miR-519d在肿瘤细胞周期调控中的作用及其分子机制研究

Recent studies have identi?ed speci?c microRNAs that regulate the cell cycle and have documented that the loss or gain of miRNA-mediated cell-cycle control contributes to malignancy. MiRNAs regulate classic cell-cycle control pathways by directly targeting proteins such as E2F transcription factors, cyclin-dependent kinases (Cdks), cyclins and Cdk inhibitors. If miRNAs control the highly orchestrated patterns of gene expression that occur during cell cycle progression, it seems likely that miRNAs themselves might be subject to cell-cycle dependent regulation. Thus, it is hypothesized that expression of specific miRNAs oscillates orderly along with cell cycle progression. However, the oscillated expression patterns of many candidate miRNAs have yet to be determined. Our previous study described the miRNA expression profiling in double-thymidine synchronized HeLa cells as cell cycle progresses and suggested that twenty-five miRNAs, such as miR-519d, exhibit characteristic cell cycle stage-specific oscillations in HeLa cells. We also provided evidence that miR-519d gain of function promotes growth of HeLa cell by controlling cell cycle progression, whereas loss of function reverses this phenotype. Further study confirmed that p21 and E2F1 are direct targets of miR-519d. Although the function and mechanism of miR-519d in tumorigenesis has been investigated in only a few studies, the results remain conflicting rather than conclusive. To resolve thess conflictes, this study aims to elucidate the molecular mechanism and functional role of miR-519d in the regulation of cell cycle progression in human cancer. First, we aim to perform a time-course analysis of miR-519d expression patterns during the cell cycle of different cancer cells by Northern blot. Second, the role of miR-519d on cell cycle progression is to be investigated by combining the gain- and loss-of-function experiments and cell synchronization. Finally, to explore the molecular mechanism by which miR-519d influence the cell cycle progression, potential target genes of miR-519d will be predicted by bioinformatics analysis and identified by luciferase reported assay. To further elucidate the correlation between miR-519d and its experimentally validated targets, the expressions of miR-519d and its targets will be analyzed throughout the cell cycle by Western blot. In addition, we intend to examine whether knockdown of these genes may mimic the effect of miR-519d overexpression by RNAi technology, and enforced expression may reverse the effect, which may be help us better understand the role of these targets in miR-519d-mediated cell cycle regulation. Taken together, the present study will help to elucidate the potential role of miR-519d in cell cycle control and tumorigenesis, and implicate its potential application in cancer therapy.

microRNAs介导的细胞周期调控与肿瘤发生密切相关。前期的研究中，我们利用细胞同步化和miRNAs芯片技术对不同细胞周期时相的HeLa细胞的miRNAs表达谱进行了分析，证明了miR-519d等25种miRNAs的表达具有周期依赖性。进一步研究表明miR-519d在HeLa细胞内可能通过靶向p21和E2F1发挥癌基因的作用。然而，目前有关miR-519d在肿瘤发生中的作用及其机制仍存在一些争议。为此，本课题拟采用细胞同步化和Northern blot检测技术，以time-course的方法分析miR-519d在肝癌、胃癌等8种不同肿瘤细胞中的表达情况，运用细胞同步化和过表达/敲减实验明确其在肿瘤细胞周期调控中的确切作用，通过生物信息学分析与实验验证相结合的方法鉴定miR-519d的靶基因，并揭示其与靶基因间的相互关系，最终阐明miR-519d调控肿瘤细胞周期的分子机制。

近年来，由于microRNA在人类肿瘤发生发展中的重要作用，相关研究领域得到了科学界的广泛关注。越来越多的证据表明，一些特定的miRNA能通过直接靶向重要的细胞周期调控蛋白如Cyclins, Cdk等而调控经典的细胞周期进程。而这些miRNA的功能缺失或过表达可能通过影响细胞周期进程，而与肿瘤发生密切相关。. miR-519d-3p是人类19号染色体上的miRNA基因簇（C19MC）成员之一，主要表达于胎盘组织中。我们先前的研究结果提示其在宫颈癌细胞周期进程中存在重要作用。已有研究提示，它在诸如如肥胖、骨肉瘤、肝癌和卵巢癌等诸多病理过程中具有促进细胞增殖和转移的潜能。然而，其在宫颈癌中作用及机制尚未见报道。鉴于此，本研究拟探讨其在宫颈癌发生发展过程中的生物学功能。. 我们发现了miR-519d-3p在宫颈癌临床组织中高表达，并通过体外实验证明其在宫颈癌细胞中通过促进细胞生长、迁移和侵袭而发挥癌基因的功能。相关研究结果表明，miR-519d-3p的高表达促进宫颈癌的发生。为了更全面地揭示其作用的分子机制，我们通过基因表达谱芯片分析和生物信息学预测相结合的方法，确定了164个候选靶基因和相关信号通路（如CDKN1A、Smad7、PTEN、Rb家族成员等），并通过qRT-PCR对部分结果进行了验证。进一步，我们发现Smad7是miR-519d-3p的一个新靶标，是其促进宫颈癌进展的重要分子。Smad7的缺失表达可抵消miR-519d-3p促宫颈癌的作用。上述结果揭示了一种miR-519d-3p促宫颈癌转移的新机制。此外，我们还发现mir-519d-3p具有加速细胞周期进程、抑制凋亡的作用。然而，miR-519d-3p表达抑制的HeLa细胞中干扰Smad7并不能产生类似的抗凋亡效应，提示其调控细胞凋亡的作用可能与其他潜在靶基因相关，这将是下一阶段研究方向之一。. 综上所述，本项目揭示了miR-519d-3p下调Smad7是宫颈癌的侵袭和转移的重要机制之一，说明TGF-β信号通路活化可能与宫颈癌转移机制相关。但它在TGF-β信号转导通路及转移通路中的具体机制仍需进一步探讨。因此，本研究为靶向miR-519d-3p抑制宫颈癌转移和化疗耐受的分子靶向治疗策略提供了重要的实验依据。