Overexpression of RP11-757G1.5 in CRC associates with poor prognosis
To further ascertain lncRNAs that are differentially expressed in CRC, we primarily analyzed GSE63675 from GEO, which comprises of lncRNA data for 43 CRC tissues and 6 neighboring non-tumor control tissues. Of note, CRC tissues expressed 8 notably differentially expressed lncRNAs as compared with neighboring non-tumor tissues (Figure 1A). Amid them, lncRNA RP11-757G1.5 was chosen for further analysis. Principally, in situ hybridization (ISH) was applied to gauge level of the expression of RP11-757G1.5 in tissues. As presented in Figure 1B, RP11-757G1.5 was gradually strongly stained with staging and lymph node metastasis in CRC tissues comparing to adjacent tissues. Also, RP11-757G1.5 was intensively stained in CRC cells' cytoplasm. Next, we assessed the expression of RP11-757G1.5 in CRC tissues by RT-qPCR and it was ascertained that this lncRNA was markedly upregulated in CRC tissues relative to the non-tumor control tissue (p<0.001, Figure 1C). Successively, we appraised the link between high RP11-757G1.5 expression and clinicopathological features of the disease. Results were similar to ISH and pointed out that elevated RP11-757G1.5 expression was directly linked with significasnt lymph node metastasis and advanced TNM staging (Figure 1D, 1E, S1A and S1B). To assess the importance of this relationship, we distributed 112 CRC patients into 2 sets in relation to the extent of RP11-757G1.5 expression: RP11-757G1.5-high and RP11-757G1.5-low. Pearson chi-square or Fisher's Exact tests revealed that elevated RP11-757G1.5 levels were linked with greater tumor size (p=0.003), lymph node metastasis (p=0.008) and advanced TNM staging (p<0.001). A relationship between RP11-757G1.5 levels and other clinical features was not observed (Table 1). Kaplan-Meier analyses and Log-rank tests were subsequently carried out to establish the connection between RP11-757G1.5 and CRC survival time. Resultls of this evaluation showed that patients in the RP11-757G1.5-high group displayed a remarkably shorter survival rate relative to those in the RP11-757G1.5-low group (45.741 ± 3.539 vs 69.818 ± 3.662 months; Log rank=4.178, p=0.0047, Figure 1F). Moreover, elevated RP11-757G1.5 levels were positively associated with poor disease-free survival (Log rank=9.561, p=0.0129, Figure 1G). Univariate and multivariate analyses revealed RP11-757G1.5 expression as an autonomous prognostic sign of CRC (hazard ratio (HR) =3.441, 95% confidence interval (CI) =1.471-8.005, p=0.008; HR=2.015, 95% CI=1.018–5.856, p=0.019, Table 2). Wholly, these findings recognized that high RP11-757G1.5 levels correlate with poor CRC clinical outcomes.
RP11-757G1.5 promotes CRC cell proliferation and cell cycle progression in vitro
Next, we assessed the expression pattern of RP11-757G1.5 in standard NCM460 and CRC cell lines (HT-29, HCT-116, SW480, SW620, LoVo, Caco-2) by RT-qPCR. The outcomes revealed markedly higher levels of RP11-757G1.5 in CRC cell lines compared to NCM460 (p<0.05, Figure 2A). RP11-757G1.5 was expressed was most in SW480 (p<0.001) and least in HCT-116 (p<0.05). These 2 CRC cell lines were therefore selected for downstream experiments. In accordance, the results of fluorescence in situ hybridization (FISH) also pointed out that lncRNA RP11-757G1.5 is mainly found in the cytoplasm (Figure 2B). The possible biological function of RP11-757G1.5 in CRC was evaluated by observing the overexpression of RP11-757G1.5 in HCT-116 cells (Figure 2C), while it was knocked down in SW480 cells (Figure 2D). RT-qPCR was conducted to confirm the success of the evaluation, and Figure 2E and 2F (p<0.05) show the outcomes. To lessen the likelihood of off-targeting, 3 shRNAs (sh-RP11-757G1.5#1 (or sh-757G1.5#1) and sh-RP11-757G1.5#2 (or sh-757G1.5#2)) were used (Figure 2D). Further analyses showed that RP11-757G1.5 overexpression significantly enhanced HCT-116 colony formation and proliferation while these processes were suppressed by RP11-757G1.5 silencing in SW480 cells (p<0.05, Figure 3A-D). In addition, flow cytometry showed that RP11-757G1.5 silencing significantly hindered the SW480 cell cycle at the G1 phase (p<0.05, Figure 3F). RP11-757G1.5 overexpression in HCT-116 cells had similar results (p<0.05, Figure 3E). Furthermore, immunoblotting analysis demonstrated that overexpression of RP11-757G1.5 in HCT-116 enhanced Cyclin D1 and PCNA expression, factors known to stimulate cell proliferation (p<0.05, Figure 3G), while RP11-757G1.5 knockdown weakened their expression (p<0.05, Figure 3H). In other words, RP11-757G1.5 promotes cell proliferation of CRC by regulating Cyclin-D1 and PCNA signaling pathway. However, its specific molecular mechanism needs further study. As a whole, these datasets concluded that RP11-757G1.5 might serve as a cancer-promoting factor, which stimulates the proliferation of CRC cells in vitro.
