[Biological function and molecular mechanism of URI in HepG2 cells]
OBJECTIVE To explore the effect and molecular mechanism of the unconventional prefoldin RPB5 interactor (URI) in hepatocellular carcinoma HepG2 cells. METHODS The cDNA sequence and shRNA of URI were obtained and sub cloned into eukaryotic expression vectors. Then those vectors were transfected into HepG2 cells to obtain stable transfection cell line. The cell proliferation and anchor independent growth in URI overexpressing and knockdown HepG2 cells were determined by CCK 8 and soft agar colony assay. Flow cytometry was applied to detect the cell cycle and apoptosis of g ray irradiated cells. Apoptosis related genes were detected by Western blot. RESULTS The pCDNA3.1 URI and pGPU6 URIi eukaryotic expression vectors were constructed successfully and corresponding stable transfection cell lines were obtained. Cell proliferation rates of the HepG2, pCDNA3.1 URI HepG2 and pGPU6 URIi HepG2 cells were (588.78 32.12) (959.33 58.8) and (393.93 39.7) respectively (P 0.05) The number of cell clones of HepG2, pCDNA3.1 URI HepG2 and pGPU6 URIi HepG2 cells were 43 7, 85 5 and 20 4 (P 0.05) respectively. After g ray irradiation, the URI overexpressing cell line showed a significantly lower apoptosis rate and G(2)/M phase arrest than those in the URI depleted cell line (P 0.05) In the HepG2 cells, the relative protein expression levels of URI, Bax and Bcl 2 were 0.92 0.03, 1.11 0.13 and 0.82 0.01 (P 0.05) In the pCDNA3.1 URI HepG2 cells, the relative protein expression levels of URI, Bax and Bcl 2 were 1.79 0.12, 0.48 0.01 and 2.20 0.30 (P 0.05) respectively. In the pGPU6 URIi HepG2 cells, the relative protein expression levels of URI, Bax and Bcl 2 were 0.50 0.04, 1.52 0.20 and 0.38 0.01 (P 0.05) respectively. The expression of Bax was down regulated and Bcl 2 was up regulated in the URI overexpressing cell line. However, on the contrary, expression of Bax was up regulated and Bcl 2 was down regulated in the URI depleted cell line. CONCLUSIONS URI may promote the growth of hepatocellular carcinoma cells via inhibition of cell proliferation and reducing the apoptosis in hepatocellular carcinoma cells in vitro. After the impairment of URI expression, the proliferation ability of hepatocellular carcinoma cells is suppressed and the ability to resist g ray irradiation is reduced. URI may become a potential new target for cancer therapy of hepatocellular carcinoma.
Zhonghua zhong liu za zhi [Chinese journal of oncology]