Effect of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7.

OBJECTIVE: To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7. METHODS: Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132, respectively or together. The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry, and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR. RESULTS: Compared with the control group, the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly, and the apoptosis ratio increased distinctly (P < 0.05). Besides, the effect of the co-transfection group was better than that of the single transfection group. The protein levels of GPC3 (Glypican-3) and YAP (Yes-associated protein), the target genes transfected only by miR-520c-3p and miR-132, respectively, reduced obviously (P < 0.05), which was similar with the co-infected cells, but cells transfected by miR-132 only showed a decrease of YAP. CONCLUSIONS: The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP, enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.

Change of the peripheral blood immune pattern and its correlation with prognosis in patients with liver cancer treated by sorafenib.

OBJECTIVE: To study the change of the peripheral blood immune pattern and its correlation with prognosis in patients with liver cancer after treated by sorafenib. METHODS: Patients with advanced liver cancer admitted in our hospital were enrolled and treated with sorafenib. After two months of the treatment, their peripheral blood was collected. The immune cell subset and cytokines level were determined by flow cytometry and luminex technology. According to the reaction expressed by patients towards sorafenib, patients were divided into the response group and the no response group. The changes of the peripheral blood immune pattern and its correlation with prognosis of patients in the two groups were compared. RESULTS: Before and after treatment of sorafenib, there was no significant difference in the ratios of T cells, NK cells and their subtypes in peripheral blood of patients between the two groups; while after treatment the ratio of B cells and regulatory B cells (Breg) of patients in the response group was significant higher than that of the no response group (P < 0.05), and the prognosis conditions of patients with decreased ratio of Breg cells were better than other patients after undergoing chemotherapy. The levels of plasma cytokines IL-6, IL-10, IL-12, IL17, FIL-3L, IFN-γ, TNF-α, MCP-1 and VEGF showed no significant differences. CONCLUSIONS: After treatment of sorafenib, the prognosis conditions of patients of advanced liver cancer with a reduced Breg ratio are better than patients with an unaltered or increased Breg ratio. The ratio of Breg in peripheral blood may be considered as early biological indicator for the prediction of the curative effects of sorafenib.

Staurosporine induces apoptosis in NG108-15 cells.

AIM: To observe if staurosporine induced apoptosis in NG108-15 cells and its effect on protein expression level of several genes related to apoptosis. METHODS: Phase-contrast microscopy, fluorescence microscopy, and transmission electron microscopy were used to observe staurosporine-induced morphological changes. Agarose gel electrophoresis was used to detect DNA fragmentation. Western blots were used to measure protein expression level of several genes related to apoptosis. RESULTS: Cells treated with staurosporine 0.1 micromol/L showed typical morphological changes of apoptosis. A "ladder" pattern representing fragmentation of DNA into oligonucleosome length fragments was observed after 6 h of staurosporine treatment and sustained until 24 h. The Bax expression increased significantly at 6 h after exposure to staurosporine, peaked at 12 h compared with vehicle cultures, and decreased at 24 h. The Bcl-2 expression increased and reached the highest level at 3 h. It was then decreased gradually but still higher than normal expression level. There was an obvious caspase-3 cleavage at 6 h after exposing the cells to staurosporine. Treatment with staurosporine for 12 h markedly decreased the expression of p53 protein. Cdk5 protein expression did not have obvious changes after the cells were exposed to staurosporine. CONCLUSION: Staurosporine induced apoptotic death in NG108-15 cells. Cells might die via a pathway that is dependent on Bax expression but independent of p53, and caspase-3 cleavage was involved.

[Protective effects of tacrine and donepezil against staurosporine-induced apoptotic death].

AIM: To study whether tacrine and donepezil can prevent cell apoptosis induced by staurosporine in NG108-15 and Hela cell lines. METHODS: MTT assay was used to examine if staurosporine impairs cell metabolism. Phase-contrast and fluorescence microscope were used to examine cell morphological changes. DNA was isolated and electrophoretically separated on 1% agarose gel to observe if there were DNA fragments. Western blot was made to analyse protein levels of anti-apoptotic Bcl-2 and proapoptotic Bax. RESULTS: NG108-15 cells treated with 0.1 mumol.L-1 staurosporine for 12-24 hours exhibited marked cell death and DNA fragmentation. Pre-treatment with 0.1 mmol.L-1 tacrine provided approximately 40% protective effect and resulted in obvious inhibition or delay of DNA fragmentation. Moreover, NG108-15 cells treated with tacrine became elongated and polarized, and showed longer processes than control cells. Pretreatment with 0.1 mmol.L-1 tacrine significantly increased the expression of Bcl-2 protein level and delayed the staurosporine-induced increase of Bax protein expression. However, donepezil did not show any protective effect on the cell impairment induced by staurosporine in NG108-15 cells. In Hela cells 0.1 mumol.L-1 staurosporine also induced significant cell injury, but pretreatment with tacrine and donepezil did not provide any obvious protective effect against this cell damage. CONCLUSION: Donepezil did not provide obvious protective effect against apoptosis, and protective effects of tacrine might not be mediated through AChE inhibition. Protective effects of tacrine against staurosporine-induced injury might be selective to different cells.