The Function And Mechanism Study Of LncRNA-SNHG7 In The Regulation Of Hepatocellular Carcinoma

Background and Objective:Hepatic carcinoma has been considered the sixth most common malignant tumor and fourth highest mortality rate among cancers in the world with about 840,000 new cases diagnosed annually.Most patients with hepatic carcinoma die from the high rates of invasion,metastasis,and post-operative recurrence.However,the aetiology and exact molecular mechanism on underlying hepatic carcinoma progression have not been comprehensively illuminated yet.The carcinogenesis of hepatic carcinoma is considered to be a multifactor,multistage complex process.Recently,noncoding RNAs(ncRNAs)have caused great concerns for its complex and crucial role in cancer progression.lncRNA is generally considered to be non-coding RNA longer than 200 nucleotides,which cannot be translated into proteins but involved in various of biological processes,including transcription regulation,posttranscriptional regulation,protein modification,and tumourigenesis especially.An increasing number of studies have shown that IncRNA can interact with miRNA by acting as competitive endogenous RNAs(ceRNAs)to sponges target mRNA,thereby regulating the expression of genes that play an important role in cancer.Small nucleolar RNA host gene 7(SNHG7)is a long ncRNA with 2157 bp in length,located on chromosome 9q34.3.Recent studies have suggested that SNHG7 could promote proliferation,migration,and invasion of various cancers.At present,it is still unclear whether SNHG7 participates in the process of occurrence and development of hepatic carcinoma through the mechanism of ceRNA.This article will deeply study the relationship between SNHG7 and hepatocellular carcinoma(HCC),analyze the role of SNHG7 in the occurrence and development of HCC and related molecular mechanisms,The purpose of this article is to provide a new molecular markers and effective targets for the early diagnosis,treatment,efficacy evaluation and prognosis of hepatic carcinoma.Part ⅠMethods:The expression level of SNHG7 in 40 pairs of hepatic carcinoma tissues and corresponding relative normal tissues in the Second Affiliated Hospital of Nanchang University was detected by qRT-PCR,Analyze the correlation between the expression level of SNHG7 and the clinicopathological characteristics of patients with hepatic carcinoma.Results:The results of qRT-PCR showed that SNHG7 was significantly overexpressed in hepatic carcinoma tissue compared with adjacent normal tissue.Moreover,SNHG7 is highly expressed in liver cancer tissue samples.Patients with high SNHG7 expression have a higher TNM stage and are prone to lymph node metastasis.Conclusion:SNHG7 overexpression was observed in hepatic carcinoma tissues compared with adjacent histologically normal hepatic tissues.In addition,overexpressed SNHG7 in hepatic carcinoma patients was significantly associated with lymph node metastasis and TNM stage.SNHG7 can be used as a potential marker for poor prognosis of liver cancer.Part ⅡMethods:siRNA interfering with SNHG7 was transfected into HepG2 and HCC-LM3 cells.The knockdown efficiency of SNHG7-siRNA was confirmed by qRT-PCR assay.Detect proliferation and growth ability of cells in each group using MTT and plate clone assays.Flow cytometry was used to analyze cell apoptosis in each group.s Wound healing assay,transwell migration and invasion assay were used to evaluate the difference in cell invasion and migration ability of different groups.The expression of EMT molecular markers,like E-cadherin,N-cadherin,and MMP-9,were detected by Western blot.Results:The expression level of SNHG7 in SNHG7-siRNA transfected HepG2 and HCC-LM3 cells was drastically decreased.MTT and colony formation assay suggested that knockdown of SNHG7 significantly suppressed the proliferation of HepG2 and HCC-LM3 cells.In addition,transfection of SNHG7-siRNA induced significantly increased percentage of apoptotic cells in HepG2 and HCC-LM3 cells.After the expression of SNHG7 was downregulated,the invasion and migration ability of cells were decreased Furthermore,silence of SNHG7 led to decreased level of metastasis related protein MM P-9,N-cadherin,and increased level of cell adhesion protein E-cadherin.Conclusion:Down-regulating the expression of SNHG7 in liver cancer cells can inhibit the proliferation and metastasis of liver cancer cells and promote cell apoptosis,which provides a potential molecular target marker for targeted therapy of liver cancer.Part ⅢMethods:Bioinformatics prediction results show that miR-425 is a downstream miRNA molecule of SNHG7 and dual-luciferase reporter assay was conducted to validate the interaction between miR-425 and SNHG7 in HepG2 cells.MiR-425-mimic and miR-NC were synthetized and transfect into hepatic carcinoma cells.Detect invasion and migration ability of cells in each group using wound healing assay,transwell migration and invasion assay.Western blot was used to detect the expression of Wnt/β-catenin/EMT signaling pathway proteins.Results:Bioinformatics analysis indicated that there are putative binding sites between SNHG7 and miR-425.The results of the dual luciferase reporter gene showed that miR-425-mimic inhibited the luciferase activity of wild-type SNHG7(SNHG7-Wt),but could not inhibit the luciferase activity of mutant SNHG7(SNHG7-Mut).Wound healing and transwell assays suggested that migratory and invasive ability were significant reduced in cells transfected with miR-425-mimic.While transfected with miR-425-mimic,the protein levels of β-catenin,MMP-9,and N-cadherin were decreased markedly.In contrast,E-cadherin expression was increased notably.Moreover,downregulation of miR-425 could significantly block the antimigration effect,which is induced by SNHG7-inhi treatment inHepG2 cells.Conclusion:SNHG7 promotes metastasis of hepatic carcinoma cell via Wnt/β-catenin/EMT pathway as a miR-425 sponge.Part ⅣMethods:Subcutaneous xenograft tumour model in nude mice was built to study the relationship between SNHG7 and tumourigenesis in vivo.Examine the expression of Ki-67 and MMP-9 proteins in transplanted tumor of nude mice with immunohistochemistry.Results:Animal research results show that the interference of SNHG7 leads to a significant reduction in the tumor size and weight of nude mice compared with the control group,the expression of Ki-67and MMP-9 proteins in HepG2 xenografts was decreased.Conclusion:Animal experiments showed that knockdown of SNHG7 could impede hepatic carcinoma cell proliferation and metastasis in nude mice.