The Role And Mechanism Of LncRNA-MSC-AS1/miR-520d-3p/EIF4G2 In The Development Of Gastric Cancer

Objective:Gastric cancer is the fourth largest common malignancy in the world,ranking second in the mortality rate of malignant tumors in Asia,seriously threatening the physical and mental health of residents.The development of gastric cancer is a very complex biological process involving the regulation of multiple oncogenes or tumor suppressor genes.Therefore,studying the pathogenesis of gastric cancer has great significance for the diagnosis and treatment of gastric cancer.Long non-coding RNA(lnc RNA)is an RNA molecule of over 200 nucleotides in length.Recent studies suggest that lnc RNA is involved in multiple physiological or pathological processes,especially during the development of malignancies such as gastric cancer,and can play a biological role as an oncogene or tumor suppressor.MSC antisense RNA 1(MSCAS1)is a novel Lnc RNA,located on chromosome 8q13.3-q21.11.It has been reported that the MSC-AS1/miR-29b-3p axis regulates cell proliferation and apoptosis in pancreatic ductal adenocarcinoma cell lines via CDK14.However,the underlying mechanism of MSC-AS1 in gastric cancer is currently unknown.Therefore,the present study aims to explore elucidating the role of MSC-AS1 in gastric cancer and its mechanisms.Methods:First,we extracted total RNA from 60 surgically removed gastric cancer tissues and paired adjacent cancerous normal gastric tissues to detect MSC-AS1 expression levels by quantitative real-time PCR(RT-q PCR).We then identified experimentally with gastric cancer cell lines MKN-45 and AGS,by cell line screening and successfully constructed the MSC-AS1 interference lentivirus,with stably expressed cell lines identified after transfection.Then the proliferation ability of gastric cancer cells was measured by CCK-8 and cell clone formation experiments,cell scratch tests the migration ability after knockdown MSC-AS1,transwell tests the invasion ability after MSC-AS1 knockdown,and then evaluates the desiccability of gastric cancer cells after MSC-AS1 knockdown,initially exploring the possible mechanism of MSC-AS1 affecting the occurrence and development of gastric cancer.We next used the same gastric cancer cell lines MKN-45 and AGS as the previous fraction as a cell model,predicted data predicting miRNA,potentially complementary to MSC-AS1 and m RNA molecules potentially targeted by bioinformatics analysis,and verified the above relationships by double luciferase reporter and RNA pull-down experiments.Then,by transient transfection,MKN-45 and AGS,were established: empty plasma,MSC-AS1,MSC-AS1+sh-NC and MSC-AS1+sh-EIF4G2.The mechanism by which MSC-AS1/miR-520d-3p/EIF4G2 regulates gastric cancer cells in vitro was explored by cytology experiments to further clarify and refine the role of the MSC-AS1/miR-520d-3p/EIF4G2 signaling axis in regulating the proliferation,invasion,migration,and stemness maintenance of gastric cancer cells.Finally,to verify the function of MSC-AS1 in gastric cancer in vivo,we established a nude mouse tumor-bearing model.To verify the effect of MSC-AS1 knockdown on the tumorigenicity of gastric cancer cells in nude mice.We then examined the expression levels of MSC-AS1,EIF4G2 and miR-520d-3p in GC tumorigenesis in two nude mice.The expression levels of tumor Ki-67,vimentin,SOX2 and E-cadherin expression levels were then examined by immunohistochemistry,exploring in nude mouse models,After the knockdown of the MSC-AS1,epicortical stromal transformation occurs against gastric cancer.Results:Detection of clinical tissue samples revealed that MSC-AS1 was highly expressed in gastric cancer tissue as compared to paired normal gastric mucosal tissue.In the cell assay,RT-q PCR examined MSC-AS1 expression in MKN-45,MKN-7,BGC-823,AGS gastric cancer cells as well as the normal gastric mucosal cell line GES-1,and found that MSC-AS1 was significantly highly expressed in gastric cancer cell lines compared to GES-1 cells,and AGS cells in the four gastric cancer cells.Through CCK-8 and clonforming experiments,we found that gastric cancer cell proliferation and clonal formation ability were significantly inhibited after MSC-AS1knockdown;Moreover,scratch experiments and transwell chamber experiments showed that MSC-AS1 knockdown significantly inhibited the migration and invasion of gastric cancer cells;cell experiments confirmed that MSC-AS1 knockdown effectively inhibited the stemness of gastric cancer cell lines MKN-45 and AGS.These experiments show that knockdown of MSC-AS1 