Yin Yiran, Tang Lian, Shi Lei
Department of Orthopedics, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China.
Department of Gastroenterology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P.R. China.
Mol Med Rep. 2017 Mar;15(3):1286-1290. doi: 10.3892/mmr.2017.6109. Epub 2017 Jan 11.
The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (P<0.05). Based on these results, we showed that KISS-1 inhibited the proliferation of osteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells.
评估转移抑制基因KISS-1在骨肉瘤细胞凋亡和自噬过程中的表达。体外将MG-63骨肉瘤细胞用KISS-1过表达或KISS-1敲低表达载体转染，并与用空载体转染的细胞系进行比较。细胞培养12、24、48和72小时后，检测细胞增殖情况。采用MTT法通过流式细胞术检测细胞凋亡，并通过RT-PCR评估凋亡和自噬标志物caspase-3、Bcl-2、Bax、LC3和Beclin1的mRNA水平。我们的结果表明，对照组和低表达组的细胞在72小时的细胞培养期内持续增殖，而过表达组的细胞数量逐渐减少。此外，低表达组的增殖率显著高于对照组。对照组和低表达组中caspase-3和Bax mRNA的相对mRNA表达水平无变化（低表达组表达最低）。此外，两个细胞组中Bcl-2的mRNA水平均升高。过表达组中caspase-3和Bax的mRNA表达水平升高，Bcl-2水平显著降低。同时，对照组和低表达组中LC3和Beclin1 mRNA的相对表达水平保持不变，过表达组则升高。过表达组中LC3和Beclin1的mRNA水平最高，低表达组最低。差异具有统计学意义（P<0.05）。基于这些结果，我们表明KISS-1可能通过加速细胞凋亡和自噬过程来抑制骨肉瘤细胞的体外增殖。
