downstream genes⁵³. Among these transcription factors, TEAD1-4 are some of the most common binding molecules⁵⁴. Once bound to TEAD, YAP1 forms a YAP1/TEAD complex to initiate downstream gene transcription^53,55. Through bioinformatic analysis, we found the potential transcription factor TEAD1. Further study showed that the knockdown of either YAP1 or TEAD1 reduced the expression of SNHG16 (Fig 6D). And evidently, the inhibitory effect on the expression of SNHG16 was most significant when we simultaneously knocked down both YAP1 and TEAD1 (Fig 6D). To investigate whether YAP1 and TEAD1 act synergistically, we overexpressed TEAD1 in the Lv-anti-YAP1 cell line and did not find a significant change in the expression of SNHG16 (Fig 6D). Similarly, when we overexpressed TEAD1 after YAP1 knockdown, the expression level of SNHG16 was not rescued (Fig 6D). To further verify the cross-talk between TEAD1 and YAP1, coimmunoprecipitation (Co-IP) analysis was applied. Co-IP confirmed that YAP1 could interact with TEAD1 directly or indirectly in HCT116 cells (Fig 6E). To further investigate whether YAP1 interact with TEAD1 through direct binding or not, a GST pulldown assay was performed. After GST-tagged YAP1 (Fig 6F) and His-tagged TEAD1 (Fig 6G) were successfully induced and purified from Escherichia coli, we incubated these two proteins at 4℃ for 4-8 h. Finally, western blotting was performed to examine the expression of these two proteins. GST pulldown further demonstrated the direct interplay between YAP1 and TEAD1 (Fig 6H). Collectively, these experiments confirmed that YAP1 could directly interact with TEAD1 and form a complex, that regulated the expression of SNHG16. However, whether YAP1/TEAD1 binds to the promoter of SNHG16 remains largely unclear.

We employed JASPAR (http://jaspar.genereg.net) and TRANSFAC (http://gene-regulation.com), we did not find possible YAP1 binding sites within 2000 bp upstream of the transcriptional start of SNHG16; however, there were three possible TEAD1 binding sites (Fig 6I). Therefore, the interplay between the YAP1/TEAD1 complex and SNHG16 was further assessed. Significantly, dual-reporter luciferase assays showed that overexpression of either YAP1 or TEAD1 in HCT116 cells stimulated the promoter activity of SNHG16 (Fig 6J). And evidently, the promoter activity was most significant when we simultaneously overexpressed both YAP1 and TEAD1 (Fig 6J). To verify the binding of SNHG16 and TEAD1, we generated a series of 5’ deletion constructs of the SNHG16 promoter. According to the results of the luciferase assay, the regulatory region between -1794 and -1357 was responsible for YAP1/TEAD1-mediated promoter regulation (Fig 6K). As shown in Figure 6I, two binding sites are located in this region. Thus, luciferase assays were further performed. YAP1/TEAD1 failed to stimulate mutants of both predicted sites in the promoter region of SNHG16 in 293T cells, while it did affect the WT construct (Fig 6L). Furthermore, in the chromatin immunoprecipitation (ChIP) assay, we designed two primer sets, one that contained site 1 and one that contained site 2. Then, we used the primers and purified DNA from the ChIP assay to amplify part of the promoter region. The ChIP assays revealed that TEAD1 directly bound to both site 1 and site 2 of the SNHG16 promoter in HCT116 cells (Fig 6M).

The results indicated that YAP1 could directly bind with TEAD1 to form a complex, which could, in turn, bind to the promoter of SNHG16 and regulate its transcription. During this process, YAP1 is indispensable for the function of TEAD1, and these two acts synergistically.

The effect of the YAP1-SNHG16 positive feedback loop on the progression of tumors

We demonstrated that SNHG16 was a direct target of the YAP1/TEAD1 complex, and positive feedback regulation existed between SNHG16 and YAP1. As mentioned above, YAP1 could regulate the expression of SNHG16 and promote tumor progression. To investigate the role of the YAP1/TEAD1-SNHG16 positive feedback loop in tumor progression, subsequent experiments were performed. Colony formation, Transwell and Wound healing assays revealed that the overexpression of YAP1 significantly promoted tumor proliferation, migration and invasion, whereas the enhanced proliferation, migration and invasion of HCT116 cells was impaired by simultaneous knockdown of SNHG16 (Fig S6A and Fig S6C). Similarly, SNHG16 overexpression rescued the impaired proliferation, migration and invasion of Lv-anti-YAP1 stable cell line (Fig S6B and Fig S6D). In addition, SNHG16 rescued the effect of YAP1 on CRC EMT (Fig 6N, Fig S6E and Fig S6F).

