LINC02535 Interacts With ADAR2 To Regulate The Effect Of P53 Signaling Pathway In Gastric Cancer

As one of the malignant tumors,gastric cancer has seriously threatened people’s health.Since there are no obvious markers in the early stage of gastric cancer,most patients have reached the middle and late stage when they are diagnosed.Although the diagnosis and treatment methods are constantly updated,the survival rates for patients with gastric cancer are still low.Therefore,looking for more effective markers and analyzing the specific mechanism of gastric cancer will have a positive effect on the diagnosis and treatment of gastric cancer.LncRNA is a non-coding RNA with a length of more than 200 nt,mainly expressed in the nucleus and cytoplasm.LncRNA plays a very important role in the occurrence and development of tumors.It interacts with mRNA,DNA and proteins and participates in various transcription and translation processes,and then affect the proliferation,migration,invasion and drug resistance of tumor cells.Recent studies show that LINC02535 as a kind of non-coding RNA can affect the proliferation,migration and invasion of cervical cancer cells by regulating related genes and signal pathways.However,the role of LINC02535 in the occurrence and development of gastric cancer is unclear.In this study,through whole transcriptome sequencing,it was found that LINC02535 was highly expressed in patients with gastric cancer,and the difference was extremely significant.And through in vivo and in vitro experimental studies,it is found that LINC02535 can promote the proliferation,migration and invasion of gastric cancer cells and inhibit the apoptosis of gastric cancer cells.In addition,molecular mechanism studies have found that LINC02535 interacts with ADAR2 to jointly regulate the p53 signaling pathway and affect the occurrence and development of gastric cancer.Part Ⅰ:Sequencing analysis of whole transcriptome of gastric cancer samples and functional study of LINC02535 in gastric cancer cellsPurpose:Whole transcriptome sequencing analysis of gastric cancer samples and in vivo and in vitro study of the effects of LINC02535 on the proliferation,migration,invasion and apoptosis of gastric cancer cells.Method:Whole transcriptome sequencing was performed by collecting samples of gastric cancer tissue and corresponding normal tissues adjacent to the cancer.Fluorescence quantitative PCR was used to detect the expression level of LINC02535 in human gastric cancer cell lines(SGC7901,MGC-803,HGC-27 and BGC823)and normal gastric mucosal epithelial cell lines(GES-1).Construct a plasmid that interferes with LINC02535 and overexpresses LINC02535.By transfecting the interfering LINC02535 plasmid in the cell lines SGC7901 and MGC803 with high expression of LINC02535,down-regulating the expression of LINC02535,and transfecting the over-expressing LINC02535 plasmid in the cell lines HGC27 and BGC823 with low expression of LINC02535,and after up-regulating the expression of LINC02535,CCK-8 test was adopted.and clone formation test to detect the proliferation ability of gastric cancer cells,scratch test to detect the migration ability of gastric cancer cells,Transwell test to detect the invasion ability of gastric cancer cells,flow cytometry to detect the apoptosis of gastric cancer cells,WB test to detect migration and apoptosis related The expression of protein,elucidating the specific mechanism of LINC02535 on the migration and apoptosis of gastric cancer cells.In addition,by subcutaneously injecting si-LINC02535 and the control NC SGC7901 stably transfected cell line into BALB/c-nu male nude mice,and then observing the tumor growth rate and the expression of related proteins in the tumor,further exploring the effects of LINC02535 on gastric cancer in vivo and in vitro Developmental impact occurs.Results:Whole transcriptome sequencing showed that a total of 499 upregulated DEGs and 627 downregulated DEGs were identified between peritumoral and gastric cancer tissues.The cell cycle,cAMP signalling pathway,p53 signaling pathway and homologous recombination were enriched in the KEGG pathway analysis.Compared with the normal gastric mucosal epithelial cell line(GES-1),the expression of LINC02535 in human gastric cancer cell lines(SGC7901,MGC803,HGC27 and BGC823)was significantly up-regulated.The cell function study results showed that compared with the control,the gastric cancer cell lines SGC7901 and MGC803,which interfered with LINC02535,had a significant decrease in the proliferation,migration and invasion abilities,an increase in the rate of cell apoptosis,and a corresponding increase in the expression of migration and apoptosis-related proteins.Increase or decrease;On the contrary,the overexpression of LINC02535 gastric cancer cell lines HGC-27 