Smad3通过p38/MAPK信号通路调控细胞自噬及凋亡促进胰腺癌进程

Smad3 promotes pancreatic cancer progression by regulating autophagy and apoptosis through p38/MAPK signaling pathway

目的观察Smad3对胰腺癌细胞Panc-1、BxPC-3凋亡、自噬的影响,初步探讨Smad3表达水平影响胰腺癌进程的可能机制。方法基于GEPIA2数据库进行生物信息学分析,探究Smad3与胰腺癌发生、发展及生存的相关性。利用CRISPR/Cas9系统以及短发卡RNA(ShRNA)敲除及敲减Smad3的表达,采用Real-Time quantitative PCR(qPCR)和Western blot检测细胞中Smad3 mRNA和蛋白水平变化。实验组转入ShSmad3-1、ShSmad3-2序列,将不靶向任何基因的Sh-Scramble序列作为对照组(Scr)。将转入Smad3 guide RNA(gRNA)序列作为CRISPR/Cas9的实验组,转入空载质粒的作为CRISPR/Cas9的对照组。采用Western blot检测细胞中LC3、p62、p38以及p-p38蛋白变化情况,同时在敲减Smad3的胰腺癌细胞用PI-Annexin V试剂盒染色并进行流式分析。利用pLVX-Smad3质粒进行回补实验,并用p38抑制剂SB203580处理细胞检测相关蛋白的变化情况。结果GEPIA2数据库及临床标本分析结果显示,Smad3在胰腺癌组织中高表达(P<0.05)。在胰腺癌细胞BxPC-3中转入ShSmad3-1、ShSmad3-2后,Smad3的mRNA和蛋白水平均显著降低(P<0.05)。同时在Panc-1中转入Smad3 guide RNA筛选到2个Smad3敲除细胞系。与对照组相比,敲减Smad3胰腺癌细胞凋亡显著增加(P<0.05)。Western blot实验发现:Smad3敲减及敲除后,p-p38的蛋白水平显著增加(P<0.05),LC3、p62水平显著降低(P<0.05)。进一步通过回补实验及利用SB203580处理胰腺癌细胞,发现p62、LC3蛋白水平能回复(P<0.01)。结论干扰Smad3促进胰腺癌细胞过度自噬,进而导致细胞凋亡增加,可能与激活p38/MAPK信号通路有关。

Objective To investigate the effect of Smad3 on the apoptosis and autophagy of pancreatic cancer cells Panc-1 and BxPC-3,in order to preliminarily verify the effect of Smad3 expression in the progression of pancreatic cancer.Methods Bioinformatics analysis was performed based on GEPIA2 database to explore the correlation of Smad3 with the occurrence,development,and survival of pancreatic cancer.The CRISPR/Cas9 system and short hairpin RNA(sh-RNA)were used respectively to knock out or reduce the expression of Smad3.Real-time quantitative PCR(qPCR)and Western blotting were subsequently adopted to detect the changes of Smad3 at mRNA and protein levels.The sh-RNA experimental groups were transferred with ShSmad3-1 and ShSmad3-2 sequences,and the CRISPR/Cas9 experimental group were transferred with Smad3 guide RNA(gRNA)sequence,using Sh-Scramble sequence(Scr)and empty plasmid as the corresponding control groups,respectively.Western blotting was applyed to determine the protein changes of LC3,p62,p38 and p-p38 in the cells.Meanwhile,Smad3 knockdown pancreatic cancer cells were stained with PI-Annexin V kit and then analyzed by flow cytometry.To further prove the effect of Smad3 on pancreatic cancer cells,the pLVX-Smad3 plasmid was used to perform a rescue experiment,and in another assay,the cells were treated with p38 inhibitor SB203580 to observe the changes of related proteins.Results The results of GEPIA2 database and clinical specimen analysis showed that Smad3 was highly expressed in pancreatic cancer tissues(P<0.05).After transfection of ShSmad3-1 and ShSmad3-2 into pancreatic cancer cells BxPC-3,both the mRNA and protein levels of Smad3 were significantly decreased(P<0.05).Meanwhile,2 Smad3 knockout cell lines were screened out after transferring gRNA into Panc-1.As compared with the control group,Smad3 knockdown remarkably increased the apoptosis of pancreatic cancer cells(P<0.05),and Western blotting also indicated that after Smad3 knockdown or knockout,the protein level of p-p38 was up-regulated(P<0.05),while those of LC3 and p62 were down-regulated(P<0.05).Further study found that the protein levels of p62 and LC3 were restored by the rescue assay as well as by the treatment of pancreatic cancer cells with SB203580(P<0.01).Conclusion Interference in Smad3 promotes excessive autophagy in pancreatic cancer cells and leads to increased apoptosis,possibly through activation of p38/MAPK signaling pathway.