Alteration of lncRNA SNHG16 expression influenced CRC tumorigenesis and^MCTC generation in vivo

To verify the above results in vivo, a nude mouse xenograft experiment was performed. The stable cell lines HCT116/ Lv-anti-SNHG16-NC, HCT116/ Lv-anti-SNHG16 + Lv-Oe-YAP1 NC, and HCT116/ Lv-anti-SNHG16 + Lv-Oe-YAP1 were injected into the flanks of nude mice separately. During the period of tumor growth, both the tumor weight and tumor volume were measured. Evidently, knockdown of SNHG16 clearly inhibited the growth of tumors. Furthermore, the inhibitory effect of SNHG16 on tumor growth was rescued by YAP1 overexpression (Fig 7A, B and C).

Immunohistochemistry (IHC) was performed to detect the expression of YAP1 and EMT-related factors in xenograft tumor, and the results was also represented by integral optic density (IOD) (Figure S7A). Significantly, Ki67 staining was decreased in the Lv-anti-SNHG16 group, indicating inhibited proliferation of tumor. Moreover, overexpression of YAP1 significantly attenuated the inhibition of Ki67 expression (Fig 7D and Fig S7A). Importantly, IHC staining also demonstrated the in vitro result that SNHG16 could regulate the expression of YAP1 and the process of EMT. Moreover, YAP1 overexpression rescued SNHG16 knockdown-mediated inhibition of EMT (Fig 7D and Fig S7A). Through the process of EMT, cancer cells can shed from tumors and invade into the blood, thereby forming CTCs and facilitating tumor metastasis. Thus, we detected CTCs from mouse blood (Fig 7E). We then detected representative markers of EMT on CTCs. As shown in Figure 7F, SNHG16 regulated the expression of YAP1 and EMT markers, and YAP1 overexpression rescued SNHG16 knockdown-mediated inhibition of EMT marker expression. Obviously, Lv-anti-SNHG16 significantly decreased the rate of ^MCTC generation, compared with that in the negative control group (Fig 7G); moreover, YAP1 overexpression rescued SNHG16 knockdown-mediated inhibition of ^MCTC generation (Fig 7G).

The stable cell lines HCT116/Lv-anti-SNHG16 NC, HCT116/Lv-anti-SNHG16 + Lv-Oe-YAP1 NC, and HCT116/Lv-anti-SNHG16 + Lv-Oe-YAP1 were separately injected into the tail vein to confirm the effect of SNHG16 on tumor metastasis. Representative images of CRC liver metastasis and lung metastasis are presented in Fig 7H and 7I, respectively. Further analysis revealed that the knockdown of SNHG16 significantly inhibited the metastasis of tumors. In addition, the overexpression of YAP1 rescued the inhibitory effect of SNHG16 knockdown on tumor metastasis (Fig 7H and 7I). Collectively, these results demonstrated that SNHG16 could promote tumor growth, metastasis, and ^MCTC generation. And what’s more, YAP1 could rescue the effect of SNHG16 on tumor progression.

We summarized our mechanistic findings in a schematic diagram (Fig 7I). Our study illustrated a positive feedback loop between lncRNA SNHG16 and YAP1/TEAD1 complex. Mechanistically, SNHG16 could act as miRNA sponge to sequester miR-195-5p on Ago2, thereby protecting YAP1 from repression. Moreover, YAP1 could also combine with TEAD1 to form a YAP1/TEAD1 complex, which could bind with the promoter of SNHG16 to regulate its transcription. By this positive feedback loop, SNHG16 promotes CRC migration, invasion, ^MCTC generation, lung metastasis, and liver metastasis.

Extensive efforts in the past have been made to explore the cellular and molecular mechanism of CRC liver metastasis, one of the main causes of treatment failure for advanced CRC. Recently, lncRNAs have gained a great deal of attention as critical regulators of tumor invasion and metastasis^56,57. For example, Tang et al. found that Rho/ROCK signaling-associated lncRNAs are useful for combating cancer metastasis⁵⁸. These researches indicates that lncRNAs play vital roles in cancer metastasis, and the research for lncRNA in CRC has great significance. In the present study, we discovered a positive feedback loop involving lncRNA SNHG16-YAP1/TEAD1 in CRC. Generally, lncRNA SNHG16 could act as a miRNA sponge to sequester miR-195-5p on Ago2, thereby protecting YAP1 from repression. Moreover, YAP1 could also combine with TEAD1 to form a YAP1/TEAD1 complex, which in turn binds with the promoter of SNHG16 to regulate its transcription. Through this positive feedback loop, SNHG16 could promote CRC cell proliferation, migration, invasion, EMT, ^MCTC generation, and liver metastasis.

