Tet1蛋白对HEK293T细胞增殖、周期及凋亡的影响

Effect of Tet1 protein on HEK293T cell proliferation,cell cycle and apoptosis

目的构建可诱导Tet1CD过表达的稳转细胞系,探究Tet1蛋白对细胞增殖、周期及凋亡的影响。方法构建重组质粒p TRE-Tight_Tet1CD_IRES_RFP_RPBSA_M2rtTA_Puro,然后与表达转座酶的"睡美人"质粒通过脂质体法共同转染到HEK 293T细胞,通过嘌呤霉素筛选和多西环素诱导构建诱导表达型的Tet1CD稳转细胞系293T(Tet1CD)。通过Western blot检测Tet1蛋白的表达;通过DNA dot blot检测细胞基因组中5hmC的含量,通过流式细胞术检测Tet1CD蛋白对细胞周期和凋亡的影响;通过细胞计数检测Tet1CD蛋白对细胞增殖的影响。结果成功建立了可诱导Tet1CD过表达的HEK293T细胞系293T(Tet1CD);Western blot结果显示诱导后细胞内Tet1CD蛋白表达上升;DNA dot blot结果显示过表达Tet1CD蛋白细胞内5hmC含量升高;流式细胞术结果显示,Tet1CD过表达促进了细胞凋亡(P<0. 05)及增加G2时期细胞比例(P<0. 05);细胞计数结果显示,Tet1CD过表达抑制细胞增殖(P<0. 0001)。结论 Tet1蛋白可能通过促进细胞凋亡及阻滞细胞周期最终抑制细胞增殖。

Objective To explore the effects of Tet1 protein on cell proliferation,the cell cycle and apoptosis,an inducible Tet1CD overexpression cell line was established.Methods A recombinant pTRE-Tight_Tet1CD_IRES_RFP_RPBSA_M2rtTA_Puro plasmid was constructed,then the recombinant plasmid and the“Sleeping Beauty”plasmid expressing a transposase were cotransfected into HEK293T cells using lipofectamine.The inducible stable 293T(Tet1CD)cell line was selected using puromycin.Tet1 protein expression was detected via western blot,and the 5hmC content in the genome was detected using DNA Dot Blot.The effect of Tet1CD protein on the cell cycle and apoptosis was detected using flow cytometry,and the effect of Tet1CD protein on cell proliferation was detected by cell counting.Results The HEK293T(Tet1CD)cell line with inducible Tet1CD expression was successfully established.Western blot and DNA Dot Blot assays showed increased amounts of Tet1CD proteins and increased 5hmC content in inducible cells.Flow cytometry result demonstrated that Tet1CD expression promoted apoptosis(P<0.05)and increased the proportion of cells in G2 phase(P<0.05).Cell counts showed that Tet1CD overexpression inhibited cell proliferation(P<0.0001).Conclusions Tet1 protein may inhibit cell proliferation by promoting apoptosis and delaying the cell cycle.