The Mechanism Of LncMIR31HG In Regulating Proliferation,migration And Invasion Of Gastric Cancer Cells

Gastric cancer is one of the most common malignant tumors in the world,ranking second in cancer-related deaths worldwide,and is one of the main causes of cancerrelated deaths.Gastric cancer originates from the surface of gastric epithelial cells,it can occur in every part of the stomach.Gastric cancer in the antrum,pylorus,and cardia has the highest incidence,and the incidence of the stomach body is slightly lower.In recent years,with the development of endoscopic and surgical techniques,the 5-year mortality rate of early gastric cancer has been significantly reduced.However,for advanced gastric cancer,the 5-year mortality rate is still 30%-50%.The occurrence of gastric cancer is a complex and gradual development process involving multiple factors,multiple steps,coding and non-coding genes.Therefore,it is of great significance to explore the progress mechanisms of proliferation,growth,migration,invasion and apoptosis of gastric cancer.Lnc RNA is a type of non-coding RNA with a length of more than 200 nt.According to its position and direction,lnc RNA can be divided into intron lnc RNA,intergenic lnc RNA,sense or antisense lnc RNA,retrotransposons.Functionally,lnc RNA can regulate gene expression at any level,including chromatin modification,transcription,and post-transcriptional levels.Numerous studies have found that lnc RNA plays an important role in many disease processes,including the progression of cardiovascular disease and cancer.Numerous studies have shown that non-coding RNA plays an important role in the progression of gastric cancer.MIR31HG(LOC554202)is a newly discovered lnc RNA located at 9p21.3 and 2166 bp in length.Recent studies have confirmed that MIR31 HG is up-regulated in many cancers compared to normal tissues.These studies have shown that MIR31 HG is associated with the occurrence and development of multiple tumors.It has been reported to be highly expressed in multiple tumors and can be used as a marker of poor prognosis.There is no research on the mechanism of MIR31 HG in gastric cancer.This article focuses on exploring the effect of MIR31 HG on the proliferation and migration of gastric cancer cells and the regulation mechanism of β-catenin,in order to provide new ideas for the clinical treatment of gastric cancer.The main experimental results in this article are as follows ::1.MIR31 HG promotes the proliferation of gastric cacner cellsWe first designed the interference primers for MIR31 HG,and we interfered with MIR31 HG in gastric cancer cell lines MKN45 and SGC7901 by RNA interference technology.At the same time,compared with the control group,the morphology of gastric cancer was changed and the number of cells was significantly decreased.Then we performed MTT and Brd U experiments,and the results showed that downregulating MIR31 HG could inhibit the proliferation of MKN45 and SGC7901,and then we found that MIR31 HG knockdown induced the MKN45 and SGC7901 cells arrest at the G0/G1 phase by using flow cytometry,the protein expression of CDK2,CDK4 and cyclin D1 decreased,while the levels of p53 protein were up-regulated.We subsequently overexpressed MIR31 HG in MKN45 and SGC7901,cell proliferation was promoted by MTT and Brd U assays,and the expression of cycle-related protein was up-regulated as expected.Collectively,these results demonstrate that MIR31 HG is necessary for cell growth and proliferation of gastric cancer cells.2.MIR31 HG promotes the clonogenicity of gastric cancer cells in vitro and vivoTo investigate the effect of MIR31 HG on the clonogenicity of gastric cancer cells in vitro and in vivo,we performed soft agar and xenograft tumor growth assays,knochdown of MIR31 HG results in a significant reduction in the number and size of clones formed by cancer cells.Moreover,MKN45 and SGC7901 cells with MIR31 HG knockdown were injected into nude mice to form xenograft tumors,it is obvious that the volume and weight of the transplanted tumor were smaller than the control group.In addition,immunohistochemical staining demonstrated that the number of Ki67-positive cancer cells was decreased after MIR31 HG knockdown.These results indicate that MIR31 HG knockdown can inhibits the self-renewal capability of gastric cancer cells.3.MIR31 HG promotes migration and invasion of gastric cacner cellsTo explore the influence of MIR31 HG on gastric cancer cell migration ability,we performed Wound-healing and Transwell assays,the results showed that the ability of migration and invasion of gastric cacner cells was decreased significantly in MIR31 HG silenced cells.At the same time,the protein expression of β-catenin,N-cadherin,MMP9,Vimentin,Slug levels were decreased after MIR31 HG knockdown.After MIR31 HG overexpression,these proteins were up-regulated,demonstrating that MIR31 HG can influences migration and invasion of gastric cacner cells4.MIR31 HG promotes the stability of β-cateninBased on the results above,we found that the protein expression levels of β-catenin and its downstream genes(c-MYC and cyclin D1)were inhibited after knockdown of MIR31 HG,and their protein levels were up-regulated after overexpression of MIR31 HG.In order to further study the relationship between MIR31 HG and β-catenin,we added Wnt/β-catenin pathway inhibitor FH535 based on overexpression of MIR31 HG.MTT,Transwell and Western blot results showed that FH535 could inhibit the proliferation,migration and invasion of gastric cancer cells induced by MIR31 HG.Subsequently,we down-regulated MIR31 HG and detected the m RNA levels of β-catenin,and found no significant decrease in m RNA levels.We performed MG132,CHX and IP assays,these results indicated that MIR31 HG can stabilize β-catenin.5.MIR31 HG promotes the stability of β-catenin through c-CBLAfter we interfered with MIR31 HG,we tested the expression level of β-catenin E3 ligase and found that the expression level of c-CBL was significantly up-regulated,but the c-CBL RNA level was not significantly up-regulated.We speculate that MIR31 HG has an effect on the protein levels of c-CBL.In order to further prove that MIR31 HG stabilizes the expression of β-catenin by c-CBL,we interfered with c-CBL on the basis of interfering with MIR31 HG,and found that the expression of β-catenin has recovered,MTT and Transwell experiments proved that interference with c-CBL can restore the inhibitory effect of MIR31 HG down-regulation on the proliferation and migration of gastric cancer cells.In summary,the final experimental results in this article show that MIR31 HG can promote the proliferation and migration of gastric cancer cells,and may promote the stability of β-catenin through c-CBL.This study focuses on exploring the proliferation of gastric cancer cells by MIR31 HG and the ability of migration and invasion and the regulation of β-catenin may better reveal the mechanism of the occurrence and development of gastric cancer,and provide new strategies for clinical treatment of this disease.