The Function And Mechanism Study Of Long Non-Coding RNA SNHG6 In Hepatocellular Carcinoma

Part I The expression of long non-coding RNA SNHG6 in hepatocellular carcinoma and its effect on the tumor biological behaviorObjective To investigate the expression level of long noncoding RNA SNHG6 in hepatocellular carcinoma and its effect on the tumor biology behavior of HCC cell lines.Methods The expression levels of SNHG6 in HCC tissues or HCC cell lines were detected by RT-qPCR. Then the expression level of SNHG6 was further confirmed by In situ hybridization (ISH) and fluorescent in situ hybridization (FISH). Combined with the clinical and survival datas of HCC patients, we then analysed the correlation between the expression of SNHG6 with clinical and pathological data. Kaplan-Meier analysis showed the relationship between the SNHG6 expression level and survival rate and recurrence-free survival. COX regression model was established and single multi-factor find the independent risk factor for HCC prognosis. Gene gain-loss assays was used to silence or overexpression SNHG6, flow cytometry analisis detect the cell cycle and apoptosis of HCC cell lines. Cell scratches, Transwell assay were used to observe the migration and invasion of HCC cell lines. CCK8 and EdU assay were uesd to detect the differences in the activity of HCC cells. In vivo assay was uesd to find the differences ability of hepatoma cells into tumors and lung metastasis following SNHG6 was silenced.Results Here, we report that lncRNA-SNHG6 is overexpressed in HCC tissues and hepatoma cells and is closely associated with histologic grade, Hepatitis B virus (HBV) infection, Barcelona Clinic Liver Cancer (BCLC) stage and portal vein tumor thrombus (PVTT). HCC patients with a higher SNHG6 expression level has a lower 3-year overall survival and recurrence rate than those with a lower SNHG6 expression level. Furthermore, SNHG6 was an independent risk factor for the prognosis of HCC. In particular, SNHG6 promotes the growth, migration, and invasion of HCC cell lines in vitro. In addition, SNHG6 promotes HCC cell line growth and metastasis in vivo.Conclusion SNHG6 maybe an oncogene in hepatocellular carcinoma and a potential warning indicator for poor prognosis of HCC patients.Part ? SNHG6 regulates ZEB1 expression by competitively binding miR-101-3p and induces EMT in hepatocellular carcinomaObjective To explore the possible molecular mechanism of long non-coding RNA SNHG6 acts as an ceRNA promotes HCC invasion and metastasis.Methods The target miRNA or gene of SNHG6 or miR-101-3p was predicted by bioinformatics software. RT-qPCR was uesd to dectect the expression level of miR-101-3p and the relationship between SNHG6 and miR-101-3p or ZEB1 was also analysed. Luciferase reporter gene assay further verify SNHG6 and miR-101-3p interaction target, luciferase reporter gene assay confirm the regulation of SNHG6 to ZEB1 3'UTR. Design point mutation SNHG6 overexpression vector, Knock down SNHG6 then transfection mutation and wild type SNHG6 overexpression vector respectively, the ZEB1 generation was observed by Western blotting. Cell morphology was decteced by optical microscope. The protein and mRNA level of EMT markers(E-cadherin, Claudin, Vimentin, Fibronection) was observed by Western blotting, immunofluorescence and RT-qPCR.Results miR-101-3p was significantly down-regulated in HCC, and was negatively correlated with SNHG6. SNHG6 was negative regulated by miR-101-3p. ZEB1 was one of target genes downstream of miR-101-3p. The 212bp-234bp of SNHG6 was the site of action of miR-101-3p coupled with each other. SNHG6 may act as an ceRNA by competing binding miR-101-3p thereby releasing the inhibition role of miR-101-3p to ZEB1, thus regulating ZEB1 mRNA 3' UTR activity, and ultimately regulates the expression level of ZEB1. Site-to-site mutation assays confirmed that the regulation role of SNHG6 to ZEB1 dependent on the binding sites between SNHG6 and miR-101-3p. The regulation role disappear when the of binding sites was mutated. Furthermore, SNHG6 promotes EMT by upregulating ZEB1 and leads to invasion and metastasis of hepatocellular carcinoma.Coclusion Upregulation of SNHG6 exhibits an oncogenic effect, regulating ZEB1 expression by competitively binding miR-101-3p and induces EMT in hepatocellular carcinoma. Our research provides new views and landscapes to understand the complex signaling network in HCC, and potential new strategies for the prognosis and therapy of HCC.Part III Long noncoding RNA SNHG6 promotes tumorigenesis by binding UPFl and then regulates TGF-?/Smad pathwayObjective To clearify the possible molecular pathway in HCC tumorigenesis which involved in SNHG6 and its RNA-binding protein.Methods The RNA-binding protein (UPF1) which bind to SNHG6 was predicted by bioinformatics software., RNA immunoprecipitation-sequencing (RIP-seq) to further validate the binding between them. The protein and mRNA level of UPF1 in HCC tissues or HCC cell lines was deteced by Immunohistochemistry, Western blotting and RT-qPCR, the relationship between SNHG6 and UPF1 was also analysed. Knock down SNHG6 by using RNAi technology, the expression level of UPF1 and Smad7 was observed by Western blotting, immunofluorescence and the relationship between Smad7 and UPF1 was also analysed. The expression level of key molecules (Smad2/3, p-Smad2/3) on TGF-?/Samd pathway was detected by using Western blotting following SNHG6 silenced or overexpressed.Results UPF1 expression was significantly down-regulated in HCC tissues and HCC cell lines, UPF1 regulated Smad7 expression in HCC. SNHG6 was able to bind to UPF1 and reduce its stability and downregulated its protein level. SNHG6 could regulate the Smad7 level by binding to UPF1 and then affect the key molecular (p-Smad2/3) on the TGF-?/Smad pathway.Conclusion SNHG6 regulates HCC tumorigenesis by binding to RNA binding protein UPF1 and then affects the TGF-?/Smad pathway.