Peg10基因在黏着斑激酶介导的肝癌细胞耐药中的作用

Role of Peg10 in FAK-mediated CAM-DR in hepatocellular carcinoma

目的初步探讨Peg10基因与黏着斑激酶(FAK)介导的肝癌细胞耐药(CAM-DR)的关系及分子机制。方法采用MTT法检测5-氟尿嘧啶(5-Fu)、阿霉素(ADR)对LO2细胞、ADR耐药的人肝癌细胞BEL-7404/ADR(7404/ADR)和Peg10基因沉默的siRNA-BEL-7404/ADR(siRNA-7404/ADR)细胞增殖的影响;建立7404/ADR和siRNA-7404/ADR裸鼠荷瘤模型,48只裸鼠随机分为6组,每组8只,A、C、D组注射7404/ADR细胞,B、E、F组注射siRNA-7404/ADR细胞;A、B组尾静脉注射生理盐水,C、E组给予5-Fu,D、F组给予尾静脉注射ADR,测量瘤块体积,RT-PCR检测Peg10基因的表达,Western Blot法检测PEG10、p-FAK、p-JNK、p-ERK和p38MAPK蛋白的表达。两组间比较采用t检验,多组间比较采用单因素方差分析。结果 ADR和5-Fu对siRNA-7404/ADR 24 h的IC50值均较7404/ADR的降低,差异均具有统计学意义(t值分别为7.641,7.560,P值均<0.01)。B、C、D组肿瘤体积较A组小,差异均有统计学意义(P值均<0.05),E、F组肿瘤体积较B组小,差异均具有统计学意义(P值均<0.01);E组与C组比较,F组与D组的肿瘤体积差异亦均有统计学意义(P值均<0.05)。B、E、F组Peg10的mRNA极少表达,C、D组Peg10的mRNA表达较A组有所降低。B组PEG10蛋白极少表达,与A组比较,差异具有统计学意义(P<0.01),其pFAK、p-JNK、p-ERK和p38MAPK蛋白表达与A组比较,差异均有统计学意义(P值均<0.05)。C、D组PEG10、p-FAK、pJNK、p-ERK和p38MAPK蛋白的表达较A组均降低,差异均有统计学意义(P值均<0.05)。E、F组p-FAK、p-JNK、p-ERK和p38MAPK蛋白表达与B组比较,差异均有统计学意义(P值均<0.01),而C组与E组、D组与F组相比,PEG10、p-FAK、p-JNK、p-ERK和p38MAPK蛋白的表达差异亦均有统计学意义(P值均<0.01)。结论 Peg10基因失活后可增加ADR耐药的BEL-7404细胞株对5-Fu和ADR的敏感性,其作用机制可能与其下调p-FAK、p-JNK、p-ERK和p38MAPK蛋白的表达有关。

Objective To preliminarily investigate the relationship of PeglO with focal adhesion kinase （FAK） - mediated cell adhesion - mediated drug resistance （CAM -DR） in hepatocellular carcinoma （HCC）, and to explore the underlying molecular mechanism. Methods The effects of 5 -fluorouracil （5 -Fu） and adriamycin （ADR） on the proliferation of LO2 cells, ADR -resistant human HCC cells BEL -7404/ADR （7404/ADR）, and PeglO -silenced cells siRNA -BEL- 7404/ADR （siRNA -7404/ADR） were examined by MTY assay. To build a tumor -bearing nude mouse model, 48 rats were randomly divided into six groups （n = 8 each） ： groups A, C, and D were injec- ted with 7404/ADR ceils; groups B, E, and F were injected with siRNA -7404/ADR cells. For experimental treatments, groups A and B revived saline by intravenous injection through the tail vein ; groups C and E were given 5 - Fu ; groups D and F received ADR by intravenous injection through the tail vein ; tumor mass volume was measured after injection. PeglO mRNA expression was assayed by RT - PCR, and PEG10, p - FAK, p - JNK, p - ERK, and p - P38 protein expression was assayed by Western blotting. Results The 24 - h IC50 values of ADR and 5 - Fu on SiRNA -7404/ADR were both significantly reduced compared with those on 74（M/ADR （ t =7. 641 and 7. 560, respec- tively; both P 〈 0.01 ）. Significantly smaller tumor mass volume was observed in groups B, C, and D than in group A （P 〈 0.05 ） , and in groups E and F than in group B （ P 〈 0.01 ）. Additionally, tumor mass volume was significantly different between groups E and C as well as between groups F and D （ P 〈 0. 05 ）. PEGIO mRNA was rarely expressed in groups B, E, and F. Compared with that in group A, PEGIO mRNA expression in groups C and D was relatively reduced. PEG10 protein was rarely expressed in group B and its expression level signifi- cantly differed from that in group A （P 〈0.01 ）. Additionally, there were statistically significant differences in p - FAK, p - JNK, p - ERK, and p38MAPK protein expression between groups B and A （P 〈 0.05 ）. In groups C and D, PEG10, p -FAK, p -JNK, p -ERK, and p38 MAPK protein expression was significantly reduced compared with that in group A （ P 〈 0.05 ）. Significant differences in p - FAK, p -JNK, p -ERK, and p38MAPK protein expression also occurred in groups E and F compared with group B （P 〈 0.01 ）. Moreover, there were significant differences in PEG10, p - FAK p - JNK, p - ERK, and p38MAPK protein expression between groups C and E as well as between groups D and F （ P 〈 0.01 ）. Conclusion The inactivation of PEG10 can increase the sensitivity of ARD - resistant cell line BEL -7404 to 5 - Fu and ARD. The underlying mechanism is possibly related to down - regulation of p - FAK, p - JNK, p - ERK, and p38MAPK expression.