益肾通癃汤通过上调miR-145-5p抑制TLR4/p38 MAPK/NF-κB信号通路抗前列腺癌作用机制研究

Mechanism of Yishen Tonglong Decoction inhibiting TLR4/p38 MAPK/NF-κB signaling pathway against prostate cancer via upregulating miR-145-5p

目的探讨益肾通癃汤(YSTLD)通过上调miR-145-5p抑制TLR4/p38 MAPK/NF-κB信号通路抗前列腺癌的作用机制。方法借助miRNA芯片技术检测YSTLD处理前列腺癌PC-3细胞后的miRNA表达谱的变化,筛选miRNA芯片结果中差异显著的miRNA,并通过实时荧光定量聚合酶链反应(qRT-PCR)进行验证。慢病毒转染miR-145-5p入前列腺癌PC-3细胞,CCK8法及划痕实验检测miR-145-5p对前列腺癌PC-3细胞增殖与迁移作用;qRT-PCR法及Wstern blot法检测miR-145-5p对TLR4/p38 MAPK/NF-κB信号通路及凋亡相关基因caspase3、TNF-α、Bax、Bcl-2表达的影响;qRT-PCR及Wstern blot法检测YSTLD含药血清对miR-145-5p、TLR4/p38 MAPK/NF-κB信号通路及凋亡相关基因caspase3、TNF-α、Bax、Bcl-2表达的影响。结果miRNA基因芯片检测发现YSTLD处理前列腺癌PC-3细胞后存在35种miRNA表达水平与对照组存在显著差异,其中以miR-145-5p差异最为显著,同时qRT-PCR验证发现YSTLD处理的PC-3细胞中的miR-145-5p水平显著高于DMSO对照组(P<0.05)。慢病毒转染miR-145-5p入前列腺癌PC-3细胞后,发现miR-145-5p能抑制前列腺癌PC-3细胞的增殖与迁移。过表达miR-145-5p可上调前列腺癌PC-3细胞caspase3、TNF-α及Bax mRNA表达水平,下调p38 MAPK、p65 NF-κB及Bcl-2的mRNA表达水平(P<0.05),同时上调前列腺癌PC-3细胞caspase3蛋白表达水平,下调TLR4、p38 MAPK、p65 NF-κB的蛋白表达水平(P<0.05);YSTLD含药血清在干预前列腺癌PC-3细胞后,可上调前列腺癌PC-3细胞caspase3、TNF-α及Bax mRNA表达水平,下调p38 MAPK、p65 NF-κB、Bcl-2、TRAF1的mRNA表达水平(P<0.05),同时上调前列腺癌PC-3细胞caspase3蛋白表达水平,下调TLR4、p38 MARK、p65 NF-κB、TRAF1的蛋白表达水平(P<0.05)。结论YSTLD可通过上调miR-145-5p的表达水平,抑制TLR4/p38 MAPK/NF-κB信号通路促进前列腺癌PC-3细胞凋亡,这可能是YSTLD抗前列腺癌的重要机制。

Objective To investigate the mechanism of Yishen Tonglong Decoction(益肾通癃汤,YSTLD)inhibiting the toll-like receptor 4/p38 mitogen activated protein kinases/nuclear factor kappa-B(TLR4/p38 MAPK/NF-κB)signaling pathway against prostate cancer by up-regulating mi R-145-5p.Methods miRNA microarray technology was used to detect the changes of mi RNA expression profile in prostate cancer PC-3 cells treated with YSTLD,and mi RNAs with marked differences in mi RNA microarray results were screened and validated by real-time polymerase chain reaction(q RT-PCR).Lentiviral transfection of mi R-145-5p into prostate cancer PC-3cells,Cell Counting Kit-8(CCK8)assay,and scratch assay were adopted to detect the effects of miR-145-5p on prostate cancer PC-3 cell proliferation and migration.q RT-PCR and Western blot were employed to detect the effects of mi R-145-5p on TLR4/p38 MAPK/NF-κB signaling pathway and the expression levels of apoptosis-related genes caspase3,tumor necrosis factor-α(TNF-α),Bax,and Bcl-2.q RT-PCR and Western blot were used to detect the effects of serum containing YSTLD on mi R-145-5p,TLR4/p38 MAPK/NF-κB signaling pathway,and the expression levels of apoptosis-related genes caspase3,TNF-α,Bax,and Bcl-2.Results The expression levels of 35 mi RNAs in prostate cancer PC-3 cells treated with YSTLD were significantly different from those in the control group,with mi R-145-5p being the most significantly different;q RT-PCR validation revealed that the mi R-145-5p levels in prostate cancer PC-3 cells treated with YSTLD were significantly higher than those in the DMSO control group(P<0.05).After lentiviral transfection of mi R-145-5p into prostate cancer PC-3cells,mi R-145-5p was found to inhibit the proliferation and migration of prostate cancer PC-3cells.Overexpression of mi R-145-5p up-regulated expression levels of caspase3,TNF-α,and Bax m RNA,and down-regulated expression levels of p38 MAPK,p65 NF-κB,and Bcl-2 m RNA in prostate cancer PC-3 cells(P<0.05),while up-regulated caspase3 protein expression levels in prostate cancer PC-3 cells and down-regulated expression levels of TLR4,p38 MAPK,and p65 NF-κB protein(P<0.05).Serum containing YSTLD could up-regulate the expression levels of caspase3,TNF-α,and Bax m RNA,and down-regulate the m RNA expression levels of p38 MAPK,p65 NF-κB,Bcl-2,and TNF receptor-associated factor 1(TRAF1)in prostate cancer PC-3 cells after intervening prostate cancer PC-3 cells(P<0.05).Simultaneously,it upregulated the expression levels of caspase3 protein and down-regulated the protein expression levels of TLR4,p38 MARK,p65 NF-κB,and TRAF1 in prostate cancer PC-3 cells(P<0.05).Conclusion YSTLD can promote apoptosis of prostate cancer PC-3 cells by up-regulating the expression level of mi R-145-5p and inhibiting TLR4/p38 MAPK/NF-κB signaling pathway,which may be an important mechanism of YSTLD against prostate cancer.