LncRNA（RP13-650J16.1）调控PIA-PCa恶性转化及其RAC3依赖机制研究

Prostate cancer (PCa) poses a significant threat to life quality and expectancy of senior males. Proliferative inflammatory atrophy (PIA) has been proposed as a novel mechanism of carcinogenesis in the prostate. Long non-coding RNAs (lncRNAs) has been implicated in the occurrence, progression and metastasis of PCa, however, they have not yet been reported to be involved in the malignant progression of PIA to PCa. We previously established a tight connection between PIA and PCa. Through chip analysis, the expression level of both lncRNA RP13-650J16.1 and RAC3 gene raised from normal tissue, PIA, to PCa, a phenomenon that was subsequently validated in 20 clinical PCa samples. Through loss-of-function assay, knockdown of RP13-650J16.1 was found to repress RAC3 expression, as well as proliferation and invasion in PCa cells. As such, we hypothesized that RP13-650J16.1 may promote PIA-PCa malignant transformation through RAC3 dependent mechanisms. In this study, we will elucidate the underlying mechanism of the regulatory role of RP13-650J16.1 in PIA-PCa transformation through RAC3, and evaluate the potential of RP13-650J16.1 as a screening marker and therapeutic target for PCa, providing new insights to the early diagnosis and treatment of PCa.

前列腺癌(PCa)是影响老年男性生活质量和预期寿命的重要疾病。研究提示增生性炎性萎缩(PIA) 可能恶性进展为PCa。长链非编码RNA(lncRNA)参与调控PCa发生、发展及转移，是目前研究热点。但目前尚无lncRNA参与PIA-PCa恶性转化相关报道。申请人前期研究证实PIA与PCa密切相关；通过基因芯片分析及大样本组织验证发现正常组织、PIA和PCa组织中lncRNA（RP13-650J16.1）和RAC3基因表达梯度上调；敲低RP13-650J16.1可下调RAC3表达，并抑制PCa细胞增值与侵袭。因此申请人提出假设，RP13-650J16.1可能通过上调RAC3表达促进PIA-PCa恶性转化。本研究拟明确RP13-650J16.1调控PIA-PCa恶性转化及其RAC3依赖机制，验证RP13-650J16.1作为PCa早期筛查指标及治疗靶点可行性，为PCa早期诊治提供新思路。

主要研究项目 1：利用高通量测序获得在前列腺癌（PCa）组织与癌旁正常组织中差异表达的长链非编码lncRNA；验证其RP13-650J16.1及其临近表达基因 RAC3的差异表达情况，通过生物信息学分析探索RP13-650J16.1对临近基因 RAC3的调控作用；明确RP13-650J16.1是否与前列腺癌的进展预预后相关。测序数据结果表明RP13-650J16.1在癌组织上调，邻近RAC3也已经被报道参与前列腺癌的发生发展，与前列腺癌预后相关。因此我们选定RP13-650J16.1作为研究对象，研究其对RAC3的调控机制。通过qRT-PCR验证其在癌与癌旁组织中的表达情况，然后构建siRNA干扰序列进行试验，敲减RP13-650J16.1后检测其对RAC3蛋白质表达的影响，检测敲减RP13-650J16.1后对前列腺癌细胞增殖、迁移及侵袭的影响；对小鼠皮下成瘤的影响。结果：qRT-PCR结果表明RP13-650J16.1与RAC3在癌组织表达水平高于癌旁正常组织；敲减RP13-650J16.1后RAC3蛋白质表达水平降低；体外细胞和体内动物实验试验表明干扰RP13-650J16.1后前列腺癌细胞增殖、迁移及侵袭能力被抑制。.主要研究项目 2：根据高通量测序结果进行差异基因分析中我们发现LINC00467在前列腺癌组织中表达量高于癌旁PIA组织和癌旁正常组织，这表明LINC00467可能参与前列腺癌的发生发展。因此我们对LIN00467调控前列腺癌发生发展的潜在机制进行了研究。首先构建LINC00467干扰序列，并检测敲减LINC00467后对体内动物实验及体外细胞实验对前列癌细胞生物学行为的影响。通过生物信息学分析寻找潜在的LINC00467调控机制。结果：通过qRT-PCR发现LINC00467相对正常前列腺上皮细胞在癌细胞系中高表达，癌组织表达量高于癌旁组织；敲减LINC00467抑制了前列癌细胞的增殖、迁移及侵袭的能力，在小鼠皮下成瘤实验中，敲减LINC00467明显抑制了体内前列腺癌组织的增殖能力；此外干扰LINC00467明显抑制了M2巨噬细胞标志物的水平，表明LINC00467 可以促进M2巨噬细胞极化，调控前列腺癌细胞的生物学行为；最后通过生物信息学及实验分析发现LINC00467调控miR-494-3p进而调控是stat3来促进前列腺癌的恶性进展。