LncRNA SNHG5 promotes the proliferation and cancer stem cell-like properties of hepatocellular carcinoma by regulating UPF1 and Wnt signaling pathway

Background: It have been revealed that lncRNAs had a critical role in human cancers, including HCC. Our previous study found a lncRNA SNHG5 related to the development of HCC. However, the role and exact mechanism of SNHG5 in HCC proliferation and the liver CSC-like properties is still unclear. Materials and Methods: We investigated the HCC cells proliferation and CSC-like properties in vitro and in vivo by knockdown or upregulate SNHG5, and detected underlying mechanisms by qRT-PCR, Western blotting, bioinformatic analysis, luciferase reporter assay, RNA immunoprecipitation. Results: Knockdown of SNHG5 repressed HCC cells proliferation and CSC-like properties, while overexpression of SNHG5 promoted cells growth. At the same time, CSCs markers ( CD44, CD133 and ALDH1) and related transcription factors (OCT4, SOX2 and NANOG) were downregulated when SNHG5 knockdown. Further investigations showed that SNHG5 regulated the proliferation and CSC-like properties of HCC by binding UPF1 and regulating Wnt/β-catenin pathway. Conclusions: SNHG5 plays critical role to promote HCC cells proliferation and cancer stem cell-like properties via UPF1 and Wnt/β-catenin pathway.

which is a kind of aggressive cancer and the primary histological type of liver malignancies 1,2 . Despite the prevention, diagnosis and treatment of HCC have obtained greatly development, the rates of recurrence and metastasis are still high, and the patients with HCC have a poor long survival 3,4 . Thus, it is significantly important and urgent to find out the mechanism of HCC progression, metastasis and recurrence, and 3 investigate the new therapy method.
Heterogeneity is a characteristic and hallmark of tumor cells, which may be one of causes of tumor growth, metastasis and recurrence. It has been revealed that heterogeneity may result from groups of stem-like cells, which are regarded as the cancer stem cells (CSCs) 5 . CSCs have the abilities to self-renew, differentiate, keep uncontrollable growth, give rise to a new tumor in local or distant organs and have the interconversion with non-CSCs 6,7 . These features may mainly contribute to the progression, metastasis and recurrence of cancers 8 . The existence of liver CSCs has been verified. This group of cells has been distinguished, and they are characterized by several makers, such as CD133, CD13, CD90, and EpCAM 9− 12 . It has been revealed that several biological mechanisms are responsible to the functions of hepatic CSCs, such as AKT, transforming growth factor-beta (TGF-β) and so on.
Long noncoding RNAs (lncRNAs) called a kind of "dark matter" in human diseases are longer than 200nt, which could not code proteins 13 . More and more researches have revealed that lncRNAs could bind to DNA and RNA by a complementary sequence, playing critical roles in gene, such as transcription, mRNA splicing, RNA decay and translation 14 .
And the posttranslational modification of proteins can be modulated by lncRNAs. LncRNAs play important roles in the proliferation, apoptosis and metastasis of cancers, which involved in different approaches, such as epigenetic silencing, splicing regulation, lncRNA-miRNA interaction, lncRNA-protein interaction and genetic variation, including HCC 15 .
LncRNAs are contribute to liver cancer pathology, progression, outcomes and the maintenance of biological properties of liver CSCs, such as LncTCF7, lncBRM and lncCAMTA1 16 − 19 .
It has been found that lncSNHG5 takes part in regulating different cancers. In this study, 4 we aim to investigate the effect of lncSNHG5 on HCC and liver cancer stem cell-like properties.

Cell lines and cell culture
The human HCC cell line HepG2 and Huh7 were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cells were cultured in the DMEM/ High Glucose (hyclone, USA) in a humidified incubator at 37 °C temperature and 5% CO 2 concentration.
10% FBS (Fetal bovine serum, Gibco USA) and Penicillin-Streptomycin (100 U/mL and 100μg/mL respectively) supplementations were added in the DMEM/ High Glucose medium prior to culture. Colony formation assay After routine incubation, transfected cells were trypsinized, centrifuged, counted and replated at a density of 500 cells/6 cm plate. After 12 days, the cell colonies (one colony containing at least 50 cells) were fixed with 3.7% methanol, stained with 0.1% crystal violet and counted.