and BGC823 have a significant increase in the proliferation,migration and invasion abilities,a decrease in the rate of cell apoptosis,and the expression of migration and apoptosis-related proteins.Increase or decrease.In vivo experiments found that compared with the control NC’s SGC7901 stably transfected cell line,the si-LINC02535 cell line was injected subcutaneously in nude mice,and the tumor volume and weight were significantly reduced.Conclusion:The high expression of LINC02535 in gastric cancer tissues affects the occurrence and development of gastric cancer by promoting the proliferation,migration and invasion of gastric cancer cells and inhibiting the apoptosis of gastric cancer cells.Part Ⅱ:The interaction between LINC02535 and ADAR2 and the function of ADAR2 in gastric cancer cellsPurpose:Explore the mechanism of the interaction between LINC02535 and ADAR2 and the effect of ADAR2 on gastric cancer.Method:Through RIP assay to verify the mutual combination of LINC02535 and ADAR2.Fluorescence quantitative PCR was used to detect the expression level of ADAR2 in human gastric cancer cell lines SGC7901,BGC823,MGC803,HGC27 and normal gastric mucosal epithelial cell line GES-1.Construct an overexpressing ADAR2 plasmid.By transfecting the over-expressing ADAR2 plasmid in the low-expressing ADAR2 cell lines HGC27 and MGC803,the CCK-8 test and the clone formation test were used to detect the proliferation ability of gastric cancer cells;the scratch test was used to determine the migration ability of gastric cancer cells;the Transwell test was used to detect the gastric cancer cells.Invasion ability;flow cytometric detection of apoptosis of gastric cancer cells;WB test to detect the expression of migration and apoptosis-related proteins to illustrate the specific mechanism of ADAR2 on the migration and apoptosis of gastric cancer cells.In in vivo experiments,the MGC803 cell line with ADAR2 overexpression and control NC was subcutaneously injected into BALB/c-nu male nude mice,and then the tumor growth rate and the expression of related proteins in the tumor were observed to further explore the effect of ADAR2 on gastric cancer cells.Results:Compared with the normal gastric mucosal epithelial cell line(GES-1),the expression of ADAR2 in human gastric cancer cell lines(SGC7901,BGC823,MGC803 and HGC27)was down-regulated.The in vitro results showed that compared with the control,the proliferation,migration and invasion ability of the HGC27 and MGC803 cell lines overexpressing ADAR2 gastric cancer decreased significantly,the apoptosis rate increased,the expression of apoptosis-related protein increased,and the expression of migration-related protein decreased.In vivo experiments found that compared with the control NC MGC803 stably transfected cell line,the tumor volume and weight decreased significantly after the ADAR2 overexpressed cell line was injected subcutaneously into nude mice.Conclusion:LINC02535 and ADAR2 can bind to each other;ADAR2 is lowly expressed in gastric cancer tissues,and ADAR2 affects the occurrence and development of gastric cancer by inhibiting the proliferation,migration and invasion of gastric cancer cells and promoting the apoptosis of gastric cancer cells.Fart Ⅲ:The molecular mechanism of LINC02535 and ADAR2 co-regulated p53 signaling pathway influencing the occurrence of gastric cancerPurpose:Explore how the interaction between LINC02535 and ADAR2 co-regulates the p53 signaling pathway and affects the occurrence of gastric cancerMethod:KEGG database was used to analyze the differentially related genes expressed in p53 signaling pathway.Then the fluorescence quantitative PCR experiment was used to detect the expression changes of these differential genes in the LINC02535 interference cell line and the ADAR2 overexpression cell line.The differences in the expression changes of these genes were detected again in gastric cancer tumor tissues and normal tissues.Results:KEGG database analysis results showed that in the p53 signaling pathway,8 genes with significant differences in expression(BID,RRM2,CDK1,CCNB1,CCNE2,CHEK1,CCNB2,GTSE1)were up-regulated,and 2 genes with significant differences in expression(GADD45B and RPRM)were down-regulated.Fluorescence quantitative PCR experiments showed that compared with the control group,the expression of genes BID,RRM2,CDK1,CCNB1,CCNE2,CHEK1,CCNB2,and GTSE1 were significantly inhibited in the LINC02535 interference cell line and the ADAR2 overexpression cell line,while the expression of genes GADD45B and RPRM expression increased significantly.In gastric cancer tumor tissue,the expression of these 10 genes is consistent with the results of KEGG analysis.Conclusion:LINC02535 and ADAR2 jointly regulate the p53 signaling pathway and affect the occurrence of gastric cancer.