RP11-757G1.5 promotes CRC cell migration and invasion in vitro
The role of RP11-757G1.5 in CRC metastasis was determined by cell invasion and migration assays. The results show that RP11-757G1.5 overexpression facilitated HCT-116 cell migration and incursion (p<0.05, Figure 4A, 4C). On the contrary, RP11-757G1.5 knockdown suppressed these processes in SW480 cells (p<0.05, Figure 4B, 4D). So, it is suggested that RP11-757G1.5 is involved in cancer cells dissemination in vitro. Further, we executed western blot to estimate whether RP11-757G1.5 controls epithelial-mesenchymal transition (EMT) in CRC cells. The outcomes illustrated that RP11-757G1.5-overexpression leads to a reduction in the expression of epithelial cell marker E-cadherin and an augmention in the expression of another epithelial cell marker N-cadherin (p<0.05, Figure 4E, 4F). Therefore, we initially verified RP11-757G1.5 promotes metastasis through affecting the EMT process of CRC cells. Moreover, to further verify the cancer-promoting effect of RP11-757G1.5 in CRC. We selected SW620 (High metastasis) and HT-29 (Low metastasis) two cell lines of CRC, then successfully performed RP11-757G1.5 knockdown and overexpression, respectively. The results confirmed that RP11-757G1.5 acts as an oncogene which could promote metastasis of CRC (p<0.05, Figure S4A, S4B). Collectively, these results strongly propose that the RP11-757G1.5 can prompt migration and invasion of CRC cells in vitro.
LncRNA RP11-757G1.5 acts as a molecular sponge of miR-139-5p to regulate YAP1 expression in CRC cells
To comprehend the mechanisms by which RP11-757G1.5 fuels CRC growth, we appraised its subcellular locality in HCT-116 and SW480 and perceived that it principally resided in the cytosol (Figure 5A). From this we can deduce that it may function at the post-transcriptional level. Various studies have informed that lncRNAs may act as a sponge by binding competitively to miRNA, and in this manner modulating gene expression by making it impossible for target miRNA responsive elements (MREs) to bind with the miRNAs. To determine whether RP11-757G1.5 also plays this role, we cloned the full-length RP11-757G1.5 comprising the presumptive miR-139-5p binding sites in order to examine potential binding sites between miR-139-5p and RP11-757G1.5 (Figure 5B). This analysis was done with the knowledge that miR-139-5p and RP11-757G1.5, express opposing functions in CRC. We therefore created a luciferase reporter plasmid (pLuc) containing RP11-757G1.5 (pLuc-RP11-757G1.5-WT) or a mutant bearing mutation in the miR-139-5p seed sequence (pLuc-RP11-757G1.5-Mut). Through luciferase reporter assays, we observed the link bewteen miR-139-5p and RP11-757G1.5. Results showed that overexpressing miR-139-5p (or its miR-139-5p mimic) markedly suppressed pLuc-RP11-757G1.5-WT activity relative to the mutant reporter (p<0.05, Figure 5C). This confirmed that miR-139-5p binds and inhibits RP11-757G1.5. To investigate whether this interaction was physical, we carried out RNA immunoprecipitation (RIP) with an anti-Ago2 antibody. As a result, we found enrichment for both RP11-757G1.5 and miR-139-5p in the Ago2 complex (Figure 5E). In the meantime, miR-139-5p was pulled down by biotin-labeled RP11-757G1.5-WT (p<0.01, Figure S5A), while mutagenesis of the binding sites for miR-139-5p in RP11-757G1.5-Mut disrupted the interaction between RP11-757G1.5 and miR-139-5p (n.s, Figure S5A).
