沉默PRRX1基因表达可增强前列腺癌耐药细胞株PC-3/DTX对多西他赛的敏感性

doi:10.6040/j.issn.1671-7554.0.2020.1656

摘要/Abstract

摘要： 目的探讨配对相关同源框1(PRRX1)基因沉默对前列腺癌耐药细胞多西他赛(DTX)敏感性的影响及可能机制。方法临床收集35例DTX化疗敏感患者(DTX-S)和29例DTX化疗耐药患者(DTX-R)的癌组织。采用qRT-PCR法检测癌组织中PRRX1 mRNA表达水平。体外培养人前列腺癌PC-3细胞,采用DTX浓度递增间断刺激法建立DTX耐药细胞株PC-3/DTX。采用qRT-PCR法和Western blotting法检测耐药株PC-3/DTX及其亲本PC-3细胞中PRRX1 mRNA和蛋白表达水平;通过siRNA沉默PC-3/DTX细胞PRRX1基因表达,采用qRT-PCR和Western blotting检测转染后PC-3/DTX细胞中PRRX1 mRNA和蛋白表达水平;DTX干预转染后PC-3/DTX细胞24 h,采用MTT法检测细胞增殖活性,计算半数抑制浓度(IC50)和耐药指数(RI);采用流式细胞术检测细胞凋亡情况;采用Western blotting法检测细胞中cleaved caspase-3、p-mTOR、mTOR、Beclin-1和LC-3蛋白表达水平。结果 DTX-R癌组织中PRRX1 mRNA表达水平高于DTX-S癌组织(6.59±1.49 vs 1.07±0.34),差异有统计学意义(F=656.401,P<0.05)。耐药细胞株PC-3/DTX及其亲本PC-3细胞对不同浓度DTX作用24 h后的IC50值分别为(89.88±6.86)nmol/L和(15.56±1.45)nmol/L,RI值为5.78。耐药细胞株PC-3/DTX中PRRX1 mRNA和蛋白表达水平高于其亲本PC-3细胞(mRNA:9.22±1.50 vs 1.00±0.17;蛋白:0.50±0.04 vs 0.08±0.02),差异有统计学意义(F_mRNA=59.05,F_蛋白=171.70,P<0.05)。PRRX1基因沉默后,PC-3/DTX细胞中PRRX1 mRNA和蛋白表达水平降低(mRNA:1.01±0.02 vs 0.98±0.04 vs 0.22±0.10;蛋白:0.62±0.01 vs 0.61±0.01 vs 0.11±0.01),差异有统计学意义(F_mRNA=122.01,F_蛋白=554.53,P<0.05)。沉默PRRX1基因可降低PC-3/DTX细胞对DTX的耐药性,促进细胞凋亡,并上调p-mTOR和cleaved caspase-3水平,下调Beclin-1和LC-3蛋白表达水平,差异有统计学意义(P<0.05)。结论沉默PRRX1基因表达可增强前列腺癌耐药细胞株PC-3/DTX对DTX的敏感性,其机制可能与激活mTOR信号通路进而抑制细胞自噬有关。

Abstract: Objective To explore the effects of paired-related homeobox 1(PRRX1)gene silencing on the sensitivity of drug-resistant prostate cancer cells to docetaxel(DTX)and the possible mechanism. Methods The cancer tissues of 35 patients who were DTX chemotherapy sensitive(DTX-S)and 29 patients who were DTX chemotherapy resistant(DTX-R)were collected. The mRNA expression of PRRX1 in cancer tissues was detected with qRT-PCR. Human prostate cancer PC-3 cells were cultured in vitro, and the DTX-resistant cell line PC-3/DTX was established by increasing DTX concentration intermittently. The mRNA and protein expressions of PRRX1 in the drug-resistant PC-3/DTX and the parental PC3 cells were detected with qRT-PCR and Western blotting. The expression of PRRX1 gene in PC-3/DTX cells was silenced by siRNA, and the mRNA and protein expressions of PRRX1 in PC-3/DTX cells after transfection were detected with qRT-PCR and Western blotting. After DTX intervention, PC-3/DTX cells were transfected for 24 hours. The cell proliferation was detected with MTT, and the half inhibitory concentration(IC50)and drug resistance index(RI)were calculated. The cell apoptosis was detected with flow cytometry. The protein expressions of cleaved caspase-3, p-mTOR, mTOR, Beclin-1 and LC-3 were detected with Western blotting. Results The mRNA expression of PRRX1 in DTX-R cancer was higher than that in DTX-S cancer(6.59±1.49 vs 1.07±0.34)(F=656.401, P<0.05). The IC50 values of the drug-resistant cell line PC-3/DTX and the parental PC-3 cells treated with different concentrations of DTX for 24 h were(89.88±6.86)nmol/L and(15.56±1.45)nmol/L, respectively, and the RI was 5.78. The mRNA and protein expressions of PRRX1 in the drug-resistant cell line PC-3/DTX were higher than those in the parental PC-3 cells(mRNA: 9.22±1.50 vs 1.00±0.17; protein: 0.50±0.04 vs 0.08±0.02)(F_mRNA=59.05, F_protein=171.70, P<0.05). After the PRRX1 gene was silenced, the mRNA and protein expressions of PRRX1 in PC-3/DTX cells reduced(mRNA: 1.01±0.02 vs 0.98±0.04 vs 0.22±0.10; protein: 0.62±0.01 vs 0.61±0.01 vs 0.11±0.01)(F_mRNA=122.01, F_protein=554.53, P<0.05). Silencing the PRRX1 gene reduced the resistance of PC-3/DTX cells to DTX, promoted cell apoptosis, and up-regulated the expressions of p-mTOR and cleaved caspase-3, and down-regulated the protein expressions of Beclin-1 and LC-3(P<0.05). Conclusion Silencing the expression of the PRRX1 gene can enhance the sensitivity of the prostate cancer drug-resistant cell line PC-3/DTX to DTX, and the mechanism may be related to the activation of the mTOR signaling pathway to inhibit autophagy.

Key words: Gene silencing, Paired related homeobox 1, Prostate cancer, Docetaxel, Drug resistance, Autophagy

中图分类号:

R737.1

徐兵,李勇,刘明,刘永辉. 沉默PRRX1基因表达可增强前列腺癌耐药细胞株PC-3/DTX对多西他赛的敏感性[J]. 山东大学学报 (医学版), 2021, 59(6): 103-110.

XU Bing, LI Yong, LIU Ming, LIU Yonghui. Silencing PRRX1 gene expression enhances the sensitivity of prostate cancer resistant cell line PC-3/DTX to docetaxel[J]. Journal of Shandong University (Health Sciences), 2021, 59(6): 103-110.

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