Gene Construction, Expression Of ADPN And Its Influences On Hela Cells Growth

The reduction of expression level of ADPN has been reported to be inversely associated with an increased risk of breast cancer,endometrial cancer, prostate cancer,colorectal cancer and gastric cancer.The ADPN level can be as an important index for detection of diseases.However,the relationship between ADPN and cervical cancer has not been reported.Therefore,we studied whether ADPN influences the proliferation of cervical cancer Hela cells.The ADPN gene was obtained by overlapping PCR technique.The expression vector pET22b(+)/ADPN was constructed and transformed into E.coli BL21(DE3). The ADPN protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial proteins under IPTG induction.The expression products were purified by Ni²⁺-NTA affinity column,SDS-PAGE analysis showed that the relative molecular weight of ADPN is 30kDa which was consistent with the theoretically predicted value.The MTT assay showed that ADPN can inhibit the endothelial cell proliferation at sub-physiological concentrations.To detect the mechanism of ADPN on tumor cells,we detected the expression of ADPN receptors by RT-PCR and FITC-fluorescence labeled method in MGC-803,BGC-823,HT-29,Hela cells.MTT assay analyzed the influence of ADPN on the proliferation rate of the these cancer cells,Flow cytometry analyzed the influence of ADPN on the cell cycle,RT-PCR detected the influence of ADPN on the expression of the cell cycle regulators and apoptosis gene,western blotting analyzed the phosphorylation of AMPK induced by ADPN in Hela cells.The results showed that ADPN receptors are expressed in MGC-803,BGC-823,HT-29,Hela cells. ADPN was sensitive to the Hela cells,0.1μg/ml ADPN can inhibit the growth of Hela cells and 10μg/ml ADPN induced Hela cells to apoptosis.ADPN down-regulated the expression of cell positive regulators cyclin-D1 and c-myc,up-regulated the expression of p21WAF1/CIP1 and tumor inhibitor p53,arrested cells at G0/G1 phase. ADPN reduced the expression anti-apoptotic gene Bcl-2 and increased the expression of apoptotic gene Bax.ADPN induced the phosphorylation of AMPK in 5-30min and mediated the growth of Hela cells.In our present study,we constructed the ADPN gene successfully and obtained efficient protein expression of ADPN gene in prokaryotic system and built the purification method of expressed protein.Our data showed that ADPN can mediate the expression of cell cycle regulators and apoptosis gene and inhibit the growth of Hela cells and induce apoptosis responses of Hela cells through binding with its receptors.These results would contribute to the further study of its anti-tumor biological functions.