RP11-757G1.5 modulates YAP1 expression by competitively binding miR-139-5p
Previous investigations have proved that microRNAs control CRC pathogenesis via YAP1 regulation[22-24]. By means of bioinformatics analysis, miRNAs sequences were found to be compatible with recognition sequences on RP11-757G1.5 and the 3'-UTR of YAP1. This suggested that miR-139-5p posssibly explicitly interacted with the RP11-757G1.5 sequence and its 3'-UTR (Figure 5B). Following this, we attempted to elucidate whether the effects of RP11-757G1.5 on CRC pathogenesis are facilitated by the miR-139-5p/YAP1 pathway. Post evaluating the relationships between RP11-757G1.5, miR-139-5p, and YAP1 by luciferase assays, it was seen that, in comparison to the empty vector control, RP11-757G1.5 overexpression reversed the miR-139-5p-mediated inhibition of pLuc-NOTCH1-3'UTR luciferase output (p<0.05, Figure 5D), thus indicating that RP11-757G1.5 suppresses miR-139-5p-mediated inhibition of YAP1 by competitively binding miR-139-5p. An elevation of YAP1 in CRC tissues and cell lines was further observed (p<0.01, Figure 5G, 5H). RP11-757G1.5 knockdown significantly suppressed endogenous YAP1 levels in CRC cells (p<0.05, Figure 5F, 5L). Counter wise, YAP1 expression was raised by RP11-757G1.5 overexpression in CRC cells (p<0.05, Figure 5F, 5L). We then analyzed our RT-qPCR results to gauge the relationship between RP11-757G1.5, miR-139-5p, and YAP1 expression. The results indicated that RP11-757G1.5 expression negatively correlated with miR-139-5p levels (r=−0.402, p=0.003), but positively correlated with YAP1 levels (r=0.380, p=0.006). Furthermore, a negative relationship was discovered between miR-139-5p and YAP1 expression in our RT-qPCR dataset (r=−0.297, p=0.004) (Figure 5I-K). We investigated the possibility of the effects of RP11-757G1.5 on YAP1 levels depended on miR-139-5p by co-transfecting HCT-116 cells with the miR-139-5p mimic and pcDNA3.1-RP11-757G1.5 (or pcDNA3.1-757G1.5) and the effects of this approach on YAP1 were assessed. Results exhibited significantly raised protein levels of YAP1 in HCT-116 cells in relation to cells transfected with miR-139-5p mimic. RP11-757G1.5 knockdown distinctly reversed the inhibitory effects of miR-139-5p on YAP1 expression in SW480 cells, as revealed by western blot analysis (p<0.05, Figure 5M).
RP11-757G1.5 promotes tumor progression in CRC via miR-139-5p/YAP1 axis
Based primarily on the above experiment, we provided strong evidence which indicated RP11-757G1.5 as a key controller of the expression of YAP1 by sponging miR-139-5p in CRC. In formerly published studies, miR-139-5p and YAP1 have been described to regulate CRC development[15, 25-27]. Therefore we speculated the possibility of RP11-757G1.5 regulating the pathogenesis of CRC via the miR-139-5p/YAP1 axis. To examine this, we assessed how miR-139-5p and YAP1 affect RP11-757G1.5-driven cell proliferation. Results of our investigation demonstrated that miR-139-5p overexpression or YAP1 knockdown obstructed RP11-757G1.5-driven CRC cell proliferation (p<0.05, Figure 6A-D). Subsequently, western blotting was performed in order to evaluate whether miR-139-5p and YAP1 have an effect on Cyclin D1 and PCNA levels in the context of RP11-757G1.5-driven cell proliferation. Results revealed the effects of RP11-757G1.5 overexpression on Cyclin D1 and PCNA expression were markedly reversed by miR-139-5p expression or YAP1 knockdown (p<0.05, Figure 6E, 6F). Following this, we assessed how the miR-139-5p/YAP1 axis affected RP11-757G1.5 driven migration and infiltration of CRC cells. The outcome showed that miR-139-5p expression or YAP1 silencing attenuated cell migration and invasion as a result of RP11-757G1.5 overexpression in CRC cells (p<0.05, Figure 6G-J). Meanwhile, we evaluated whether YAP1 can affect miR-139-5p-driven cell proliferation and metastasis in CRC. This analysis exposed the overexpression of YAP1 as the cause of the restoration of miR-139-5p-driven CRC cell proliferation and metastasis (p<0.05, Figure S6A-H). This was consistent with previous studies. Comprehensively, data demonstrated that RP11-757G1.5 behaves as an oncogene in CRC to promote proliferation and metastasis, partly by sponging miR-139-5p, thereby regulating YAP1 levels.
Deregulation of lncRNA RP11-757G1.5 suppresses cell proliferation and invasion in CRC orthotopic xenografts
In this section, we explored the role of RP11-757G1.5 in CRC in vivo by building orthotopic xenograft mouse models. In short, HCT-116 cells overexpressing RP11-757G1.5 or with SW480 knockdown were xenografted subcutaneously into mice. The stimulating effect of RP11-757G1.5 knockdown on tumor evolution and migration was inspected by use of the IVIS imaging system. Results of this investigation showed that tumor luciferase activity in the pcDNA3.1-757G1.5 expressing cells was elevated in those transfected with an empty vector (Figure 7A). Meanwhile, RP11-757G1.5-silencing exhibited approximately opposite effects (Figure 7F). Moreover, we discovered that RP11-757G1.5 overexpression enhanced tumor growth (Figure 7A-E). RP11-757G1.5 knockdown hindered tumor growth (Figure 7F-J). In addition, overexpression of RP11-757G1.5 stimulated metastasis to the liver and spleen. However, when interfering with RP11-757G1.5, the metastasis of tumor cells to organs in the liver and spleen was significantly reduced (Figure 7K-O). Evaluating the results, we could see that the elevation of RP11-757G1.5 behaves as an oncogene and contributes to tumorigenesis and liver, spleen metastasis of CRC in vivo.