let-7b对肝癌细胞增殖的影响及机制研究

Malignant cell proliferation of hepatocellular carcinoma (HCC) is one of the critical reasons during carcinogenesis and development of HCC. So it is very important to study the molecular mechanism related with HCC cell proliferation. Recent researches suggested that although miRNAs played an important role in HCC cell proliferation, the mechanism was still unclear. Our preliminary studies revealed that let-7b expression in HCC tissue and cell lines were both significantly lower than that in normal tissue and cell lines, and let-7b overexpression can inhibit cell proliferation of HCC significantly. Bioinformatics analysis showed that CDKN1A, CDKN1B, TGFB3 and CCND1 may be potential candidate target genes of let-7b. However, it was unclear about the mechanism that let-7b affected HCC cell proliferation through signaling pathways associated with target genes. We planned to identify downstream target genes of let-7b by using luciferase carrier experiment, to observe effects of let-7b on cell proliferation in vivo and in vitro through regulating target genes, and thus to confirm the molecular mechanism that let-7b regulated target genes to influence proliferation of HCC. Finally with the help of clinical data, we analyzed the correlation of expression of let-7b and its downstream target genes with clinical pathological characteristics and prognosis of HCC. This study can help us to understand the mechanism of HCC cell proliferation and provide new theory and therapeutic targets for clinical treatment.

肝癌细胞恶性增殖是肝癌发生发展的本质原因之一，因此研究肝癌增殖相关分子机制非常重要。新近研究表明miRNA在肝癌细胞增殖中发挥重要作用，但相关机制仍无定论。我们前期研究发现，let-7b在肝癌组织及细胞系中表达均显著下调，过表达let-7b显著抑制肝癌细胞增殖。生物信息学分析提示，CDKN1A、CDKN1B、TGFB3及CCND1可能是let-7b潜在候选靶基因。然而let-7b通过靶基因相关信号通路影响肝癌细胞增殖的机制尚不明确。本研究拟利用荧光素酶载体实验明确let-7b下游靶基因，再通过调控靶基因表达，在体内外实验中观察let-7b对肝癌细胞增殖的影响，从而明确let-7b调控靶基因影响肝癌增殖的分子机制；最后结合临床资料分析let-7b及其下游相关基因的表达与肝癌临床病理指标及预后的相关性。本研究结果有助于认识肝癌细胞恶性增殖相关机制，为肝癌临床治疗提供新的理论依据和治疗靶点。

背景:肝细胞癌(HCC)是目前发病率和致死率非常高的恶性肿瘤之一。既往研究证实许多miRNAs在HCC的发生发展中起重要作用,为了更好地了解miRNAs在生理和病理环境中的功能，识别这些miRNAs及其靶基因变得越来越迫切。既往研究证实，在肿瘤发生过程中，let-7表达经常下调，提示let-7可能作为癌基因参与了这一过程。方法:采用免疫组化法观察肝细胞癌组织中let-7b的表达。采用CCK-8检测let-7b在HCC细胞增殖中的作用。流式细胞仪检测HCC细胞的细胞周期。采用BALB鼠成瘤实验检测HCC细胞的成瘤能力;采用Western blot和realtime PCR方法观察p21在HCC细胞中的表达情况。结果:我们前期在东方肝胆外科医院国家肝癌组织标本库中对HCC组织标本进行研究和检测，发现let-7b表达异常，且明显下调。根据我们的发现，let-7b在HCC肿瘤中表达缺失，其低表达与HCC预后不佳有关。在进一步的实验中，我们发现let-7b通过上调p21来抑制HCC细胞的增殖。结论:我们的研究结果提示let-7b可能通过上调p21来抑制肝癌细胞的增殖，这可能为开发新的HCC治疗策略提供宝贵的信息。