Sphere-formation assays
Ultra-low attachment culture dishes (Corning, USA) were used to culture HepG2 and Huh7 cells with DMEM/F12 (Gibco, USA) added with 1% FBS, 20 ng/mL epithelial growth factor, and 20 ng/mL fibroblast growth factor for two weeks. The formation and the number of spheroids were detected by a stereomicroscope (Olympus, Japan).

RNA isolation and quantitative real-time PCR
The total RNA from the cultured cells and collected HCC tissues were extracted form Trizol

Western blot analysis
The total protein from the cultured HCC cells and the tissue samples were isolated by RIPA (Beyotime, Haimen China) supplemented with proteinase and phosphatase. BCA detection 6 kit (Keygen, Nanjing, China) was used for qualification according to the manufacture's protocol. For electrophoresis 5% gel was used for concentration and 10% for separation.
Following electrophoresis the proteins were transferred on a PVDF membrane (Merck Milipore) and were blocked by 5% non-fat milk for 1 hour. Then the PVDF membrane was incubated overnight at 4 0 C with the primary antibodies (Supplementary Table 2). On the next day the secondary antibody (Zhuangzhi Biology, China) was diluted 1:5000 ratio in TBST, and the membrane was re-incubated for 1 hour. The protein bands were evaluated by ECL immunoblotting kit following the manufacture's protocol ( Milipore, USA).

Immunofluorescence (IF)
Cells were fixed in 4% paraformaldehyde at room temperature for 15 min after culturing on glass coverslips for 24 h. Then washed by PBS. The adherent cells were permeabilized using 0.5% Triton X-100, blocked with 10% goat serum in for 1h. Then, incubated with primary antibody 4 °C overnight and secondary antibodies with an appropriate dilution.
After washing three times with PBS, coverslips were stained with DAPI and imaged with a invert fluorescent microscope (Nikon Eclipse Ti-S).

Tumor formation in BALB/c nude mice
4 weeks aged BALB/c nude mice were randomly divided into two groups, which were purchased from the Central Laboratory of Animal Science, Xi'an Jiaotong University, China.
The mice were kept under Sterile Specific-Pathogen free (SPF) environment. Then, 5 ×

Luciferase reporter assay
Before transfection the HCC cells were placed and cultured in a 96 well plate til 60-80% proliferation was achieved. Wild and mutant reporter plasmids of SNHG5 (SNHG5-WT-luc, SNHG5-MuT-luc) were co-transfected with UPF1 plasmid respectively. The alteration in the luciferase activity was devaluated in each group by Dual Luciferase Assay Kit and as a control Renilla luciferase activity was employed.

Immunoprecipitation (RIP)
Millipore EZ-Magna RIP RNA Binding Protein Immunoprecipitation kit (Millipore) was applied to performed RIP assays according to the manufacturer's protocol. Rabbit polyclonal IgG (Millipore) and antibodies to UPF1 (Abcam) were applied in RIP assays.
Then, RIP-PCR was performed, and total RNA was used as input controls.
Statistical analysis SPSS 23.0 (IBM, SPSS, Chicago, IL, USA) and GraphPad Prism V7.0 (GraphPad Software, CA, USA) were used to analyzed the results. Student's t-test was done to evaluate the difference between two groups. A two-tailed P< 0.05 was considered as statistically significant and P < 0.01 was very significant.

Upregulation of SNHG5 promotes HCC cells proliferation in vitro
The effect of SNHG5 on HCC cells in vitro was investigated by upregulating the expression of SNHG5. In order to perform this, we conducted the pCMV-SNHG5 expression plasmid, and transfected it into HepG2 and Huh7 cells. As a result, comparing with the empty vector, the expression of SNHG5 dramatically increased after transfection with the pCMV-SNHG5 vector in HepG2 and Huh7 cells ( Figure 1A). Then growth curve of MTT assay showed that overexpression of SNHG5 could promote HepG2 and Huh7 cells proliferation ( Figure 1B). Next, both the result of Edu assay and plate colony formation assays revealed the same growth trend of HCC cells with upregulation of SNHG5 ( Figure 1C, D).

