| 摘要: |
| 目的 观察和比较在人肝癌与癌旁组织中蛋白磷酸酶1调节亚基16A(protein phosphatase 1 regulatory subunit 16A,PPP1R16A)基因在基因组和转录水平的差异,探讨靶向沉默PPP1R16A基因对肝癌细胞HCC LM3增殖能力的影响。方法 收集106例肝癌患者的肿瘤组织及癌旁组织,采用实时荧光定量PCR技术检测PPP1R16A基因的基因组和转录水平。体外合成PPP1R16A序列特异性小干扰RNA(siRNA),使用脂质体法转染肝癌细胞株HCC LM3。实验分si-16A组(针对PPP1R16A的特异性 siRNA转染的HCC LM3细胞)、NC组(非特异性siRNA转染的HCC LM3细胞)、空白组(未转染siRNA的HCC LM3细胞),用实时荧光定量PCR检测PPP1R16A基因拷贝数和转录水平,用蛋白质印迹法检测PPP1R16A蛋白表达,用CCK-8实验、克隆形成实验检测细胞增殖和克隆形成能力,用流式细胞术检测细胞周期。结果 人肝癌组织中PPP1R16A基因的拷贝数和转录表达水平均高于癌旁组织(P<0.01),且两者具有相关性(P=0.015)。CCK-8实验和克隆形成实验显示,转染siRNA后,si-16A组细胞增殖低于NC组和空白组(P<0.001)。流式细胞术结果显示,si-16A组较NC组和空白组细胞周期被抑制。结论 肝癌组织中PPP1R16A的拷贝数发生扩增,基因转录上调。靶向沉默PPP1R16A能抑制肝癌细胞HCC LM3的增殖能力。提示PPP1R16A基因在肝癌中发挥癌基因的作用。 |
| 关键词: 肝肿瘤 肝细胞癌 PPP1R16A 细胞增殖 拷贝数变异 |
| DOI：10.16781/j.0258-879x.2016.05.0529 |
| 投稿时间：2016-01-11修订日期：2016-03-06 |
| 基金项目:国家自然科学基金(81330037). |
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| RNAi-mediated PPP1R16A gene silencing suppresses proliferation of hepatocellular carcinoma cells |
| BI Feng-rui,ZHOU Chuan-chuan,YUAN Ji-hang,SUN Shu-han* |
(Department of Medical Genetics, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China
*Corresponding author) |
| Abstract: |
| Objective To observe and compare the genomic and transcription level of protein phosphatase 1 regulatory subunit 16A (PPP1R16A) gene in human hepatocellular carcinoma (HCC) tissues and adjacent tissues, and to explore the effect of specific sliencing of PPP1R16A on the proliferation of HCC LM3 cells. Methods Quantitative reverse transcription PCR (qRT-PCR) was used to assess genomic and transcription level of PPP1R16A gene. The specific small interfering RNA (siRNA) of PPP1R16A was synthetized in vitro, and was transfected into HCC LM3 cells with liposome. The experiment was divided into the following three groups, namely, PPP1R16A-siRNA (si-16A) transfected group, non-specific siRNA (NC) transfected group and blank control group. The genomic and transcription level of PPP1R16A gene was detected by qRT-PCR. The expression of PPP1R16A protein was detected by Western blotting analysis. CCK-8 and clone formation assay were used to investigate the proliferation ability of transfected cells. Cell cycle was investigated by flow cytometry. Results The genomic level (P<0.001) and transcription level (P<0.001) of PPP1R16A gene in human HCC tissues were significantly increased compared with those in the adjacent liver tissues; and the genomic level was found significantly correlated with transcription level of PPP1R16A gene (P=0.015). The results of CCK-8 and clone formation experiment in vitro showed that the cell proliferation of si-16A group was significantly inhibited compared with NC group and blank control group (P<0.001). Flow cytometry showed that cell cycle was suppressed in si-16A group. Conclusion The genomic and transcription levels of PPP1R16A gene are increased in HCC tissues. The proliferation of HCC LM3 cells is suppressed by inhibiting the PPP1R16A gene transcription, which suggests that PPP1R16A gene functions as an oncogene in HCC. |
| Key words: liver neoplasms hepatocellular carcinoma PPP1R16A cell proliferation copy number variation |
