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

涂雅玲; 刘德果; 羊羡; 李博; 陈其华

doi:10.1016/j.dcmed.2023.02.008

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

摘要

摘要:
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。

Abstract:
ObjectiveTo 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 miR-145-5p.
MethodsmiRNA microarray technology was used to detect the changes of miRNA expression profile in prostate cancer PC-3 cells treated with YSTLD, and miRNAs with marked differences in miRNA microarray results were screened and validated by real-time polymerase chain reaction (qRT-PCR). Lentiviral transfection of miR-145-5p into prostate cancer PC-3 cells, 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. qRT-PCR and Western blot were employed to detect the effects of miR-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. qRT-PCR and Western blot were used to detect the effects of serum containing YSTLD on miR-145-5p, TLR4/p38 MAPK/NF-κB signaling pathway, and the expression levels of apoptosis-related genes caspase3, TNF-α, Bax, and Bcl-2.
ResultsThe expression levels of 35 miRNAs in prostate cancer PC-3 cells treated with YSTLD were significantly different from those in the control group, with miR-145-5p being the most significantly different; qRT-PCR validation revealed that the miR-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 miR-145-5p into prostate cancer PC-3 cells, miR-145-5p was found to inhibit the proliferation and migration of prostate cancer PC-3 cells. Overexpression of miR-145-5p up-regulated expression levels of caspase3, TNF-α, and Bax mRNA, and down-regulated expression levels of p38 MAPK, p65 NF-κB, and Bcl-2 mRNA 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 mRNA, and down-regulate the mRNA 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 up-regulated 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).
ConclusionYSTLD can promote apoptosis of prostate cancer PC-3 cells by up-regulating the expression level of miR-145-5p and inhibiting TLR4/p38 MAPK/NF-κB signaling pathway, which may be an important mechanism of YSTLD against prostate cancer.

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