| Objective: To explore the regulatory mechanism effect of long noncoding RNA gastric carcinoma high expressed transcript 1(lncRNA GHET1)on proliferation and invasion of gastric cancer AGS cells.These results may provid an insight for the targete therapy of gastric cancer.Methods:(1)The expression of GHET1 in normal gastric epithelial cell GES-1 and differentiated gastric cancer BGC-823,AGS,SGC-7901 and HGC-27 cells was detected by qRT-PCR to select the proper model for GHET1 in vitro.(2)A cell model was constructed by RNA interference,transfection efficiency was detected by si-FAM,and the interference efficiency was detected by qRT-PCR.(3)The cell vability was detected by CCK-8 assay.The morphological impact of apoptosis was observed by hoechst33342 staining assay.The cell cycle and apoptosis was detected by flow cytometry assay.The expression of cell cycle-related proteins such as P21,cyclin E,cyclin D,CDK2,CDK4,CDK6 were detected by western blot assay.Finally,the cell migration and invasion were detected by wound healing assay and transwell assay.Results:(1)Compared with normal gastric epithelial cell GES-1,GHET1 was over-expressed in gastric cancer cells compared to GES-1,the expression of GHET1 was 3.20±0.31 in AGS(P<0.001).(2)The transfection efficiency of si-FAM was approximately 85% detected by fluorescence microscopy,at the same time,the interference efficiency of si-GHET1 was(58.02±6.33)% detected by qRT-PCR(P<0.01).The experiment was divided into three groups: control group,negative control group(si-NC)and interference group(si-GHET1).(3)The cell viability was decreased after transfection with si-GHET1 in 48 h and 72 h determined by CCK-8 assay(P<0.001).The level of PCNA was decreased in AGS cells after transfection with si-GHET1 in 48h(P<0.01).Flow cytometry results indicated that AGS cell cycle was arrested in G0/G1 phase(P<0.01).The level of P21 was up-regulated(P<0.001)and the level of cyclinD,cyclinE,CDK4,CDK6,CDK2 were down-regulated(P<0.05)after transfection with si-GHET1 in western blot analysis.(4)The result of Hoechest33342 staining assay showed that there was no difference in apoptosis after knockdown of GHET1(P>0.05),which was consistent with the flow cytometry(P>0.05).(5)Wound healing assay and transwell migration assay without matrigel showed that knockdown of GHET1 inhibited the migration of AGS cells(P<0.01).Transwell invasion assay with matrigel show that knockdown of GHET1 inhibited the invasion of AGS cells(P<0.01).Conclusions:(1)Knockdown of GHET1 inhibited cell viability.Cell cycle was arrested in G0/G1 phase.(2)Knockdown of GHET1 inhibited migration and invasion of AGS cells in wound healing assay and transwell assay.It suggests that the high level of GHET1 may be related to the invasion of gastric cancer cells.(3)GHET1 may inhibite cell proliferation through up-regulated the level of P21 and down-regulated the level of cyclinD,CDK4,CDK6,cyclinE,and CDK2 in AGS cells.It is suggested that GHET1,as one of important regulating factors of gastric cancer cell proliferation,may promote the proliferation of gastric cancer cells by changing the expression of cell cycle-related proteins to accelerate the progression of gastric cancer. |
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