The Expression And Function Of Circ-sirt1 In Gastric Cancer

Part One The expression of circ-sirt1,sirt1 and mi R-132-3p/mi R-212-3pin gastric cancer tissues and serumsObjective: To detect the expression of circ-sirt1,sirt1 and mi R-132-3p/mi R-212-3p in gastric cancer tissues and adjacent tissues,as well as in the serums of gastric cancer patients and normal human serums.Then the relationships between the expressions of these genes in gastric cancers were analyzed.Methods: The real-time fluorescence quantitative PCR(RT-q PCR)technology was used to detect the relative expression of circ-sirt1,sirt1,mi R-132-3p/mi R-212-3p in gastric cancer tissues and adjacent tissues,as well as in the serums of gastric cancer patients and normal human serums.Results:1.The levels of circ-sirt1 in gastric cancer tissues were significantly lower than that in adjacent tissues(P<0.05).The levels of circ-sirt1 in the serums of gastric cancer patients were significantly lower than that in normal human serums(P<0.05).2.The m RNA levels of sirt1 in gastric cancer tissues were markedly lower than that in adjacent tissues(P<0.05).3.The levels of mi R-132-3p/mi R-212-3p in gastric cancer tissues were higher than those in adjacent tissues(P<0.01,P<0.01).The levels of mi R-132-3p/mi R-212-3p in the serums of gastric cancer patients were significantly higher than those in normal human serums(P<0.001,P<0.01).Conclusions: The circ-sirt1 and sirt1 were hypo-expressed in cancer tissues and serums of patients with gastric cancer.The mi R-132-3p/mi R-212-3p were hyper-expressed in gastric cancer tissues and serums.The suggested that circ-sirt1,sirt1 and mi R-132-3p/mi R-212-3p might have related to gastric cancer to a certain extent.Part Two The effect of circ-sirt1/mi R-132-3p/mi R-212-3p/sirt1 axis onbiological functions of gastric cancer cellsObjective: To explore the effects of overexpression of circ-sirt1 on the proliferation,migration and invasion in gastric adenocarcinoma cells by cell experiment,and to elucidate whether those function of circ-sirt1 is mediated by mi R-132-3p/mi R-212-3p and sirt1.Methods:1.The levels of circ-sirt1 in human gastric adenocarcinoma cell lines(AGS-1 and BGC-823),as well as in normal human gastric mucosal cell lines GES-1 were detected by RT-q PCR.2.The circ-sirt1 plasmid containing circ-sirt1 sequence were constructed and the band digested with restriction enzyme were verified by DNA sequencing.3.BGC-823 and AGS-1 cell lines were transfected with pc DNA3.1,circ-sirt1 plasmid,ASO-mi R-132-3p+ASO-mi R-212-3p and ASO-NC respectively.The transfection effects were verified by RT-q PCR.4.The proliferation abilities of gastric adenocarcinoma cells in each group were detected by plate cloning assay.5.The migration and invasion abilities of gastric adenocarcinoma cells in each group were detected by transwell migration assay and transwell invasion assay.6.The levels of mi R-132-3p/mi R-212-3p in gastric adenocarcinoma cells were detected by RT-q PCR.7.The proliferation abilities of gastric adenocarcinoma cells in each group were detected by plate cloning assay.8.The migration and invasion abilities of gastric adenocarcinoma cells in each group were detected by transwell migration assay and transwell invasion assay.9.The levels of m RNA and protein of sirt1 in gastric adenocarcinoma cells BGC-823 were detected by RT-q PCR and western blot.Results:1.The expressions of circ-sirt1 in human gastric adenocarcinoma cells AGS-1 and BGC-823 cells were significantly lower than that in normal gastric mucosal cells GES-1(P<0.05,P<0.05).2.The expressions of circ-sirt1 in AGS-1 and BGC-823 cells in circ-sirt1 groups were markedly higher than that in blank groups and pc DNA3.1 