First, bioinformatics analysis demonstrated that lncRNA SNHG16 was one of the most upregulated lncRNAs in CRC. Next, the result was further demonstrated by qRT-PCR analysis of our cohort and the TCGA cohort. Clinically, elevated levels of lncRNA SNHG16, which were mainly located in CRC cytoplasm, were positively correlated with TNM stage, LVI, PNI, distant metastasis, and a poor prognosis for cancer patients, indicating its potential regulation of CRC dissemination and invasion. Recently, accumulating evidence has revealed that lncRNAs can regulate the EMT of CRC cells, thus promoting tumor migration and invasion^59,60. In this study, our results potently demonstrated that SNHG16 exerted an oncogenic role in CRC by promoting cell proliferation, migration, invasion, and EMT. CTCs, which are considered as the precursors of metastases, play vital roles in the process of metastasis¹⁰. According to recent research, CTCs shed from primary tumors could gain more mesenchymal traits via EMT program, which increases their invasive ability and metastatic ability⁶¹. Qi et al. also demonstrated that an ^MCTC percentage ≥ 2% prior to resection was significantly associated with early recurrence, multi-intrahepatic recurrence, and lung metastasis in hepatocellular carcinoma (HCC)⁶². Currently, our result revealed that the ^MCTC ratio was significantly downregulated after SNHG16 knockdown. Meanwhile, SNHG16 knockdown significantly inhibited the EMT of CTCs and the liver metastasis and lung metastasis of CRC. Our results indicate that, SNHG16 could regulate the EMT state of CTCs, thus facilitating the liver metastasis and lung metastasis of CRC. Given the key role of EMT activation in cancer metastasis, we screened a panel of EMT-related transcription factors. Among them, YAP1 was the most significantly changed in the loss- and gain-of-function experiments for SNHG16. According to previous research, YAP1 plays vital roles in the proliferation, invasion, EMT, and metastasis of many types of cancers^63-66. In addition, YAP1 is implicated in the EMT of mammary epithelial cells^67,68. In our recent research, we also demonstrated the role of miR-195-5p/Hippo-YAP in EMT activation³³. Thus, we hypothesized that SNHG16 could affect the process of EMT through YAP1. Consistent with previous studies^69,70, our study clearly demonstrated that YAP1 could significantly promote the proliferation, migration, invasion, and EMT of CRC cell. Next, we also found a positive correlation between SNHG16 expression and YAP1 expression in CRC tissue, indicating that SNHG16 could regulate YAP1 expression. Subsequently, we further demonstrated the positive regulation between SNHG16 and YAP1 in a CRC cell line. Finally, functional analysis revealed that YAP1 could rescue the SNHG16-mediated effect on CRC metastasis in vitro and in vivo. To our knowledge, this study provides the first evidence that SNHG16 could regulate the process of EMT through YAP1, thus facilitating ^MCTC generation and liver metastasis of CRC.

Currently, mounting evidence suggests that lncRNAs can act as sponges of miRNAs to impact the expression of mRNA⁴⁰. For instance, lncRNA-ATB could act as a miRNA sponge to inhibit miR-200, thus regulating the expression of ZEB1 and inducing EMT⁷¹. In addition, lncRNA-HOXD-AS1, which is highly expressed in HCC, could act as a ‘sponge’ to prevent SOX4 from miR-130-3p-mediated degradation, leading to HCC metastasis⁵⁷. Subsequent ISH assays and Subcellular distribution assays in our study showed that SNHG16 is mainly located in the cytoplasm of CRC, indicating a potential role in posttranscriptional regulation. In our previous research, YAP1 was identified as a target gene of miR-195-5p in CRC³³. Intriguingly, there exists same miR-195-5p response elements between SNHG16 and YAP1. Thus, we speculated that SNHG16 could act as a miRNA sponge to protect YAP1 from miR-195-5p-mediated inhibition. Here, the inverse correlation between SNHG16 expression and miR-195-5p further indicated the potential targeted regulatory relationship between SNHG16 and miR-195-5p. Using bioinformatic analyses, biotin-labeled miR-195-5p pulldown assay, anti-Ago2 RIP assays, and luciferase assays, we clearly demonstrated that SNHG16 could directly bind to and negatively regulate miR-195-5p expression. Both lncRNA SNHG16 and YAP1 interact with miR-195-5p, indicating that SNHG16 may regulate YAP1 expression by competing with miR-195-5p. Unsurprisingly, our research revealed that miR-195-5p could rescue the effect of SNHG16 on the 3’-UTR luciferase activity of YAP1, the expression level of YAP1, and tumor progression. For the first time, we strongly confirmed that SNHG16 could act as a miRNA sponge to sequester miR-195-5p, thereby protecting YAP1 from repression and facilitating tumor progression. Based on our previous research³³, this research further illuminates the role and molecular mechanism of CTC EMT in liver metastasis of colorectal cancer. Interestingly, Chen et al and He et al also reported that lncRNA SNHG16 could promote CRC progression through miR-124-3p and miR-132-3p^72,73, respectively. In contrast to these two studies, we comprehensively described the upstream and downstream factors of SNHG16. Moreover, we further revealed a positive feedback loop between SNHG16 and the YAP1/TEAD1 complex, which could facilitate ^MCTC generation and CRC liver metastasis. However, it remains unclear which mRNAs affect most of these three microRNAs in CRC. In addition, considering the complex function of lncRNAs in cancer^34,37,74, it remains unclear whether SNHG16 could bind certain proteins or regulate other pathways to exert its effect on CRC progression. These issues should be further investigated in future research. Collectively, these findings indicate that the crosstalk between RNAs (e.g., lncRNAs and mRNAs) facilitates the formation of a large-scale regulatory network involving different pathway. Thus, ceRNA networks may pave a new way for tumor therapeutic regimens.