can effectively inhibit the proliferation,invasion,and migration of gastric cancer cells at the cellular level,and also inhibit the maintenance of its stemness.We subsequently passed through the literature as well as through the online database(Starbasev3.0 http://starbase.sysu.edu.cn/)Analysis predicted that miR-520d-3p may be one of the main targets of MSC-AS1 and miR-520d-3p may bind to EIF4G2 to play downstream regulatory roles,to confirm this hypothesis,we verified these two targeting relationships using the double luciferase reporter experiment and the experiments of RNA pull-down,respectively,to explore the mechanism by which MSC-AS1/miR-520d-3p/EIF4G2 regulates gastric cancer cells.The results were found that,in the gastric cancer cell lines MKN-45 and AGS transfected with miR-520d-3p mimic,MSC-AS1 can interact with miR-520d-3p through the miR-520d-3p domain with its own 3’UTR,miR-520d-3p can inhibit the amount of fluorescence of psi-CHECK-MSC-AS1-WT(P<0.05),However,there was no inhibitory effect on psi-CHECK-MSC-AS1-mut(P>0.05).Then,to validate MSC-AS1 binding to miR-520d-3p,we performed an RNA-pull-down assay to detect the amount of miRNA,and found that miR-520d-3p binding to Bio-miR-520d-3pWT increased significantly,confirming that miR-520d-3p binds to MSC-AS1 and is one of the main targets of MSC-AS1.In terms of EIF4G2 interaction with miR-520d-3p,we made it clear that EIF4G2 interacts with miR-520d-3p through its own luciferase miR-520d-3p domain possessed by the luciferase reporter assay,which thus suppresses the fluorescence amount of EIF4G2-WT(P<0.05).However,without an inhibitory effect on EIF4G2-mut(P>0.05),RNA-pull-down experiments showed that bio-miR-520d-3pWT alone was able to pull down EIF4G2 expression,thus verifying the mutual binding of EIF4G2 to miR-520d-3p.To test the effect of EIF4G2 on MKN-45 and AGS cells under the regulation of MSC-AS1,the gastric cancer cell line MKN-45 was examined using the CCK-8 assay.Cells were grouped into empty plasmid,MSC-AS1,MSC-AS1+sh-NC,and MSC-AS1+sh-EIF4G2 groups.The CCK-8 results show that,sh-EIF4G2 inhibits the proliferation of MKN-45 cells upregulated by MSCAS1.The results of the Transwell invasion experiments indicate that,overexpression of MSC-AS1 promotes the migration and invasion ability of MKN-45 cells.Knockdown of EIF4G2 restored the migration and invasion ability of gastric cancer cells.To investigate whether EIF4G2 affects the stem cell-like properties of gastric cancer cells,we transfected the sh-EIF4G2 plasmid in MKN-45 from gastric cancer cells that had been slowly transfected with MSC-AS1,a cellular model of EIF4G2 silencing was established,After the balloon formation assay,we observed that the MSC-AS1-induced balloon formation capacity was inhibited by sh-EIF4G2.The western blot results show that,upregulation of MSC-AS1 caused increased expression levels of Nanog,SOX2 and OCT4.However,EIF4G2 downregulation reduces the effect of MSC-AS1 overexpression on the above stemness markers,Suphibiting its expression.Experimental results in in vivo graft tumor models in nude mice showed reduced expression levels of MSC-AS1 and EIF4G2 and enhanced miR-520d-3p in tumors in nude mice injected with sh-MSC-AS1 gastric cancer cells(P<0.01).Immunohistochemical experiments showed that silencing of MSC-AS1 significantly reduced the expression levels of EMT-associated proteins such as Ki-67,vimentin and SOX2,and enhanced the expression levels of E-cadherin.It is suggested that MSC-AS1 may regulate the biological activity of gastric cancer cells by regulating tumor EMT-associated protein expression.Conclusions:In conclusion,the expression level of MSC-AS1 was significantly higher in gastric cancer tissues or cells,compared with normal gastric mucosal tissues or gastric mucosal epithelial cells.Reducing the expression level of MSC-AS1 in gastric cancer cells suppresses the proliferation,migration and invasion of gastric cancer cells and prevents the maintenance of stemness.In vitro and external experimental studies show that this regulation mechanism is carried out through the interaction between MSC-AS1/miR-520d-3p/EIF4G2.By exploring the role of MSC-AS1 in gastric cancer and studying the occurrence and development mechanism of gastric cancer from the perspective of MSC-AS1/miR-520d-3p/EIF4G2 pathway regulation,the research conclusion enriches the regulatory mechanism of Lnc RNA in gastric cancer,lays a new theoretical foundation for the treatment of gastric cancer,and provides a new intervention strategy and therapeutic target.