groups(P<0.001,P<0.001).3.The proliferation abilities in AGS-1 and BGC-823 cells in circ-sirt1 groups were significantly lower than that in blank groups and pc D-NA3.1groups(P<0.05,P<0.01);The migration abilities of AGS-1 and BGC-823 cells in circ-sirt1 groups were significantly lower than that of in blank groups and pc DNA3.1 groups(P<0.05,P<0.05);The invasive abilities of AGS-1 and BGC-823 cells in circ-sirt1 groups weresignificantly lower than that of in blank groups and pc DNA3.1 groups(P<0.01,P<0.001).4.The levels of mi R-132-3p/mi R-212-3p in BGC-823 and AGS-1 cells in circ-sirt1 groups were significantly lower than those in blank groups and pc DNA3 groups(P<0.05,P<0.01).5.The levels of mi R-132-3p/mi R-212-3p in BGC-823 and AGS-1 cells in ASO groups were significantly lower than those in blank groups and ASO-NC groups(P<0.01,P<0.01).6.The expressions of mi R-132-3p/mi R-212-3p in BGC-823 and AGS-1cells in ASO groups were markedly lower than those in blank groups and ASO-NC groups(P<0.01,P<0.01).7.The proliferation abilitlies of AGS-1 and BGC-823 cells in ASO groups were significantly lower than those in blank groups and ASO-NC groups(P<0.01).The migration abilities of AGS-1 and BGC-823 cells in ASO groups were significantly lower than those in blank groups and ASO-NC groups(P<0.001).The invasive abilities of AGS-1 and BGC-823 cells in ASO groups were significantly lower than those in blank groups and ASO-NC groups(P<0.001).8.The m RNA and protein levels of sirt1 in AGS-1 and BGC-823 cells in ASO groups were markedly higher than those in blank groups and ASO-NC groups(P<0.01,P<0.01).Conclusions:1.The overexpression of circ-sirt1 can inhibit the proliferation ability,migration ability and invasion ability of gastric adenocarcinoma cells.2.The overexpression of circ-sirt1 could reduce the levels of mi R-132-3p/mi R-212-3p and up regulate the levels of sirt1.3.mi R-132-3p and mi R-212-3p mediate the regulation of circ-sirt1 on sirt1.Part Three The effect of circ-sirt1 overexpression on the growth of gast-ric cancer cells in nude miceObjective: To detect the effects of circ-sirt1/mi R-132-3p/ mi R-212-3p/sirt1 axis on the proliferation of gastric adenocarcinoma cells in nude mice.Methods:1.The BGC-823 cells stable transfected with pc DNA3.1 plasmids containing with circ-sirt1(BGC-823-circ-sirt1)sequenceand blank pc DNA3.1respectively(BGC-823-pc DNA3.1)were screened by G418.The transfection was verified by RT-q PCR.2.Tumorigenesis in nude mice: The female BALB/c-nu mice were injected subcutaneously with BGC-823-circ-sirt1 cells(overexpression group,n=6)and BGC-823-pc DNA3.1 cells(control group,n=6)at the axillary fossae.3.The expression levels of circ-sirt1,mi R-132-3p/mi R-212-3p and sirt1 in tumor tissues from nude mice were detected by RT-q PCR and western blot assay.Results:1.The expression levels of circ-sirt1 in BGC-823-circ-sirt1 groups were significantly higher than that in BGC-823-pc DNA3.1 groups(P<0.01).The BGC-823 cell line with stable expressing circ-sirt1 were successfully constructed.2.The tumor volumes and weights in the overexpression groups were markedly lower than those in the control groups(P<0.05).3.The expression levels of mi R-132-3p/mi R-212-3p in the overexpression groups were significantly lower than those in the control groups(P<0.05,P<0.01),and the m RNA and protein expression levels of sirt1 were significantly higher than those in the control groups(P<0.01).Conclusions:1.The overexpression of circ-sirt1 can significantly inhibit the tumorigenicity of gastric adenocarcinoma cells in vivo.2.The expressions of mi R-132-3p/mi R-212-3p were decreased,and the expressions of sirt1 were increased by circ-sirt1 overexpression in vivo.