The positive regulation of YAP1, an important transcriptional coactivator, by SNHG16 led us to investigate the role of YAP1 in SNHG16 transcription. Evidently, we demonstrated for the first time that YAP1 could activate the transcription of SNHG16. However, the molecular mechanism underlying the YAP1-lncRNA interaction remains unknown. According to previous research, YAP1 cannot regulate transcription alone but combines with downstream transcription factors^53,55,75, which mainly consist of TEAD1-4⁷⁶. Through the N-terminal TEA DNA binding domain, TEAD can bind to the C-terminal region of YAP1^77,78. Once bound to TEAD, YAP1 relies on the DNA binding domain of TEAD to initiate downstream gene transcription^53,55,75. Shen et al. found that the YAP1/TEAD1 complex could transform normal fibroblasts (NFs) to cancer-associated fibroblasts (CAFs) by regulating SRC⁷⁹. Lamar also found that, YAP1 could promote metastasis through its TEAD-interaction domain⁶⁸. In this study, we found the potential downstream transcription factor TEAD1 via UCSC, JASPAR, and TRANSFAC. We then revealed that, the knockdown of either YAP1 or TEAD1 could inhibit the expression of SNHG16, and the inhibitory effect was most significant when we knocked down both TEAD1 and YAP1 simultaneously. qRT-PCR and luciferase assays further revealed that, only when YAP1 and TEAD1 act synergistically can they regulate the transcription of SNHG16. By performing Co-IP and GST pulldown assay, we demonstrated the direct interaction between YAP1 and TEAD1. Unsurprisingly, a dual-luciferase assay revealed that the SNHG16 promoter regulatory region between -1794 and -1367 was responsible for YAP1/TEAD1-mediated transcription. What’s more, there were two predicted binding sites in this region. Compared with WT, YAP1/TEAD1 failed to stimulate the mutants of both predicted sites in the promoter region of SNHG16 in HCT116 cells. ChIP assays also showed that TEAD1 could bind to both predicted sites of the SNHG16 promoter. Herein, we potently demonstrated that YAP1 could directly combine with TEAD1, forming a complex that could bind to the promoter of SNHG16 and regulate its transcription. Finally, we revealed that YAP1 significantly promoted tumor progression and that SNHG16 rescued the effect of YAP1 on tumor progression. Therefore, SNHG16 and YAP1 form a positive feedback loop to regulate tumor progression.

Overall, we demonstrated that SNHG16 was upregulated in CRC and significantly associated with a poor prognosis in CRC patients. According to Cox-regression analysis, SNHG16 may represent an independent prognostic biomarker in CRC patients. Loss- and gain-of function analysis revealed that SNHG16 could regulate CRC proliferation, migration, invasion, EMT, ^MCTC generation, and liver metastasis. Next, we demonstrated that YAP1 was the functional mediator of SNHG16, and that SNHG16 fulfilled its oncogenic role in a YAP1 dependent-manner. Mechanistically, SNHG16 could act as a miRNA sponge to sequester miR-195-5p on Ago2, thereby protecting YAP1 from repression. Moreover, YAP1 could also combine with TEAD1 to form a YAP1/TEAD1 complex, which in turn binds with the promoter of SNHG16 to regulate its transcription. Finally, we revealed that YAP1 significantly promoted tumor progression and that SNHG16 could rescue the effect of YAP1 on tumor progression. Thus, there exists a positive feedback loop between YAP1/TEAD1 and SNHG16. Through this positive feedback loop, SNHG16 promotes CTC EMT and CRC liver metastasis. These novel results suggested that the SHNG16-YAP1 positive feedback loop may be a potential therapeutic target for CRC.

Dataset’s analysis

The CRC-related RNA-sequencing dataset GSE84984 was obtained from the Gene Expression Omnibus (GEO) database. The GSE84984 dataset contains nine tumor tissues and six normal tissues. Then, we filtered the background noise of the gene expression profile and then performed the data analysis using R software. Next, the differentially expressed lncRNAs were identified as follows: FDR (adjusted p-value) <0.05 and |log2-fold change|>1. Differential expression analysis was performed using the “limma” package in R. In addition, a volcano plot was generated to show the upregulated and downregulated lncRNAs. The top10 downregulated and upregulated lncRNAs are shown in the heatmap.

To analyze the expression of SNHG16 and the prognosis of CRC patients, we performed overall survival analysis. First, 512 cases of CRC and matched clinical information were obtained from the TCGA database (https://cancergenome.nih.gov/). The RNA-seq data were generated on the Illumina HiSeq 2000 RNA Sequencing platform. Finally, CRC patients without complete survival analysis were excluded.

