RESUMO
OBJECTIVE: To investigate the mechanism of miR-7-5p in TK6 cell damage induced by hydroquinone( HQ) by constructing stable miR-7-5p over-expressing human lymphoblastoid TK6 cell line using lentivirus. METHODS: i) The miR-7-5p over-expression lentivirus vectors were constructed,and then infected to TK6 cells. The miR-7-5p overexpression stable TK6 cell line( TK6-miR-7-5p cells) and negative control cells( TK6-NC cells) were selected with puromycin. The infection efficiency was confirmed by real-time fluorescent quantitative polymerase chain reaction assay.ii) TK6,TK6-NC and TK6-miR-7-5p cells were treated with HQ at final concentrations of 0 and 40 μmol/L for 48 hours.Cell viability was determined by CCK-8 assay. The early apoptosis rate of cells was detected by flow cytometry. The relative expression of poly( ADP-ribose) polymerase-1( PARP-1) and breast cancer susceptibility gene 1( BRCA1) proteins in 3 kinds of cells treated with HQ at the final concentration of 40 μmol/L was detected by Western blotting. RESULTS: i) The TK6 cell line with stable expression of miR-7-5p were successfully screened. Compared with normal TK6 cells,the relative expression of miR-7-5p in TK6-miR-7-5p cells increased by about 17 times( P < 0. 01) with no significant changes in cell morphology. ii) After treatment with 40 μmol/L HQ,the survival rate of TK6-miR-7-5p cells decreased compared with normal TK6 cells and TK6-NC cells( P < 0. 01),early apoptosis rate increased( P < 0. 01),the relative expression of PARP-1 and BRCA1 protein was decreased( P < 0. 05). CONCLUSION: MiR-7-5p may lead to the increase of early apoptosis in TK6 cells induced by HQ through inhibiting the DNA damage repair capacity related to PARP-1 and BRCA1.
RESUMO
Objective To investigate the dose-effect relationship of mRNA level of sensitive mitochondrial genes in human lymphoblastoid cells induced by ionizing radiation.Methods Seven human sensitive genes,including ND3,Cyt b,COX Ⅰ,COX Ⅱ,COX Ⅲ,ATPase6 and ATPase8 were chosen.Changes of mRNA level of these genes were detected by RT-PCR and Real-Time PCR at 24 h after irradiation in human lymphoblastoid cells,which were exposed to 0 - 15 Gy of 60 Co γ-rays.Results The expression of these 7 genes at mRNA level was up-regulated 24 h after irradiation in human lymphoblastoid cells.The level of gene expression of COX Ⅰ,which belongs to complex Ⅳ of mitochondrial respiratory chain,was most obvious,and the peak occurred after irradiation of 8 Gy,which was 13 times of the control group.A good dose-effect relationship was showed for COX Ⅲ gene expression at dose range of 3 -10 Gy as well as 3 - 15 Gy for other 3 genes including ND3,ATPase6 and ATPase8.Conclusions Gene expression levels of COX Ⅲ,ND3,ATPase6 and ATPase8 24h post-irradiation at certain irradiation dose range could be used for radiation damage biomarkers.