线粒体蛋白酶LONP1在前列腺癌生物学行为中的作用机制研究

Mechanism of mitochondrial protease LONP1 in the biological behavior of prostate cancer

目的:探讨线粒体离子肽酶1(lon peptidase 1,mitochondrial,LONP1)在去势抵抗性前列腺癌(castration-resistant prostate cancer,CRPC)进展中的作用机制。方法:采用蛋白质印迹法检测前列腺增生细胞BPH-1、雄激素依赖性前列腺癌细胞LNCa P和雄激素非依赖性前列腺癌细胞(即CRPC细胞)PC3中LONP1、N-myc下游调节基因1(N-Myc downstream-regulated gene 1,NDRG1)和雄激素受体(androgen receptor,AR)蛋白的表达水平。采用慢病毒感染的方法将携带有LONP1全基因的重组载体转入LNCa P细胞,构建稳定过表达LONP1(overexpression LONP1,OE-LONP1)的LNCa P细胞(以OE-NC作为阴性对照);将特异性针对LONP1基因的sh RNA(sh LONP1)转入PC3细胞,构建稳定沉默LONP1表达的PC3细胞(sh NC作为阴性对照);分别通过CCK-8法、集落形成实验和Transwell小室侵袭实验检测细胞的增殖和侵袭能力。通过免疫共沉淀和染色质免疫沉淀法检测LONP1对AR/NDRG1信号轴的影响,并采用CCK-8法和Transwell小室实验检测在沉默LONP1表达的PC3细胞中进一步沉默NDRG1表达对PC3细胞增殖及侵袭能力的影响。将沉默LONP1表达的PC3细胞及其阴性对照(PC3-sh NC细胞)接种于BALB/c裸鼠皮下构建移植瘤模型,并分别采用DMSO和AR拮抗剂恩杂鲁胺(enzalutamide,ENZ)进行治疗;最后,分别采用免疫组织化学法检测肿瘤组织中Ki-67的表达水平,TUNEL法评估肿瘤组织中肿瘤细胞的凋亡情况。结果:相较于前列腺增生细胞BPH-1细胞,LNCa P细胞中LONP1的蛋白表达水平相对较低(P<0.05),PC3细胞中LONP1蛋白的表达水平相对较高(P<0.05)。LNCa P细胞中LONP1过表达抑制了AR和NDRG1的表达水平(P均<0.05),而PC3细胞中下调LONP1的表达水平可上调AR和NDRG1的表达水平(P均<0.05)。LNCa P细胞中LONP1过表达促进了细胞的增殖和侵袭能力(P均<0.05),PC3细胞中敲低LONP1表达抑制了细胞的增殖和侵袭能力(P均<0.05)。LONP1直接与AR结合并识别NDRG1中的雄激素反应元件(androgen response element,ARE),并且其通过抑制AR/NDRG1信号通路促进前列腺癌的进展。小鼠移植瘤治疗实验结果显示,沉默LONP1表达和ENZ治疗都可以抑制肿瘤的生长,以沉默LONP1表达联合ENZ治疗作用最好;免疫组织化学法和TUNEL法检测结果提示,sh LONP1+ENZ组肿瘤组织中表现出更低水平的Ki-67染色和更高水平的细胞凋亡(P均<0.001)。结论:LONP1在雄激素非依赖性细胞PC3中高表达,并通过抑制AR/NDRG1信号转导促进前列腺癌的进展。

Objective:To ex plore the mechanism of lon peptidase 1,mitochondrial(LONP1)in the progression of castration-resistant prostate cancer(CRPC).Methods:The expression levels of LONP1,N-Myc downstream-regulated gene 1(NDRG1)and androgen receptor(AR)proteins in benign prostatic hyperplasia cell line BPH-1,androgen-dependent human prostate cancer cell line LNCaP and castration-resistant prostate cancer(CRPC)cell line PC3 were examined by Western blotting.Recombinant vector carrying full-length LONP1 gene was delivered into LNCaP cells via lentiviral infection to establish stable LONP1-overexpressing(OE-LONP1)cell line,and LNCaP cells transfected with empty vector was used as the corresponding negative control(OE-NC).shRNA specifically targeting LONP1 gene(shLONP1)was delivered into PC3 cells to establish stable LONP1-silencing cell line,and PC3 cells transfected with empty shRNA vector(shNC)was used as the negative control.The effect of LONP1-overexpression and silencing on cell proliferation and invasion was analyzed by CCK-8 assay,colony formation assay and Transwell invasion assay in OE-LONP1 and shLONP1 cell lines.The effect of LONP1 on the AR/DNRG1 signaling axis was analyzed by co-immunoprecipitation assay and chromatin immunoprecipitation assay.The effect of NDRG1-silencing on the proliferation and invasion of shLONP1-expressing PC3 cells was evaluated by CCK-8 assay and Transwell assay.Tumor xenograft model was established by subcutaneously inoculating shLONP1-expressing PC3 cells and the negative control cells(PC3-shNC cells)into BALB/c nude mice.The mice were treated with DMSO or AR antagonist enzalutamide(ENZ).Then,the expression level of Ki-67 in tumor tissues was examined by immunohistochemical staining,and the cell apoptosis in tumor tissues was evaluated by TUNEL assay.Results:Compared with BPH-1 cells,LNCaP cells had lower(P<0.05)while PC3 cells had higher(P<0.05)expression level of LONP1 protein.The expression of AR and NDRG1 were inhibited in LNCaP cells when LONP1 was overexpressed(P<0.05),whereas the expression of AR and NDRG1 were increased in PC3 cells when LONP1 was silenced(P<0.05).The proliferation and invasion of LNCaP cells were promoted when LONP1 was overexpressed(P<0.05),whereas the proliferation and invasion of PC3 cells were suppressed when LONP1 was knocked-down(P<0.05).LONP1 can directly bind with AR to recognize the androgen resp onse element(ARE)of NDRG1,which further inhibits the AR/NRDG1 signaling pathway to promote the progression of prostate cancer.The treatment of xenograft tumors in mouse models showed that both LONP1-silencing and ENZ application could inhibit tumor growth,and the best inhibitory effect was observed in mice treated with LONP1-silencing in combination with ENZ.The results of immunohistochemical staining and TUNEL assay indicated that the tumor tissues in the shLONP1+ENZ group had lower level of Ki-67 expression and higher level of cell apoptosis(P<0.001).Conclusion:LONP1 is highly expressed in CRPC cell line PC3,and promotes prostate cancer progression by inhibiting AR/NDRG1 signaling transduction.