| Objective: To investigate the expression of long non-coding RNA(LncRNA)in gastric cancer and its effect on the malignant biological behavior of gastric cancer cells through competitive binding with miR-206 and its related mechanism.Methods: The expressions of LncRNA HOTAIR and miR-206 in gastric cancer tissues and adjacent tissues,gastric cancer cell lines(SGC-7901,AGS)and normal gastric epithelial cells(GES-1)were detected by real-time fluorescent quantitative PCR.By constructing stably silenced HOTAIR expression(si-HOTAIR)and its negative control(si-NC)plasmid or over-expressing miR-206 and its control group(miR-206 mimics / NC)plasmid,human gastric cancers were cultured in vitro.Cell lines SGC-7901 and AGS were transfected.After down-regulating HOTAIR expression or over-expression of miR-206,the cell proliferation ability of each group was tested by CCK8 test,cell clone formation test,and EDU experiment;cell scratch test and Transwell invasion test were used to detect the cell migration and invasion ability of each group;Annexin V-FITC flow cytometry was used to detect the apoptosis rate of cells in each group after transfection.Bioinformatics software was used to predict the possible binding sites of HOTAIR and miR-206 online,and then the two luciferase reporter gene experiments were used to verify the binding and interaction of the two.Animal experiments were used to observe the growth of subcutaneous tumor tissues in nude mice after inoculation of gastric cancer cells that down-regulated HOTAIR expression.Real-time PCR,Western blot and immunohistochemical methods were used to detect the expression of CDK9 at the mRNA and protein levels after downregulated the HOTAIR expression in human gastric cancer cell lines and nude mice.Results:(1)The expression of HOTAIR in gastric cancer tissues and gastric cancer cell lines SGC-7901 and AGS was higher than it in adjacent cancer tissues and normal gastric epithelial cells(GES-1).Compared with the control cells,the proliferation,migration,and invasion capacity of gastric cancer cells in the experimental group after down-regulating HOTAIR expression was reduced,and the number of apoptotic cells was increased.(3)The binding site of HOTAIR and miR-206 was predicted by bioinformatics software,and verified by double luciferase reporter gene experiment.At the same time,inhibit the expression of HOTAIR in gastric cancer cells,the expression of miR-206 was increased by qRT-PCR.Contrarily,the expression of HOTAIR decreased when miR-206 overexpressed.(4)The expression of miR-206 in gastric cancer tissues and gastric cancer cell lines SGC-7901 and AGS was lower than that in adjacent cancer tissues and normal gastric epithelial cells(GES-1).When miR-206 overexpressed,the proliferation,migration,and invasion ability of gastric cancer cells were reduced compared with the control group,and the number of apoptotic cells was increased compared with the control group.(5)Downregulate the expression of HOTAIR in animals,the weight and volume of tumor in nude mice were reduced,compared to the control group.(6)The expression of CDK9 mRNA and protein levels was reduced after inhibition of HOTAIR in gastric cancer cells by qRT-PCR and Western blot assays.Immunohistochemistry was used to detect the expression of CDK9 protein in nude mice tumor tissues and its expression suppressed.Conclusion: HOTAIR has a significantly higher expression level in gastric cancer tissues,while miR-206 has a lower one.After down-regulating its expression,it can inhibit the expression of CDK9 by reducing the binding with miR-206,and inhibit the malignant biological behavior of gastric cancer cells and inducing apoptosis. |
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