Role of Peg10 in FAK-mediated CAM-DR in hepatocellular carcinoma
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摘要:
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<0.0...
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关键词:
- 肝肿瘤 /
- 黏着斑蛋白酪氨酸激酶类 /
- Peg10基因 /
- 抗药性,肿瘤 /
- RNA,小分子干扰
Abstract:Objective To preliminarily investigate the relationship of Peg10 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 Peg10- silenced cells siRNA- BEL- 7404 / ADR( siRNA- 7404 / ADR) were examined by MTT 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 injected with 7404 / ADR cells; 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. Peg10 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 7404 / ADR( t = 7. 641 and 7. 560,respectively; 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). PEG10 mRNA was rarely expressed in groups B,E,and F. Compared with that in group A,PEG10 mRNA expression in groups C and D was relatively reduced. PEG10 protein was rarely expressed in group B and its expression level significantly differed from that in group A( P < 0. 01). Additionally,there were statistically significant differences in p- FAK,p- JNK,p-ERK,and p38 MAPK 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 p38 MAPK 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 p38 MAPK 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 p38 MAPK expression.
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