ABSTRACT
Objective To observe Hedgehog signaling pathways of liver cancer cell growth and the influence of the metastatic potential targeted inhibit Hedgehog.Methods Construction of Smo shRNA plasmid,The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened after lipofection.The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened,The cell cycle,apoptosis,invasion and metastasis of QGY-7701 cells were detected by Western blot,flow cytometry,CCK8 and transwell assay.Subcutaneous implantation of hepatocarcinoma cells in nude mice.Study on the growth and metastasis of hepatocarcinoma cells with low expression of Smo in.The ultrastructural changes of hepatoma cells with low expression of Smo were observed under electron microscope.Results RT-PCR and Western blot showed stable shR-Smo cell line was successfully constructed.Cell cycle test showed that compared with the control group,G0/G1 cells increased in shR-Smo,cells in S phase decreased;apoptosis,CCK8 and Transwell tests showed that Smo-gene silencing could significantly increase the apoptosis percentage of the hepatic cancer cells to (5.46％ ± 1.46％),proliferation activity decreasedand and the migration rates reduced to (7.82％ ±2.14)％;nude mice model showed that Smo-gene silencing could inhibit the growth of hepatocellular carcinoma cells in vivo,electron microscopy revealed that lysosomes increased significantly in Smo-gene silence cells.Conclusions Blocking Hh signaling pathways,liver cancer cells in vitro malignant degree of decline.Hedgehog in treating liver cancer have hidden meaning.
ABSTRACT
Objective@#To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma (HCC) cells.@*Methods@#The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore, the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated by immunohistochemical staining.@*Results@#Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9, conjugated SUMO1, free SUMO1 and AKT1 in HCC cells (P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%, 54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were (59.47±4.66) μm and 89.44±8.36, (56.56±5.37) μm and 93.84±8.79, as well as (34.57±6.61) μm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were (3.78±0.69) g, (3.72±0.72) g and (2.09±0.61) g, respectively. The corresponding apoptotic cell rates were (7.79±2.21)%, (6.45±2.48)% and (33.59±5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group (P<0.05).@*Conclusions@#Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.