Patient samples

The Research Ethics Committee approved this study of Wuhan University (Wuhan, Hubei, PR China). Informed consent was obtained from all participating patients. The enrolled patients were diagnosed with primary CRC by histopathologic diagnosis, underwent surgeries and had complete prognostic information. No patients received local or systemic neoadjuvant radiotherapy or/and chemotherapy and targeted therapy. One hundred and eleven human CRC tissues and PANTs (distance to cancer > 5 cm) were randomly obtained from patients in the Zhongnan Hospital of Wuhan University between January 2014 and December 2015. Each sample was snap-frozen in liquid nitrogen and then stored at -80°C.

CTC isolation and identification

The isolation and enrichment of CTCs were performed by a CTCBIOPSY device (Wuhan YZY Medical Science and Technology Co., Ltd., Wuhan, China), which was described in our previous research⁸⁰. According to the manufacturer’s instructions, we diluted 1 ml mouse blood into 5 ml with 0.9% sodium chloride solution, and the total liquid was then transferred to ISET tubes with an eight μm diameter aperture membrane. Through positive pressure from 12 to 20 mmHg in ISET tubes, candidate CTCs were adhered to the ISET tube membrane and identified by three-color immunofluorescence.

Cell culture and treatment

The human colon cancer cell lines DLD1, Caco2, SW480, SW620, HT29, lovo, and HCT116 and the normal intestinal epithelium cell line NCM460 were obtained from the Cell Bank of Wuhan University. Cells were maintained in an incubator at 37℃ and 5% CO₂.Cells were cultured in DMEM (Gibco, USA) supplemented with 2 mmol/L glutamine and 10% fetal calf serum (Gibco, USA).

Transient transfection and stable transfection

To induce miR-195-5p overexpression and inhibition, cells were transfected with miR-195-5p mimics and inhibitors (RiboBio Co. Ltd., China) using Lipofectamine 2000. To verify the transfection efficiency, miR-195-5p mimic negative control and miR-195-5p inhibitor negative control were included in each transfection experiment. For SNHG16, a pool of three siRNAs was purchased from GenePharma and transfected into cells supplemented with Lipofectamine 2000 to select the best knockdown effect.

To construct stable transfection cell lines, anti-SNHG16 lentiviral vectors (Lv-anti-SNHG16) and their nonspecific lentiviral vectors (Lv-anti-SNHG16 NC) containing green fluorescent protein (GFP) were constructed by Genepharma (Shanghai, China). In addition, SNHG16-overexpression lentiviral vectors (Lv-Oe-SNHG16) and their nonspecific lentiviral vectors (Lv-Oe-SNHG16 NC) containing mCherry were purchased from GeneCopoeia (Wuhan, China). We also induced the overexpression and inhibition of YAP1 by transfecting cells with lentiviral vectors (Lv-Oe-YAP1/Lv-Oe-YAP1 NC, Lv-anti-YAP1/Lv-anti-YAP1 NC) from GeneChem Chemical Technology (Shanghai, China). According to the manufacturer’s instructions, we obtained the multiplicity of infection (MOI) values of HCT116 (MOI = 10) and DLD1 (MOI = 20) cells. For virus infection, virus and polybrene (final 7 μg/ml, Sigma Aldrich, Cat# 107689) were added to cells with 25% confluence. Fresh media was added 16 h after infection. The medium was changed to medium containing appropriate antibiotics 48 h after infection. After puromycin selection, the cells were maintained for at least one day without drug for further experiments.

qRT-PCR and subcellular fractionation

Total RNA was extracted from tissue samples and cell lines using a the TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. A Nanodrop2000 was used to quantify RNA concentration, and 1μg of total RNA was reverse transcribed into cDNA. We performed cDNA synthesis from mRNA by using the PrimeScript™ RT Reagent Kit (Vazyme, Nanjing, China). cDNA synthesis for microRNA detection was carried out using miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). cDNA was used for subsequent qRT-PCR using SYBR-Green PCR Master Mix (Vazyme, Nanjing, China). Each reaction was run on a BioRad IQ5 Real-time PCR instrument (BioRad, USA). Relative expression was calculated using the 2^-ΔΔCt method. The primer sequences are listed in Table S1 (supplementary Table 1).

According to the manufacturer’s instructions, cytosolic and nuclear fractions of HCT116 and DLD1 were isolated by the Nuclear/Cytoplasmic Extraction Kit (Cayman, USA). Subsequently, the RNA levels of a nuclear control transcript (U6), cytoplasmic control transcript (GAPDH), and SNHG16 were analyzed by qRT-PCR.

Western blotting and antibodies

Cells were lysed using RIPA buffer supplemented with complete proteinase inhibitor cocktail (Thermo Scientific, USA). Then, the lysate was homogenized using sonication and quantified using the BCA reaction. Total proteins were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Millipore, USA). Prior to incubation with diluted primary antibodies and HRP-conjugated secondary antibodies, blots were blocked in nonfat milk for an hour. The primary antibodies and dilution ratios were as follows: anti-E-cadherin (1:1000; CST, USA), anti-Vimentin (1:1000; Proteintech, China), anti-YAP1 (1:1000; Proteintech, China), and anti-GAPDH (1:5000; CST, USA). Quantitation on all western blots has been performed by Image J, and the results were normalized by the blank group. Finally, all the bar graphs of protein quantitation were placed on a supplementary file.

