小RNA如何调控互为反义的CDK4/TSPAN31和CCND1/LOC15基因的表达及肝癌细胞的增殖与凋亡

Our study of the effect of cannabinoids on hepatoma cells ( HCC ) found that CDK4 has two kinds of 40kD and 33kD proteins, but small RNA for CDK4 decreased the expression of 33kD protein, but increase the expression of 40kD protein. The change should be related to CDK4/TSPAN31 overlap . Our preliminary study shows the overlap region is longer than the NCBI database show. CCND1(cyclin D1), acts by forming a complex with CDK4. the human genomic regions that harbor the CCND1 and CDK4 genes are special, because while one DNA strand encodes CCND1, the opposite strand encodes two other genes, LOC100996515 (abbreviated as LOC15) and DNCC1. Similarly, the DNA strand opposite to the CDK4-coding one encodes another gene called TSPAN31. when the two DNA chains are transcripted into mRNA, how does small RNA determine which RNA to degraded as the target sequence? Therefore, we hypothesize that in the particular situation in which both DNA strands encode mRNAs, small regulatory RNAs, including double-stranded shRNA and single stranded siRNA and antisense, work differently from the canonical situation wherein only one strand of the DNA double helix encodes mRNA. Since CCND1 and CDK4 belong to these special genes and are known to play important roles in HCC, we will use these two genes, along with the genes encoded by their opposite DNA strand, as a model system to address my hypothesis in HCC. So firstly we will use RT-PCR to determine whether the CDK4 and TSPAN31 mRNAs, as well as the CCND1, DNCC1 and LOC15 mRNAs, are co-expressed, mutually excluded, or independent of each other in HCC cell lines. And then , using molecular cloning, hybridization, IP and cDNA Protection analysis to determine whether and how CDK4-targeting shRNA, siRNA and antisense affect not only CDK4 but also TSPAN31 expression, while those targeting TSPAN31 also affect CDK4 expression, and to study CCND1, DNCC1 and LOC15 too. Lastly,the effect of small RNA on the proliferation of hepatocellular carcinoma (HCC) cells will be studied.In order to accomplish this project, We have established several novel cloning strategies and methods that allow us to clone both ends and the full-length of an mRNA without mixing up one strand with its opposite one. We will use human HCC cell lines to study the effects of the single or double stranded regulatory small RNAs on the five genes, i.e. CCND1, LOC15, DNCC1, CDK4 and TSPANS31 in HCC. The results of this project will reveal how this kind of genes influence HCC at the RNA level, but also Lay the foundation for further study on the mechanism of cannabinoid on HCC.

我们在研究大麻素对肝癌细胞（HCC）CDK4的影响时发现，CDK4存在40kD和33kD 的两种蛋白，而针对CDK4的小RNA降低其33kD 的蛋白表达，却上调了40kD 的蛋白表达。推测这种变化与CDK4/TSPAN31之间的重叠有关。我们前期研究显示这种重叠区域比NCBI数据库显示的更长。CDK4基因负链编码CDK4、正链编码TSPAN31，当这两条DNA链都转录mRNA，小RNA如何确定去降解靶序列的哪条RNA呢？为此，我们拟使用链特异性的RT-PCR研究互为反义的mRNA是共表达，相互排斥还是彼此间独立的表达？然后用IP、cDNA保护分析及我们新建的克隆方法研究以这类基因为目标的小RNA对自身及其反义基因的表达是否有影响及如何影响？最后检测这类小RNA对HCC增殖与凋亡的影响。这个项目的结果将揭示这类基因如何在RNA水平影响HCC, 也为深入研究大麻素对HCC影响的机制奠定基础。

大麻素可以有效抑制肝癌细胞CDK4的表达。CDK4与TSPAN31在3’UTR存在517 nt反义互补。它们到底是共表达，相互排斥还是彼此间独立的表达？不清楚。为此，我们设计了不同的siRNA，并分别转染不同的肝癌细胞。Western和qPCR显示，TSPAN31siRNA上调了CDK4 mRNA和蛋白水平，CDK4 siRNA却下调了TSPAN31 mRNA和蛋白水平，这表明CDK4与TSPAN31基因间可能存在单向调控关系，TSPAN31作为CDK4的反义转录物，以discordant manner调控CDK4表达。我们进一步构建了pEGFP-TSPAN31-3’UTR载体并转染BEL-7402细胞。结果显示，过表达TSPAN31-3’UTR显著下调了CDK4 mRNA和蛋白水平。同时，过表达TSPAN31-3’UTR下调了由TSPAN31 knockdown上调的CDK4表达水平。这些研究结果表明TSPAN31重叠区域可能作为新的调控序列，对CDK4表达产生调控作用。我们的结果揭示基因下游序列如何调控mRNA水平的新机制。.为进一步了解TSPAN31对CDK4发挥调节作用区域，我们构建了3个过表达质粒结果显示，Hela细胞过表达TSPAN31-3’UTR及全长均能下调CDK4表达， CDS则不能；TSPAN31-siRNA上调CDK4表达，过表达TSPAN31-3’UTR及全长可以补救其诱导的CDK4上调。表明TSPAN31调控CDK4。我们还构建了CDK4-3’UTR载体，并使用双荧光素酶报告基因检测。结果下调TSPAN31表达后，CDK4-3’UTR活性上调； TSPAN31-3’UTR及全长均能下调CDK4-3’UTR活性，但是CDS则不能。表明TSPAN31通过3’UTR调控CDK4。克隆形成实验显示，抑制TSPAN31表达后，能够促进Hela与SiHa细胞株的克隆形成；然而过表达TSPAN31后，抑制Hela细胞株的克隆形成。MTS实验，抑制TSPAN31表达后宫颈癌细胞株细胞的增殖能力增强。TSPAN31-shRNA抑制TSPAN31表达后，上调其靶基因CDK4和p-RB蛋白水平。表明TSPAN31通过调节其靶基因CDK4介导细胞的增殖。 这个项目的结果揭示这类基因如何在 RNA 水平影响 HCC, 也为深入研究大麻素对 HCC 影响的机制奠定基础