MiR-181a Regulates The Proliferation,Apoptosis,Invasion And Migration Of Gastric Cancer Cells Through Caprin-1

Objective:to observe the expression of mir-181a and target gene caprin-1 in gastric cancer tissues and gastric cancer cell lines.Mir-181a and caprin-1 were up-regulated and down-regulated in the cell line SGC7901,and the effects of mir-181a and caprin-1 on the proliferation,apoptosis,invasion and migration of gastric cancer cells were determined.Further observation Caprin-1 in the mRNA and protein level of change,through the luciferase test agent miR-181a regulation function of Caprin-1for miR-181 a and Caprin-1 in the role and mechanism of gastric cancer,to provide theoretical basis for the clinical treatment of gastric cancer.Methods:(1)the specimen collection and cell lines to buy:collection of weifang medical school pathology,the first affiliated hospital of 90 cases of gastric cancer tissue,in normal tissue adjacent to carcinoma specimens by contrast,MKN45 gastric cancer cells,SGC-7901,MGC803,BGC-823 and normal gastric epithelial cells are bought from GES-1 FengHui biotechnology LTD.(2)target gene prediction:The target genes of mir-181a downstream were predicted by miRanda,TargetScan and Microcosm.(3)the detection of mir-181a was used to detect the expression changes of mir-181a in gastric cancer tissues and gastric cancer cell lines,as compared with normal tissue samples and normal gastric epithelial cells.(4)target genes Caprin-1detection:application of RT-pcr and Western blot technique to detect Caprin-1 in gastric cancer tissue and the change of expression in gastric cancer cell lines,cell culture and transfection.(5)SGC7901 cryopreserved cells from liquid nitrogen tank recovery to cultivate in a bottle;The medium was replaced 24 hours later;The cells that grew to 80%to 90%were discarded in the old culture medium,and 2 mL sterilized d-hank's solution was added,and the washing cells were shaken twice,then the supernatant was discarded.Add 1 mL of 0.25%trypsin digestive juices(in the37?incubator),the cells of the pancreatic enzyme digestion,made from cell suspension;Will be 1x10~5 cell suspension inoculation in 6-well culture plate,37?and 5%CO2 incubator culture to the cell density reached about 60%;Replace the medium in the 6-hole culture plate with the OPTI-MEM of 800 L.Each hole was transfted with 4 g plasmid,8 L Lipofectamine 2000,respectively dissolved in 200 L OPTI-MEM,5 min at room temperature,then mixed with the two,then at room temperature for 25 min.Will join the rest of the 6 orifice mixture,37?and 5%CO2incubator in training after 6 h,supernatant,add complete medium.(6)after the intervention in miR-181a in gastric cancer cells and gastric cancer cell biology behavior detection:because of miR-181a in gastric cancer SGC-7901 cells expressing the most obvious,so the selection of gastric cancer SGC-7901 cells,corotating miR-181a up plasmid and miR-181a down plasmid,EDU,CCK8experimental tests changes in cell proliferation,with tunel and Western blot technique to detect apoptosis changes,wound healing experiment test cell migration ability,transwell invasion experiment test cell invasion ability,with empty vector as control Caprin-1.(7)mir-181a regulatory detection of caprin-1 in SGC-7901 cells:gc-7901 gastric cancer cells were taken,respectively transfected with mir-181a up plasmid and mir-181a down plasmid,and the expression of caprin-1 in mRNA and protein levels was detected.(8)intervention in gastric cancer cells after gastric cancer cell biology behavior detection:because Caprin-1 cut the most obvious in gastric cancer SGC-7901 cells,selection of gastric cancer SGC-7901 cells,transfection pEGFP-siRNA and Caprin Caprin-1,EDU,CCK 8 experimental tests changes in cell proliferation,with tunel and Western blot technique to detect apoptosis-changes,wound healing experimental detection cell migration ability,transwell invasion experiment test cell invasion ability,with empty vector as control.(9)the luciferase report gene:293t cells and SGC-293 cells for the tool,respectively Caprin 1 3'UTR plasmid Caprin-1 and 3'UTR-Mut plasmid and miR-181a-up,miR-181a-down with their own controls plasmid transfection into 293t cells and gastric cancer SGC-7901 cells.The experiment was divided into eight groups:Caprin-1,3'UTR+mir-181a up,caprin-1 3'UTR-MUT+mir-181a up,caprin-13'UTR+mir-181a-up-control,caprin-1 3'UTR-MUT+mir-181a-up-control,Caprin-13'UTR+mir-181a-down,caprin-1 3'UTR-MUT+mir-181a-down,caprin-1 3'UTR+mir-181a-down-control,Caprin-1 3'UTR-MUT+mir-181a-down-control,Caprin-13'UTR-MUT+mir-181a-down-control.Results:(1)the miR-181a and Caprin-1 in gastric cancer tissue and the expression changes of gastric cancer cell lines:RT-qPCR results show that compared with normal tissue adjacent to carcinoma,miR-181a expression level increased significantly,the difference is statistically significant(P<0.05);MKN45,gastric cancer cell line SGC-7901,MGC803,BGC-823 compared with normal gastric tissue cells GES-1,miR-181a higher expression level are different degree,statistically significant difference(P<0.05)and(2)prediction of target genes:by miRanda,TargetScan and Microcosm software to predict miR-181a downstream target genes,learned that Caprin-1 is a target genes of miR-181a.