Downregulation of SNHG5 represses HCC cells proliferation in vitro
To further evaluate the role of SNHG5 on HCC cells proliferation, lentivirus-mediated short hairpins RNA (shRNA) was used to silence the SNHG5 (SNHG5-shRNA). The result of qRT-PCR showed that the expression of SNHG5 reduced highly in HepG2 and Huh 7 (Figure 2A).
MTT, Edu assay and plate colony formation assays showed that knockdown of SNHG5 impaired the HepG2 and Huh 7 proliferation ( Figure 2B, C, D). These results suggest that SNHG5 is critical to HCC cells proliferation.

Knockdown of SNHG5 represses tumor growth in vivo
HepG2 and Huh 7 cells stably transfected with SNHG5-shRNA and NC were subcutaneously injected in male nude mice for 5 weeks. Compared with NC group, HCC cells transfected with SNHG5-shRNA greatly inhibited tumor growth ( Figure 3A). We also observed the tumor volume and tumor weight in two group. And the tumor volume curve and tumor weight indicated that downregulation of SNHG5 suppressed tumor growth effectively ( Figure 3B, C). Additionally, the expression of SNHG5 was detected in xenograft tumors.
The result of qRT-PCR indicated that the expression of SNHG5 greatly decreased in SNHG5-shRNA xenograft tumors ( Figure 3D).

Knockdown of SNHG5 represses the liver CSC-like properties
To analyze the role of SNHG5 in liver CSC self-renewal, SNHG5-shRNA was used to deplete the expression of SNHG5 in HepG2 and Huh7 cells. And qRT-PCR was performed to detect the expression of SNHG5 in liver CSCs, which showed that the SNHG5 expression decrease in liver CSCs ( Figure 4A). Then, the sphere-formation assays were performed in the cells with SNHG5-shRNA and sh-NC, we found that the sphere formation was dramatically impared in SNHG5-shRNA cells compared with the sh-NC cells ( Figure 4B, C). We also 9 detected the makers of stem cells and the transcription factors in two CSCs groups. The result of qRT-PCR showed that the expression of CD133, CD44, ALDH1, Oct4, Sox2 and Nanog decreased in CSCs derived from SNHG5-shRNA cells ( Figure 4D). The IF and Western Blot analysis verified the result of qt-PCR ( Figure 4E, F). These results indicated that lncSNHG5 was required in liver CSC-like properties.

Depletion of SNHG5 increases the expression of UPF1
It has been revealed that RNA-binding proteins (RBPs) play critical roles in lncRNA related pathway. And interacting with RNA-binding proteins (RBPs) is one of approached for lncRNAs involved in regulations. In order to investigate this, biological information prediction was performed including StarBase and DIANA LncBase software to find the proteins associated with SNHG5. Then, we noticed that there was a potential binding site for UPF1.
We found that the expression of UPF1 increased in HepG2 and Huh7 cells with depletion of SNHG5 ( Figure 5A). Then, qRT-PCR and Western Blot were performed to analysis the expression of UPF1in liver CSCs, revealing that knockdown of SNHG5 could upregulate the expression of UPF1 ( Figure 5B, C). Consistently, IF verified that UPF1 highly expressed in sh-SNHG5 liver CSCs ( Figure 5D). To further investigate the interaction between SNHG5 and UPF1, luciferase reporter vectors of SNHG5 were constructed. The result of luciferase reporter assay showed that luciferase reporter activity was greatly promoted by cotransfecting cells with pCMV-SNHG5-WT and UPF1 vector, while co-transfecting cells with pCMV-SNHG5-Mut and UPF1 vector inhibited luciferase reporter activity ( Figure 5E). Then, RNA immunoprecipitation (RIP) was performed to deeply detected the interaction between SNHG5 and UPF1. An antibody extracts from HCC cell lines HepG2 and HuH7, which was against UPF1. We found that SNHG5 enriched with UPF1 antibody, comparing to the nonspecific antibody (IgG control; Figure 5F). These results suggested that SNHG5 interacts with UPF1, and the expression of SNHG5 and UPF1 was opposite tendency.