Luciferase reporter assay

To evaluate whether miRNAs regulate SNHG16, the SNHG16-WT and an SNHG16-mut sequence (mutant in the miR-195-5p binding site) were amplified from the genomic DNA and subcloned into the pmiR-RB-Report plasmid (Promega, USA), we called the corresponding constructs h-SNHG16-WT and h-SNHG16-mut, respectively. To assess whether miR-195-5p regulates YAP1 directly, we incorporated YAP1-WT and YAP1-mut 3’-UTRs into pmiR-RB-Report luciferase reporter vectors (Promega, USA). For the SNHG16 promoter assay, the SNHG16 promoter (-2000/+1) and modified constructs (-1861/+1, -1794/+1, -1368/+1) were amplified from the genomic DNA and inserted into the pGL3-Basic plasmid (Promega, USA). Then, the cDNA of the SNHG16 promoter containing the predicted TEAD1 binding sites was subcloned into the pGL3-Basic vector (Promega, USA), termed PGL3-SNHG16-pro. Then, the TEAD1 binding site mutated SNHG16 promoter was synthesized by RiboBio (RiboBio Co. Ltd., China), termed as pGL3-SNHG16-pro-mut (1 and 2). According to the manufacturer's instructions, the transfection was carried out using Lipofectamine 2000, and luciferase activities were determined using the dual-Luciferase reporter system (Promega, USA).

RIP assay

According to the instructions of the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA), we performed RIP to investigate the binding of miRNA-195-5p to lncRNA SNHG16. Anti-Ago2 antibodies (CST, USA) were used for immunoprecipitation, and the species anti-IgG antibodies and total RNA (input controls) were used as controls. The co-precipitated RNAs were reverse transcribed to cDNA and detected by qRT-PCR.

Co-IP

CRC cells were lysed in RIPA buffer and the protein concentration of lysates was quantified using the BCA reaction. After impurities were removed, the clarified lysates were incubated with rabbit anti-YAP1 or anti-TEAD1 overnight with end-over-end shaking at 4°C. Subsequently, we used the Protein A/G Sepharose beads to capture the antigen-antibody complexes at 4°C for 12 h. Then, the beads were washed with PBS, and the complexes were boiled to separate antigens and antibodies from the beads. After elution from the beads, the expression levels of YAP1 and TEAD1 in the IP were analyzed by western blotting.

Pulldown assay with biotinylated miRNA

To assay whether miR-195-5p binds to lncRNA SNHG16, a biotin-avidin pulldown system was applied. Biotinylated miR-195-5p was synthesized by RioBio (RiboBio Co. Ltd., China), we called it miR-195-5p-Bio. Then, biotinylated miR-195-5p with mutated SNHG16 binding sites was also synthesized in RiboBio, termed miR-195-5p-mut-Bio. We also used NC-Bio as a negative control to ensure the accuracy of the result, and the operation process was as follows. Briefly, CRC cells were transfected with biotinylated miR-195-5p with the aid of Lipofectamine 2000 and then were rinsed and lysed in buffer 48 h later. Then, the cell lysates were treated with streptavidin magnetic beads, and the bound RNAs were isolated by TRIzol LS reagent for further RT-PCR analysis.

ChIP

We performed ChIP using the Simple ChIP ® Enzymatic Chromatin IP Kit (ChIP) Kit (Cell Signaling, #9003, USA) according to the manufacturer’s instructions. Put simply, after the cells were crosslinked and lysed, the cells became lysates. Subsequently, the lysates were sonicated into 300 bp to 600 bp fragments and immunoprecipitated with anti-TEAD1 antibody (Abcam, UK). In addition, we used normal rabbit immunoglobulin G (IgG) as a negative control. Then, we performed reverse crosslinking and DNA purification. Finally, the precipitated DNA was analyzed by qRT-PCR via SYBR-Green PCR Master Mix (Vazyme, Nanjing, China). Primers used for SNHG16 promoter regions are shown in the supplementary material.

GST pulldown assay

To test whether YAP1 could interact with TEAD1 directly, we performed the GST pull-down assay as follows. First, after the transformation, cloning and expression of the GST-YAP1 fusion protein in E. coli, the GST-tagged YAP1 fusion protein was further purified using glutathione-Sepharose 4B beads. For the pull-down assay, the GST or GST-tagged YAP1 fusion protein was incubated with purified His tagged TEAD1 from E. coli for 4 - 8 h at 4 ℃. Then, the protein-bound glutathione-Sepharose 4B beads were washed four times and the proteins were recovered. Finally, the recovered proteins were analyzed by SDS-PAGE.

