Abstract:

Objective To investigate the effects of trefoil factor family 3(TFF3) gene silencing on proliferation and cell cycle of papillary thyroid carcinoma TPC-1,BCPAP cell lines and related molecular mechanisms. Methods To obtain TPC-1 and BCPAP stable cell lines of thyroid papillary carcinoma by packaging TFF3 shRNA lentiviral vectors and virus infection;Growth curve and colony formation assay were used to detecte cell proliferation after TFF3 silencing.Flow cytometry was used to detect the effects of TFF3 gene on cell cycle of TPC-1 and BCPAP cell; Real-time PCR was used to investigate the mRNA levels of P27,P21,cyclin D1 and cyclin-dependent kinase(CDK)4,in TPC-1 and BCPAP cell. The protein expression levels of P27,P21,cyclin D1 and CDK4,protein kinase B(Akt) and phosphorylated protein kinase B(pAkt) were detected by Western blotting and immunocytochemistry. Results shTFF3-TPC-1 and shTFF3-BCPAP stable cell lines were obtained by TFF3 shRNA lentiviral packaging and virus infection. Growth curve and colony formation assay result showed that cell viability was decreased after silencing TFF3.The result of flow cytometry showed that the proportion of cells in the G₁ phase was significantly increased (*P<0.05), however the proportion of cells in the S and G₂ phase was significantly decreased in TFF3 slienced group compared with the control group (*P<0.05)；The expression of P27 and P21 mRNA and protein in TPC-1 and BCPAP cells was significantly up-regulated(*P<0.05),while the expression of cyclin D1 and CDK4 mRNA and protein was decreased in group TFF3 silenced (*P<0.05).There was no significant difference in the content of Akt protein in the four groups of cells, but the expression of pAkt was weakened after TFF3 silenced;Immunocytochemical staining showed that cyclin D1 positive protein expression was located in the nucleus of cancer cells, P27 and P21 proteins lo ated in cytoplasm and nucleus,and after silencing TFF3, the positive signals were upregulated in the nucleus, but cytoplasmic positive expression was decreased. Conclusion Silencing TFF3 can significantly prolong the cell cycle and inhibit cell proliferation，which might be related to the inhibition of phosphatidylinosital 3-kinase and protein kinase B(PI3K/Akt) pathway-related protein expression.

Key words: Trefoil factorfamily 3, Cell cycle, Lentivirus transfection, Phosphatidylinositol 3-kinase and protein kinase B, Flow cytometry, Human