RNA干扰下调SOX9表达抑制肝癌细胞的增殖

Proliferation of hepatocellular carcinoma cells is inhibited by RNA interference on SOX9

目的:分析转录因子性别决定区Y框蛋白9(sex-determining region Y box protein 9,SOX9)在肝癌组织中的表达情况,并探讨其对肝癌细胞增殖的调节作用及分子机制。方法:分析肝癌表达谱芯片数据中SOX9的表达情况,并采用实时荧光定量PCR法在32对肝癌组织及癌旁肝组织中进行验证。构建靶向干扰SOX9的慢病毒载体pGreenPuro?-SOX9-shRNA,并用慢病毒感染的方法转入肝癌细胞Huh7中;采用实时荧光定量-PCR法及蛋白质印迹法检测SOX9的干扰效果。采用CCK-8(cell counting kit-8)法、克隆形成实验、FCM法和衰老相关β半乳糖苷酶(senescence-associated beta-galactosidase,SA-β-Gal)染色等方法检测细胞的增殖能力和衰老情况。最后,采用实时荧光定量PCR法及蛋白质印迹法检测SOX9可能调节的下游基因。结果:肝癌表达谱芯片数据分析结果显示,SOX9在肝癌组织中高表达(P<0.000 1),并在32对配对的肝癌组织及癌旁肝组织中得到验证(P=0.009 5)。靶向干扰Huh7细胞中SOX9的表达可抑制细胞的增殖并促进衰老。SOX9表达下调可降低增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞周期蛋白A2(cyclin A2,CCNA2)、细胞周期蛋白E2(cyclin E2,CCNE2)和核转录因子E2F-2基因的表达;肝癌组织表达谱芯片数据分析结果显示,它们的表达与SOX9存在一定的相关性。结论:SOX9在肝癌组织中高表达,可以调节肝癌细胞的增殖和衰老,且其分子机制可能通过影响细胞周期蛋白及核转录因子E2F-2等增殖调控基因的表达而实现。

Objective： To analyze the expression level of sex-determining region Y box protein 9 （SOX9） in hepatocellular carcinoma （HCC） tissues, investigate the effect of SOX9 on the proliferation of HCC cells, and explore its possible molecular mechanism, Methods： SOX9 expression levels were analyzed in normal liver, liver cirrhosis, and human HCC tissue samples using the public clinical data sets. Then its mRNA expression levels in 32 paired samples of HCC tissue and its corresponding para-cancerous tissue were further confirmed by real-time fluorogenic quantitative-PCR. The recombinant pGreenPuro^TM-SOX9-shRNA targeting SOX9 was established. Then lentiviruses were produced and the Huh7 cells were infected. The efficiency of SOX9 interference was confirmed by real-time fluorogenic quantitative-PCR and Western blotting. The proliferation, colony-formation, cell cycle, and senescence of the transfected cells were detected by cell counting kit-8 （CCK-8 assay）, colony-forming assay, flow cytometry （FCM）, and senescence-associated beta-galactosidase （SA-β-Gal） staining, respectively. Finally, the genes potentially regulated by SOX9 were explored. Results： Elevated expression of SOX9 was found in public clinical data sets （P 〈 0.000 1）, and the result was further confirmed in 32 paired samples of HCC tissue and its corresponding para-cancerous tissue （P = 0.009 5）. The recombinant pGreenPuro^TM-SOX9-shRNA was successfullyconstructed, and the stably infected Huh7 cells were established. The abilities of proliferation, colony formation, and senescent resistance of Huh7 cells were impaired as a result of SOX9 silencing. Additionally, the genes involved in proliferation, including proliferating cell nuclear antigen （PCNA）, cyclin A2 （CCNA2）, cyclin E2 （CCNE2） and transcription factor E2F-2 were possibly down-regulated by soxg. Condusion： SOX9 expression is elevated in HCC tissues and may promote the proliferation and senescent resistance of HCC cells, and this molecular mechanism may be related to regulation of the expressions of cyclin and E2F-2.