Colony formation and wound healing assay

For colony formation detection, HCT116 cells were placed in 6-well plates at a density of 1000 cells per well. In the same way, DLD1 cells were placed at a density of 1000 cells per well. After two weeks of culture in an incubator, the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. A wound healing assay was also used to measure the migratory capacity of CRC cells. Cells were seeded in 6-well plates and allowed to grow until the confluence reached 90%. A 20 ul pipette tip was used to scrap several lines across the cell surface, and then plates were washed three times to remove nonadherent debris. Finally, the cells were incubated at 37℃ with a serum-free medium, and photos were taken every 12 hours.

Transwell migration and invasion assay

Twenty-four-well Transwell (8μm pore size; Corning, USA) were used to assess the cell migration and invasion ability of CRC cells. For migration assay, 1.0 x 10⁵cells in 0.4 ml serum-free DMEM medium were seeded on the upper surface of the well. For the invasion assay, Transwell chambers were coated with Matrigel, and 1.5 x 10⁵cells were seeded on the upper surface of the chamber in 0.4 ml serum-free medium. After 36 - 48 h of incubation, cells on the upper surface of the chamber were removed with cotton swabs, and the remaining cells on the lower surface of the chamber were fixed and stained with 0.5% crystal violet solution.

Xenograft assays

The animal experiment was approved by the ethical committee of Zhongnan Hospital of Wuhan University and performed in accordance with the Guide for the Care and Use of Laboratory Animals of Wuhan University. To perform the tumor proliferation assay, 12 BALB/c nude mice (4-6 weeks old) (Hubei Research Center of Laboratory Animals, China) were randomly divided into three groups (n = 4 per group): HCT116/ Lv-anti-SNHG16 NC, HCT116/ Lv-anti-SNHG16 + Lv-Oe-YAP1 NC, and HCT116/ Lv-anti-SNHG16 + Lv-Oe-YAP1. In total, 5 × 10⁶ HCT116 cells suspended in 200 ul medium were subcutaneously injected into the right flank of mice. After ten days, tumor size was measured by digital Vernier calipers every 1 week, and the tumor volume was calculated by the following formula: volume = 1/2 × (length × width²). After thirty days, the transplantable tumor and 1ml mouse blood were collected for further research. For the metastasis experiment, the mice were randomly divided into three groups (n = 6 per group). The stable cell line was injected into the mice via the tail vein, and metastasis was assessed 30 days after injection by dissection. Finally, the mice were euthanized and necropsied to assess metastatic burden. Then, we further examined the tumor tissues, liver and lung tissues by hematoxylin and eosin (H&E) staining, IHC staining and RT-PCR assay. Protein expression in xenograft tumor was also represented by IOD, which is normalized by negative control group.

Statistical analysis

All the data were derived from at least three independent experiments, and p values < 0.05 were considered statistically significant. Through SPSS statistical software and GraphPad Prism software, all statistical analyses were performed. To assess the association between SNHG16 expression and YAP1, and miR-195-5p expression, we used Pearson’s correlation analysis. Then, we applied the chi-square test to analyze the expression of SNHG16 and the clinicopathological status of CRC patients. Comparisons between 2 groups were performed using a 2-tailed, unpaired Student’s t-test. In addition, Kaplan-Meier survival curves and log-rank tests were used for survival analysis. Finally, univariate and multivariate Cox-regression analyses were applied to identify the independent factors of prognosis.

3′-UTR: 3’-untranslated region; CRC: Colorectal cancer; CTC: circulating tumor cell; ^ECTC: epithelial-type circulating tumor cell; ^MCTC: mesenchymal-type circulating tumor cell; EMT: Epithelial-mesenchymal transition; LncRNA-SNHG16: Long noncoding RNA SNHG16; miR: MicroRNA/miRNA; miR-195-5p: Hsa-microRNA-195-5p; NC: Negative control; PANT: Paired adjacent normal tissue; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; SD: Standard deviation; YAP1: Yes-associated protein 1; Lv-Oe-SNHG16: lentivirus mediated overexpression of SNHG16; Lv-Oe-SNHG16 NC: nonspecific Lentiviral vectors of SNHG16 overexpression; Lv-Oe-YAP1: lentivirus mediated overexpression of YAP1; Lv-Oe-YAP1 NC: nonspecific Lentiviral vectors of YAP1 overexpression; LVI: lymphovascular invasion; PNI perineural invasion; TI: tumor invasion; LNM: lymph node metastasis; TNM: tumor-node-metastasis; CA19–9: carbohydrate antigen 19–9; CEA: carcinoembryonic antigen. IHC: immunohistochemistry.

Ethics approval and consent to participate

The research protocol was reviewed and approved by the Ethics Committee of Wuhan University, and informed consent was obtained from all participated patients.

Consent for publication

All coauthors have agreed to publish the results of this study

Availability of data and materials

The data supporting our findings can be found in the supplementary data.

competing interests

The authors declared that we have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Funding

This study was supported by grants from National Natural Science Foundation of China (81872376), National Natural Science Fund Youth Fund of China (81702411), and Health Commission of Hubei Province Scientific Research Project (WJ2019H012).

