Yet, trans fection of miR 302b inhibitor can increase the expression of EGFR at protein degree, suggesting that miR302b inhibit EGFR expression at translational level but not transcription degree in SMMC 7721 cells. Curiosity ingly, as proven in Figure 2D, miR 302b expression degree in vivo was inversely correlated with EGFR mRNA expression level, which was verified by Pearsons corre lation coefficient check, suggesting that miR 302b may possibly relate to EGFR mRNA expression level. Taken together, our information demonstrated that miR 302b targeted at EFGR and suppressed its expression at translation degree in SMMC 7721 cells. The miR 302b inhibited the growth of SMMC 7721 cells by means of targeting EGFR To examine the results of miR 302b to the development of SMMC 7721 cells by focusing on EGFR, we made the siRNA for EGFR, which induced 50% lower of EGFR expression the two at the mRNA and protein ranges in SMMC 7721 cells.
Simultaneously, we transfected miR 302b into SMMC 7721 cells and observed a thirty fold grow with the miR 302b expres sion. MTT assay showed that miR 302b overexpression resulted from the suppression on the SMMC 7721cells selleckchem growth at 48 and 72 h, which was in accord with all the effect of siEGFR. To further examine the inhibitory purpose of miR 302b and siEGFR in SMMC 7721 cells, colony formation assay was employed. Notably, miR 302bsiEGFR transfected cells displayed fewer and smaller sized colonies in contrast with their respective controls. Also, miR 302b and siEGFR suppressed cell proliferation on the G0G1 phase at 24 h, 48 h and 72 h time factors. Finally, to deter mine the development fraction of HCC cells immediately after above expression of miR 302bsiEGFR, we carried out Ki 67 immunofluorescence staining. The signal of Ki 67 in the miR 302bsiEGFR transfected SMMC 7721 cells was visibly low compared with that within the cells transfected with their respective controls.
These findings demonstrated that the result of miR 302b re expression on cell proliferation was constant with that of siEGFR on SMMC 7721 cells, suggesting that miR 302b may perhaps inhibit the development of SMMC 7721 cells by targe ting EGFR. MiR 302b inhibits cell proliferation by EGFR dependent cell cycle regulation AKT could be the major molecule during the signaling pathway, that is regulated selleck chemical by EGFR. Abnormal expression of EGFR leads to a alter of AKT expression. The re expression of miR 302b decreased the expression of AKT2, pAKT2, and its downstream gene CCND1, CDK2, and up regu lated CDK inhibitor p27 in SMMC 7721 cells. Comparable final results have been proved through the therapy of siEGFR, suggesting that miR 302b might suppress the growth of SMMC 7721 cells by focusing on the EGFR AKT2CCND1 signaling pathway. Discussion HCC can be a primary lethal neoplasm in the liver and the third cause of cancer connected deaths throughout the world.