The Mechanism Of SNHG12 To Influence The Apoptosis And Invasion Of Esophageal Squamous Cell Cancer

Esophageal squamous cell carcinoma is a common human malignant digestive tract tumor,which originated in esophageal squamous epithelium.According to the latest statistics of global cancer data 2018,it ranks sixth among 36 types of cancer.The incidence of esophageal cancer in China is among the top five in the world,far higher than the world average.The number of new cases of esophageal cancer in the world was about 572,000,located as 7th among 36 cancers.The number of deaths was about 50.8.Since the early symptoms are not obvious and the course of disease progresses rapidly,most of the patients are diagnosed in the middle and late stage.Therefore,finding simple and effective early diagnosis is conducive to timely diagnosis in the early stage of disease development,which is of great significance to reduce the fatality rate and improve the prognosis of patients.Currently,the most studied serum tumor markers consist of the following four categories:DNA,miRNA,long non-coding RNA and protein.In this study,we mainly discussed long non-coding RNA(lncRNA).It was found that the expression of small nucleolus RNA 12(SNHG12)in ESCC tissues and cell lines was up-regulated compared with normal tissue cells,while the expression of mmp-2 protein and mmp-9 protein was down-regulated and correlated with the down-regulation of SNHG12,which could inhibit the proliferation and migration of tumor cells.A growing body of research now supports this.In addition,SNHG12 can also regulate the migration of endothelial cells and play an important role in cell proliferation and migration.Therefore,in this study,we screened two strains of cells with high SNHG12 expression,reduced the expression of SNHG12 by RNA interference,and then determined the effect of siRNA transfer of SNHG12 on the expression of SNHG12 in each group by qRT-PCR.The effect of SNHG12 gene expression on apoptosis of esophageal carcinoma cells was determined by flow cytometry.Cell scratch test and transwell invasion test were used to detect the infiltration and metastasis ability of esophageal cancer cells.Finally,Western blot was used to detect the expression levels of proliferation-related proteins(bcl-2 and Bax)and infiltration and metastasis related proteins(MMP2 and MMP9)in each cell group.The purpose of this study is to preliminarily explore the regulation of SNHG12 in the development and progression of esophageal squamous cell carcinoma,and to explore and confirm whether SNHG12 can be used as a new potential target for the treatment of esophageal squamous cell carcinoma.Methods(1)Detection of SNHG12 expression in esophageal squamous cell carcinoma:the expression of SNHG12 gene in different esophageal squamous cell carcinoma strains was detected by qrt-pcr,and the two cells with the highest expression levels were selected for subsequent studies.Normal esophageal epithelial cells were used as controls.(2)siRNA Transfection:NC siRNA and three SNHG12 siRNA were transfected into the above two cell lines with lipo2000,respectively,and the transfection effect was analyzed according to the results of qrt-pcr.(3)Cell proliferation detection:each cell strain was divided into three groups,one of which was transferred to siRNA,and then cultured for 0h,24h,48h and 72h.Cell proliferation rate was determined by cck-8 method.(4)Cell apoptosis detection:cells in SNHG12 si group and NC group were treated with PIPC kit,and cell apoptosis was detected by flow cytometry,and the apoptosis rates of cells in the three groups were calculated to observe the effect of SNHG12 on apoptosis of esophageal squamous cell carcinoma cells.(5)Cell migration detection:cell scratch test and Transwell test respectively measured the changes of migration and invasion ability of three groups of cells under the same conditions.(6)Mechanism:western-blotting assay was used to detect the changes of bcl-2 and Bax apoptosis-related proteins and MMP2 and MMP9 infiltrating transfer-related proteins in the two groups before and after transfection.(7)Statistical processing:Graphpad software was used for statistical processing of the data,and x2 test was used for inter-group comparison.The data of each group was represented by mean ± standard deviation(x±s),and the mean of multiple samples was analyzed by one-way ANOVA,and the test level was 0.05.Results(1)qRT-PCR results showed that the expression levels of SNHG12 in different esophageal squamous cell cells and normal esophageal epithelial cells were significantly different,in particular,the expression levels of KYSE450 cells and KYSE180 cells were the highest,so they were selected for follow-up study.(2)After transfection with SNHG12 siRNA,compared with the respective siRNA group and NC group,the expression of SNHG12 gene in the siRNA group of the two strains was significantly reduced,and snhg12-homo-354 showed the most significant knock-down effect in the three siRNA sequences,so this sequence was selected for subsequent interference RNA experiments.(3)The results of cck-8 cell proliferation assay showed that the cell proliferation rate was significantly lower than that of the NC group within 48h after transfection,and the cell proliferation rate continued to decrease at 48h and 72h,respectively.These results suggest that the expression of SNHG12 can inhibit cell proliferation effectively.(4)Apoptosis results showed that after 48h of transfection,the number of early apoptotic cells in the siRNA group in KYSE450 and KYSE180 cells accounted for 7.3%and 1.2%of the total number of cells,respectively,significantly higher than that in the control group and the blank group.The difference was statistically significant(P<0.01).In addition,the number of living cells in the NC group was 96.4%(KYSE450)and 86.1%(KYSE180),which was significantly higher than that in the siRNA group(kyse450/50.3%and kyse 180/9.9%),and the difference was statistically significant(P<0.01).(5)Cell scratch test results of KYSE450 showed that cell migration was significantly inhibited in the si group after 48h transfection compared with the blank group and the NC group,and the difference was statistically significant(P<0.01).The cell scratch test of KYSE180 was also compared,and the difference was statistically significant(P<0.01).However,there was no significant difference in cell migration distance between the blank group and the NC group.(6)The Transwell results showed that the average number of transmembranous cells in the si group of KYSE450 cells was 221.3,significantly lower than that in the blank group(389.1)and the control group(395.5).The mean number of transdermal cells in the si group of KYSE180 cells was 97.3/per field,significantly lower than that in the blank group(147/per field)and the control group(152.6/per field).(7)Western-blotting results showed that both the si group of KYSE450 and KYSE180 showed significantly lower bcl-2 protein expression than the blank control group,with statistically significant differences(KYSE450:P<0.01).KYSE180:P<0.01).The Bax protein expression level in the si group was significantly higher than that in the NC group,and the difference was statistically significant(KYSE450:P<0.01,KYSE180:P<0.01).Conclusion(1)SNHG12 may be involved in regulating the occurrence,development,invasion and metastasis of esophageal squamous cell carcinoma.(2)the down-regulation of SNHG12 gene is related to the expression of bcl-2 protein,Bax protein,MMP2 protein and MMP9 protein.