(3)Caprin-1 in gastric cancer tissue and the expression changes of gastric cancer cell lines:RT-qPCR results show that compared with normal tissue adjacent to carcinoma,gastric cancer tissues in Caprin-1 mRNA level is reduced,statistically significant difference(P<0.05);Western-blot results showed that the expression of caprin-1 protein was decreased in gastric cancer tissues compared with the normal tissue adjacent to cancer,and the difference was statistically significant(P<0.05).RT-qPCR results showed that gastric cancer cell lines MKN45,SGC-7901,MGC803 and BGC-823 were compared with GES-1 in normal gastric tissue,and the expression of caprin-1mrna was significantly reduced,and the difference was statistically significant(P<0.05).Western-blot results showed that compared with gs-1 of normal gastric tissue cells,gastric cancer cell lines MKN45,SGC-7901,MGC803 and BGC-823,and Caprin-1protein content were all decreased,and the difference was statistically significant(P<0.05).(4)Effects of miR-181a on proliferation,apoptosis,migration and invasion ability of SGC-7901:transfection of miR-181a up plasmid 48 h,and Edu results showed that SGC7901 cell proliferation was enhanced;Tunel results showed that SGC7901 cell apoptosis decreased,and the expression of apoptotic protein Caspase-3 and Caspase-9 decreased.The results of wound healing showed that overexpression of miR-181a and SGC7901 cell migration were significantly enhanced.Transwell invasion experiment found that overexpression of miR-181a,SGC7901 cell invasion ability was significantly enhanced.The difference was statistically significant(P<0.05).Transfection miR-181a down plasmid,inhibit the expression of SGC7901 cells miR-181a,for the same cell proliferation,apoptosis,migration and invasion of experiments,the results show that compared with transfection empty carrier group,SGC7901 cells proliferation ability and apoptotic cells increased,cell migration and invasion ability is restrained,also have a statistically significant difference(P<0.05).(5)the effect of miR-181a on the expression of caprin-1 in SGC-7901 cells:the results showed that the expression of the transfected miR-181a down group was higher than that of the control caprin-1 in the mRNA and protein levels,and the difference was statistically significant(P<0.05).The expression of mRNA and protein levels in the transfected miR-181a up group was lower than that in the control group,and the difference was statistically significant(P<0.05).(6)Caprin-1on SGC-7901 cell proliferation,apoptosis,invasion and migration change:transfection pEGFP-siRNA and Caprin Caprin-1,72 h after transfection,change detection of gastric cancer cell proliferation apoptosis,Western blot-change detection of apoptosis protein,in 24 h,48 h,72 h after transfection test cell migration changed,after 72 h after transfection detection of gastric cancer cell invasion.The results showed that the increased proliferation,apoptosis,invasion and migration of the gastric cancer cell SGC7901 were significantly enhanced in the transfection caprin-1 siRNA group compared with the control group,and the difference was statistically significant(P<0.05).The transfected pEGFP-Caprin-1group had the opposite experimental effect in the control group,and the difference was also statistically significant(P<0.05).(9)Dual luciferase inspection report verification Caprin-1 is the target genes of miR-181a:dual luciferase reporter gene detection shows that t cells and SGC-7901 cells in 293,a total of transfection Caprin1 3'UTR+miR-181a group up plasmid and transfection Caprin 1 3'UTR MUT+miR-181a group up,dual luciferase intensity ratio significantly decreased(P<0.01),a total of transfection Caprin-1 3'UTR+miR-181a down plasmid group and transfection Caprin-1 3'UTR MUT+miR-181a down group comparison,dual luciferase intensity ratio increased significantly(P<0.01),to determine Caprin-1 is the target genes of miR-181a.Conclusion:(1)MiR-181a and Caprin-1 have differential expression in gastric cancer tissues and gastric cancer cell lines,indicating that miR-181a and Caprin-1are closely related to the incidence of gastric cancer.(2)MiR-181a promotes the proliferation,migration and invasion of gastric cancer cells through target gene caprin-1,inhibits apoptosis of gastric cancer cells.