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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 1regulatory 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。
Objective To observe and compare the differences in the genomic and transcriptional levels of the protein phosphatase 1 regulatory subunit 16A (PPP1R16A) gene between human hepatocellular carcinoma (HCC) and paracancerous tissues, and to investigate the effect of targeting silencing PPP1R16A gene on the proliferation of HCC LM3 The impact of ability. Methods The tumor tissues and adjacent tissues of 106 HCC patients were collected. The genomic and transcriptional levels of PPP1R16A gene were detected by real-time fluorescence quantitative PCR. PPP1R16A sequence-specific small interfering RNA (siRNA) was synthesized in vitro and transfected into hepatocellular carcinoma cell line HCC LM3 by liposome. The experiment was divided into groups of si-16A (HCC LM3 cells transfected with specific siRNA targeting PPP1R16A), NC group (HCC LM3 cells transfected with nonspecific siRNA) and blank group (HCC LM3 cells without transfected with siRNA) PPP1R16A gene copy number and transcription were detected by real-time PCR. The expression of PPP1R16A protein was detected by Western blotting. The cell proliferation and colony formation ability were detected by CCK-8 assay and clone formation assay. Cell cycle was detected by flow cytometry. Results The copy number and transcript level of PPP1R16A gene in human hepatocellular carcinoma were significantly higher than those in para-cancerous tissues (P <0.01), and the correlation was significant (P = 0.015). The results of CCK-8 assay and clone formation assay showed that the proliferation of si-16A group was lower than that of NC group and blank group (P <0.001). Flow cytometry showed that the cell cycle was inhibited in si-16A group compared with NC group and blank group. Conclusion The copy number of PPP1R16A in hepatocellular carcinoma is amplified and the gene transcription is up-regulated. Targeting silencing PPP1R16A can inhibit the proliferation of HCC LM3 cells. Suggesting that PPP1R16A gene play an oncogene role in liver cancer.