MT1DP通过调控Cav1.3促进镉诱发的细胞死亡效应

MT1DP promotes cadmium-induced cell death by regulating Cav1.3

目的检测重金属镉暴露下长链非编码RNA MT1DP对钙内流和胞内镉摄入的影响,并探讨其分子机制。方法首先采用特异性钙通道抑制剂维拉帕米(VER)处理HepG2细胞,然后采用钙离子荧光探针Fluo-3AM检测镉对细胞钙流的影响,并使用电感耦合等离子体质谱(ICP-MS)测定胞内镉的含量;采用Real-timeqPCR检测镉对细胞内MT1DP表达水平的影响;采用shRNA敲低胞内MT1DP含量后检测其对镉暴露下胞内镉摄入量的影响;采用qPCR和Western blotting检测镉暴露下MT1DP对钙通道蛋白Cav1.3mRNA和蛋白表达水平的影响;采用siRNA敲低Cav1.3含量后检测镉暴露下其对细胞钙流、胞内镉的摄入量和细胞死亡效应的影响;最后采用AKT通路抑制剂检测镉暴露下AKT对Cav1.3表达水平的影响,并且过表达AKT后检测其对Cav1.3表达水平和胞内镉摄入量的影响。结果钙通道被抑制后细胞镉的摄入明显减少,在镉的暴露下肝癌细胞内MT1DP的表达显著增加,而当抑制MT1DP的表达水平后,镉诱发的胞内镉的摄入也显著降低;同时,抑制MT1DP可以降低镉暴露后细胞内Cav1.3基因和蛋白表达水平。当抑制Cav1.3的蛋白表达水平时,镉诱发的钙内流、胞内镉的摄入和细胞死亡效应也都显著降低。此外,抑制AKT的表达可以抑制Cav1.3的表达水平,而当细胞内过表达AKT后,镉暴露下Cav1.3的诱导表达水平和镉诱发的胞内镉摄入也显著增加。结论MT1DP可以通过活化AKT/Cav1.3信号通路促进钙内流和胞内镉摄入,最终诱发细胞死亡。

Objective To investigate the effect of long non-coding RNA MT1DP on heavy metal cadmium-induced Ca 2+ influx and intracellular cadmium uptake and its molecular mechanism. Methods After HepG2 cells were treated with specific calcium channel inhibitor Verapamil (VER),the effect of cadmium-induced Ca 2+ influx was detected after adding calcium fluorescence probe Fluo-3 AM and intracellular cadmium content was detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The expression level of MT1DP in cells before and after cadmium exposure was detected by Real-time qPCR (Real-time quantitative polymerase chain reaction). After HepG2 cells were transfected by MT1DP shRNA,the changes of intracellular cadmium uptake before and after cadmium exposure were detected. The effect of MT1DP on calcium channel protein Cav1.3 mRNA and protein level were detected by qPCR and Western blotting before and after cadmium exposure. After HepG2 cells were transfected by Cav1.3 siRNA,the changes of Ca 2+ influx,intracellular cadmium intake and cell death after cadmium exposure were detected. Finally,the inhibitor of AKT and over-expression of AKT were used to detect the effect of AKT on the expression level of cadmium-induced Cav1.3 expression or intracellular cadmium uptake. Results After the calcium channel was inhibited,cadmium uptake was significantly reduced,and the expression of MT1DP was significantly increased in cadmium-exposed cells. When the expression level of MT1DP was inhibited,the cadmium-induced intracellular cadmium uptake was also significantly decreased. Meanwhile,we found that inhibiting MT1DP can decrease the expression of Cav1.3 in cells after cadmium exposure. While inhibiting the expression level of Cav1.3,cadmium-induced Ca 2+ influx,intracellular cadmium uptake and cell death were also significantly reduced. Moreover,We further found that inhibition of AKT expression inhibited the expression level of Cav1.3. Conversely,when inducing AKT overexpression in cells,the induction level of Cav1.3 and cadmium-induced intracellular cadmium uptake were also significantly increased under cadmium exposure. Conclusion MT1DP could promote Ca^ 2+ influx and intracellular cadmium uptake through the activation of AKT/Cav1.3 pathway,thus inducing cell death.