| Background:hTERT is closely linked to tumor invasion and metastasis. Our previous study found that hTERT is highly expressed in tumor tissue, closely linked to the prognosis of the patients. Overexpression of hTERT can significantly enhance the osteosarcoma U2OS cells which are telomerase negative invasion and metastasis. But the mechanism of hTERT regulates tumor invasion and metastasis is not clear. The purpose of this study was to investigate the possible molecular mechanism of hTERT promoting gastric cancer metastasis and provide a theoretical basis for the prevention of gastric cancer.Methods:(1) proteome technique was used to analysis of protein expression profile of thTERT/U2OS and EGFP/U2OS cells; (2) The hTERT-regulating miRNA was screened by miRNA microarray tests; (3) Bioinformatics technique was used to screen miRNAs which can regulate the expression of ITGB1. The results were compared with the outcome of miRNA microarray tests; (4) Quantitative real-time and immunohistochemical was used to detect the expression of miR-29 and ITGB1 in the 58 cases of carcinoma tissues and noncancerous tissues respectively. Then we analyze the relation of the expression of miR-29 and ITGB1 in patients with gastric cancer with clinical pathology (5) Western blot was used to detect the expression of ITGB1 abundance after overexpression of miR-29 by miRNA mimics; (6) miR-29 targeting ITGB13’UTR was verified by dual luciferase reporter assay. (7) Transwell experiment was used to detect the ability of cancer cells invasion after downregulating hTERT, and then recovering the expression of miR-29a; (8) The ability of miR-29a inhibit cancer cells invasion was test in vivo.Results:(1) proteome technology revealed that the protein relating with cell adhesion regulation especially ITGB1 in the U2OS/hTERT group had significantly upregulated.(2) There were 153 possible miRNAs which can be regulated by hTERT. Bioinformatics software revealed that miR-29、miR-124、miR-183、miR-506 might regulate the expression of ITGB1. miR-29 was the only miRNA which could regulate ITGB1 expression. Meantime, it was down-regulation in the U2OS/hTERT cells.(3) QRT-PCR results showed that the relative content of three miRNA of miR-29 family was lower than those in paracancerous tissues adjacent to cancer. miR-29a was found closely associated with lymph node metastasis. The relation of clinical and pathological with miR-29b, miR-29c was poor. The survival analysis indicated that high expression of miR-29a in gastric cancer patients promised longer survival time;(4) The proliferation curve by MTT method showed that miR-29 inihibits, human gastric cancer SGC-7901 cells proliferated in vitro. miR-29a can inhibit the invasion and metastasis of gastric cancer cells which was injected to nude mice by tail vein.(5) Immunohistochemistry experiments confirmed the upregulation of ITGB1 in gastric cancer tissue; the survival analysis indicated that high expression of ITGB1 was closely linked to the poor prognosis of gastric cancer. The expression level of ITGB1 and miR-29a in gastric cancer tissues were negative correlation;(6) Western blot revealed that miR-29 reduced the expression level of ITGB1. The dual luciferase reporter assay also showed that miR-29 could target to 3’UTR of ITGB1.(7) Downregulating of hTERT in gastric cancer cells could promote the expression of miR-29, and could decrease the expression level ITGB1 and the capacity of gastric cancer cells invasion. Downregulating the expression of miR-29a, the expression level of ITGB1 could reply, and invasion and metastasis of gastric cancer cell also reply;(8) Upregulating of hTERT in gastric cancer cells, could inhibit the expression of miR-29, the expression level ITGB1 and the capacity of gastric cancer cells invasion was enhanced, upregulating the expression of miR-29a, the expression level of ITGB1 and the invasion and metastasis of gastric cancer cell was decreased.Conclusion:(1) After transfecting with hTERT, ITGB1 in the gastric cancer cells and U2OS cells had significantly upregulated; (2) The expression level of miR-29a in gastric cancer was significantly lower than that of adjacent tissue, which was negatively correlated with the expression of ITGB1; The expression level of miR-29a was closely linked to gastric cancer clinical pathology and prognosis; (3) Overexpression of miR-29a could reduce the proliferation ability and invasion capacity of the cancer cells; (4) miR-29a could downregulate ITGB1 in gastric cancer cells by targeting to ITGB13’UTR; (5) hTERT can downregulate the expression of miR-29a, Gastric cancer cell invasion ability and ITGB1 expression lever was reduced after recovering miR-29a expression.In summary, this study found that hTERT enhanced ITGB1 by downregulating miR-29 expression, so as to promote the gastric cancer cell invasion and metastasis. his might provide a new mechanism for the invasion and metastasis of gastric cancer. |
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