Downregulation of UPF1 increases liver CSC-like properties
To explore the role of UPF1 in the sphere-formation of liver CSCs, we depleted the expression of UPF1 by introducing two specific siRNAs (siRNA #1, #2) against UPF1 gene transcript into HepG2 and Huh7 cells. We performed sphere-formation assays, finding that the liver CSC-like properties were promoted in HCC cells with si-UPF1 ( Figure 6A). And the number of spheres per 1000 single liver CSCs increased after UPF1 knockdown ( Figure 6B).
Then, qRT-PCR was performed to detected the expression of markers (ALDH1, CD44, and CD133) and stem factors (NANOG, OCT4, and SOX2), which were upregulated with depletion of UPF1 ( Figure 6C). Meanwhile, this result was verified in Western Blot analysis ( Figure 6D). These results suggested that UPF1 played a critical role in liver CSCs formation.

Wnt/β-catenin pathway is responsible for liver CSC-like properties
It has been proved that Wnt/β-catenin pathway played a critical role in HCC, and SNHG5 took part in activation of Wnt/β-catenin pathway. Hence, we speculated that Wnt/β-catenin pathway may be a key factor to liver CSC-like properties. To investigate this, we extracted proteins from HepG2 CSCs and Huh7 CSCs and performed Western Blot analysis. The result showed that the expressions of Wnt1, Wnt3a and Wnt10a were downregulated with depletion of SNHG5, while they were upregulated with knockdown of UPF1 ( Figure 7A). The result was confirmed in qRT-PCR ( Figure 7B). β-catenin is one of the key factors in Wnt/βcatenin pathway, and we detected the expression of β-catenin in HepG2 CSCs and Huh7 CSCs with IF analysis. We observed that the expression of β-catenin increased in liver CSCs with depletion of UPF1, whileβ-catenin decreased in liver CSCs with downregulation of SNHG5 ( Figure 7C), as same result shown in qRT-PCR ( Figure 7D). Consistently, critical components of Wnt/β-catenin pathway (β-catenin, TCF4, c-myc, cyclinD1, c-Jun) were detected in HepG2 CSCs and Huh7 CSCs on protein level via Western Blot analysis. We observed that these key components were downregulated with knockdown SNHG5, while their expression increased with depletion of UPF1 ( Figure 7E). To further prove this, XAV-939,a Wnt/β-catenin pathway inhibitor was adopted. With XAV-939, the sphere-formation of liver CSCs was impared and the number of spheres per 1000 single liver CSCs decreased ( Figure 7F, G). These results suggested that SNHG5 could activated Wnt/βcatenin pathway, and the activation of Wnt/β-catenin pathway could promote the liver CSC-like properties.

Discussion
LncSNHG5 has been investigated in many solid cancers, such as colorectal cancer, gastric cancer and osteosarcoma, even myeloid leukemia 20 − 23 . And the mechanisms were detected. LncSNHG5 could promote the growth and(or) metastasis of osteosarcoma cells by suppressing the miR-212-3p/SGK3 signaling pathway, and it also plays a role of competing endogenous RNAs (ceRNAs) to regulate gastric cancer through miR-32 targeting KLF4, and contribute to the resistance of imatinib in chronic myeloid leukemia through miR-205-5p 22,23 . In our recent study, we found that knockdown of lncSNHG5 could contribute to the growth of HCC cells, while the upregulation showed the opposite tendency.
CSCs is a kind of cells with characters of self-renew and differentiation, which has been observed and isolated in many solid cancers, such as colorectal cancer, lung cancer, and breast cancer 24− 26 . Liver CSCs also was identified, which contained various subtypes characterized by different surface makers, such as CD133 + CD13 + , EpCAM + CD24 + OV6 + and CD133 + CD44 + CD24 + EpCAM + and so on 27 .
CSCs contribute to the epithelial-mesenchymal transition (EMT), metastasis, drug resistance and radio resistance though varieties of mechanisms. 26 Recently, several researches demonstrated that Wnt/β-Catenin signaling played a pivotal 13 role in cancer stem cells. For example, in glioma, the self-renewal and tumorigenicity of CSCs were regulated by dysregulated Wnt-FoxM1/β-Catenin signaling pathway 33

Conclusions
In summary, our research found that lncSNHG5 was related to the HCC cells proliferation in vitro and the tumor growth in vivo. The first time, we observed that lncSNHG5 was responsible to the sphere formation of liver CSCs and the HCC cells stem-like properties, and downregulation of UPF1 could increase liver CSC-like properties. In mechanism, the importance of Wnt/β-Catenin pathway was reconfirmed in CSCs, especially in HCC in our study. At the same time, the study demonstrated that lncSNHG5 could active the Wnt/β-