Author’s contribution

Participated in research design: Zhenxian Xiang, Bin xiong, Shuyi Wang.

Performed data analysis: Zhenxian Xiang, Bin xiong.

Experimental operation: Zhenxian Xiang, Haitao Wu, Guoquan Huang, Qiuming He, Rongzhang Dou.

Wrote or contributed to the writing of the manuscript: Zhenxian Xiang, Qing Liu, Yan Fang, Jialing Song.

Acknowledgements

The authors appreciate Wuhan YZY Medical Science and Technology Co., Ltd. (Wuhan, China) for providing equipment and excellent technical support for CTC isolation and identification. We also thank Sze Ka Lun for his helpful language revisions.

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Table 1: Correlation between the expression of SNHG16 and clinicopathologic characteristics in CRC

Paramaters	n	SNHG16 expression		p
Paramaters	n	Low	High	p
Gender
Male	63	30	33	0.784
Female	48	25	23	0.784
Age, years
<60	48	27	21	0.298
>60	63	28	35	0.298
Tumor site
<4	58	31	27	0.503
>4	53	24	29	0.503
Tumor size, cm
Colon	56	26	30	0.636
Rectal	55	29	26	0.636
Tumor differentiation
Moderate/well	81	42	39	0.523
Poor	30	13	17	0.523
LVI
Absence	59	38	21	0.002
Presence	52	17	35	0.002
PNI
Presence	56	18	38	0.001
Absence	55	37	18	0.001
TI
T1-2	15	11	4	0.056
T3-4	96	44	52	0.056
LNM
N0-1	86	52	34	0.003
N2-N3	25	3	22	0.003
TNM stage
I/II	57	48	9	0.001
III/IV	54	7	47	0.001
CA199
<27	77	42	35	0.15
>27	34	13	21	0.15
CEA
<5	59	35	24	0.037
>5	52	20	32	0.037
M
No metastasis	101	55	46	0.001
Metastasis	10	0	10	0.001
Overall	111	55	56

Table 2: Univariate and multivariate analyses of clinicopathologic parameters associated with progression-free survival and overall survival

parameters	overall survival						Progression-free survival
	univariate analysis				Multivariate analysis			univariate analysis				Multivariate analysis
	HR	95%CI	P	HR		95%CI	P		HR	95%CI	P	HR	95%CI	P
Gender Female vs Male	1.19	0.625-2.69	0.756						1.076	0.624,1.856	0.792
CA19-9 <27 vs >27	0.806	0.404-1.607	0.54						0.666	0.377,1.177	0.162
CEA (low vs high)	0.731	0.382-1.397	0.343						0.872	0.505, 1.507	0.624
PNI (low vs high)	0.738	0.387-1.408	0.229						0.625	0.363,1.074	0.259
LNM （low vs high）	0.225	0.115-0.443	0	0.51		0.224-1.143	0.06		0.32	0.173,0.591	0.001	0.819	0.399-1.681	0.586
Tumor size (<4cm vs >4cm）	0.9	0.472-1.715	0.748						0.587	0.338,1.018	0.058
Tumor site （rectal vs colon）	1.683	0.879- 3.224	0.135						1.657	0.962,2.854	0.069
Age （<60 vs >60）	0.702	0.44-1.344	0.286						1.076	0.624,1.856	0.792
TI (T1-2 vs T3-4)	0.443	0.157-1.254	0.125						0.31	0.123,0.783	0.013	0.417	0.163-1.064	0.067
Tumor differentiation poor/well vs moderate	1.18	0.583-2.390	0.645						1.243	0.691,2.236	0.467
Metastasis absence vs presence	0.031	0.011-0.086	0.001	0.03		0.009-0.102	0.01		0.02	0.005,0.077	0.000	0.028	0.007-0.113	0.000
TNM stage^a I-II vs III-IV	0.226	0.110-0.463	0.002	0.19		0.064-0.564	0.01		0.26	0.142,0.458	0.000	0.352	0.188-0.658	0.001
SNHG16 Low vs High	0.493	0.255-0.953	0.045	0.32		0.118-0.873	0.03		0.41	0.236,0.726	0.002	0.535	0.226-1.269	0.156
LVI absence vs presence	0.327	0.169-0.633	0.003	0.44		0.210-0.937	0.00		0.319	0.184,0.555	0.000	0.625	0.332-1.176	0.145

Notes: ^aThe 8th edition of the AJCC Cancer Staging Manual; Boldface indicates P < 0.05

Abbreviations: LVI lymphovascular invasion, PNI perineural invasion, TI tumor invasion, LNM lymph node metastasis, TNM tumor-node-metastasis, CA19-9 carbohydrate antigen 19–9, CEA carcinoembryonic antigen.

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LncRNA SNHG16 facilitates the liver metastasis of colorectal cancer by modulating circulating tumor cell epithelial-mesenchymal transition via a positive feedback loop with the YAP1/TEAD1 complex

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