ABSTRACT
Objective To investigate the influence of silencing Med19 on metastasis and invasion of human breast cancer MCF-7 cells. Methods lentivirus expression vector delivering small hairpin RNA (shRNA) against Med19 gene was constructed, then transfected of human breast cancer MCF-7 cell line. There were three experimental groups: non-infected (CON) group, Lv-NC-infected (NC) group, and Lv-shMed19-infected (KD) group. To determine the lentiviral infection efficiency, expression of GFP was detected with fluorescence microscopy. The mRNA and protein levels of Med19 in three groups MCF-7 cells were detected by real-time PCR and Western blotting. Scratching and transwell tests were employed to determine the ability of metastasis and invasion of cells. Results The highest infection efficiency was obtained, resulting fluorescent expression identified in more than 90%of MCF-7 cells 120 h after infection. The expression of Med19 mRNA in the KD group was dramatically decreased by 72.3%compared with the NC group, and by 72.1% compared with the CON group, respectively (P < 0.05). The Med19 protein expression in the KD group was significantly lower than that of the NC group and the CON group, with a reduction of 85.4%and 85.3%, respectively (P<0.05). No statistical significance was detected between the NC groups and the CON groups. Thus, the constructed Lv-shMed19 was demonstrated to be active and specific in inhibiting the expression of Med19. Moreover, the invasive distances of KD group, NC group and CON group MCF-7 cells were compared at 6 hour, 12 hour and 24 hour. The invasive ability of MCF-7 cells decreased significantly with the extension of time (P<0.05). The cells passed through polycarbonate membrane in KD group, NC group and CON group were 23.8 ± 4.32, 43.4 ± 3.65 and 45.8 ± 5.81 respectively. The metastatic ability of KD group was significant reduced (P<0.05). Conclusion Silencing of Med19 gene significantly decreases the ability of metastasis and invasion of human breast cancer MCF-7 cells, and then suppresses the malignant biological behavior of breast cancer.
ABSTRACT
Objective To study the role of Med19 in bladder cancer by analyzing the effects of lentivirus-mediated suppression of Med19 expression on T24 bladder cancer cells in vitro.Methods The lentivirus vectors containing a small hairpin RNA (shRNA) to target Med19 were constructed.After T24 bladder cancer cells were infected,real-time PCR and Western-blotting were used to study the Med19 expressions in the CON group (non-infected cells),the NC group (Lv-NC-infected cells) and the KD group (Lv-shMed19-infected cells).The influence of Med19 on the proliferation of bladder cancer cells were assessed using MTT,BrdU,colony formation assay and tumorigenicity experiment in mice.Cell cycle was analyzed with flow cytometry assay.Results Med19 relative mRNA level (0.35 ± 0.03) and Med19 protein expressing in the KD group were significantly inhibited (P ＜ 0.05).The KD group displayed an increased proportion of cells (77.50 ± 0.29)％ in the G0/G1 phase compared with the CON group (69.81 ± 0.81)％and NC group (67.53 ± 0.67) ％ (P ＜ 0.05).Compared with the CON group and the NC group,the KD group displayed a significant cell proliferation defect by MTT and BrdU assay and the number of colonies (91.33 ± 6.11) was significant decreased (P ＜ 0.05).On the day 24,the tumor volume (596.64 ± 485.36) mm3 and weight (0.57 ± 0.44) g of the KD group mice were decreased after inoculation into nude mice (P ＜ 0.05).Specific lentivirus-mediated knockdown of Med19 significantly impacted the cell cycle and proliferation of bladder cancer cells.Infected T24 cells nearly lost their tumorigenicity when being inoculated into nude mice.Conclusion Our results provide new evidence of an important role for Med19 in the development of bladder cancer,suggesting that lentiviruses delivering shRNA against Med19 may be a promising tool for bladder cancer therapy.