基因论文人PDLIM4基因在前列腺癌的表达调控研究费.doc

基因论文：人PDLIM4基因在前列腺癌的表达调控研究【中文摘要】前列腺癌(prostate cancer,PCa)是欧美国家常见的肿瘤,居男性肿瘤死亡原因第二位,仅次于肺癌。我国前列腺癌的发病率虽低于西方国家,但随着生活水平的提高及人口老龄化发展,其发病率正迅速上升,成为危害中老年男性健康的主要疾病。前列腺癌的发病及恶化转移机制复杂,不仅与种族,饮食、生活习惯等关系密切,并且癌基因和抑癌基因及其相应的信号通路的异常调控直接参与了前列腺癌的发生、发展。Vanaja等。研究发现PDLIM4基因在正常细胞中普遍表达,而在肿瘤细胞中常发生缺失,通过稳定转染恢复表达可以抑制肿瘤细胞增殖和集落形成,并促进细胞发生凋亡6。初步认为具有抑癌基因的功能。目前研究表明,PDLIM4氏表达与杂合子缺失、启动子区域CpG岛的异常高甲基化密切相关,且PDLIM4甲基化失活常和前列腺癌预后较差相关6，7。DNA甲基化是表观遗传学主要研究内容之一。DNA甲基化主要指基因启动子区域CpG岛中胞嘧啶C发生甲基化,由于DNA甲基化是一个可逆的过程,通过药物逆转启动子区域的甲基化可使沉默的基因恢复表达8-11。A甲基化可导致抑癌基因转录失活,进而影响肿瘤的发生发展12-15。人前列腺癌中PDLIM4基因启动子核心区域中含有多个CpG岛,已证实其CpG岛高度甲基化与PDLIM4表达下调密切相关但其调控机制尚不明确。我们首先通过免疫组化检测中国汉族人群中前列腺癌患者PDLIM4在前列腺增生组织及前列腺癌组织中的表达,已确定该基因的表达是否存在种族差异。同时检测相关的甲基转移酶(DNA methyltransferase,DNMTs)DNMT3A及DNMT3B在相应组织中的表达水平。结果显示在前列腺癌组织中PDLIM4的表达明显低于正常癌旁组织及前列腺增生组织,与国外的研究报道一致,表明该基因的表达无显著的种族差异。而DNMT3A及DNMT3B表达在增生组织及癌组织中无明显差异。为了研究PDLIM4基因在前列腺癌中低表达的调控机制,我们通过Western blotting确定前列腺癌激素依赖及非依赖细胞株中PDLIM4蛋白的表达水平。结果显示,PDLIM4在正常人前列腺永生细胞株RWPE1中表达,而在激素非依赖的前列腺癌细胞PC-3、激素依赖的LNCaP中表达沉默。经过甲基转移酶抑制剂5-aza-dc处理后,LNCaP细胞中PDLIM4的表达明显上调,而RWPE1和PC3表达未发生明显改变,表明PDLIM4的表达不仅与其启动子CpG岛甲基化有关,可能还存在其它的调控机制。由于PDLIM4的表达调控发生在转录水平,我们以基因组DNA为模板,通过PCR扩增获得PDLIM4基因启动子序列片段,插入到pGL3-Basic报告基因载体,分别得到含有3kb和1.2kb的PDLIM4基因启动子的两个报告基因质粒pGL3-PDLIM4-3kb和pGL3-PDLIM4-1.2kb。另外,pGL3-PDLIM4-1.2kb质粒运用外切酶将启动子从5端截短获得系列不同短片段的启动子序列,并构建其荧光素酶报告基因的重组质粒,得到5个含不同长度启动子序列的报告基因载体：pGL3-PDLIM4-970bp,pGL3-PDLIM4-803bp, pGL3-PDLIM4-653bp, pGL3-PDLIM4-645bp,pGL3-PDLIM4-627bpo利用PDLIM4启动子及其不同截短体报告基因转染不同组织来源细胞系及各前列腺细胞株,检测其表达情况。结果表明,PDLIM4基因长片段的3kb和1.2kb启动子荧光素酶质粒,在乳腺癌细胞株、宫颈癌细胞株、入口腔上皮肿瘤细胞株、前列腺癌细胞株中均有不同程度的表达活性,其中在前列腺癌细胞株PC3、LNCaP中表达较低。而不同长度的PDLIM4启动子报告基因质粒转染PC-3、LNCaP的结果显示,pGL3-PDLIM4-1.2kb表达活性最强,pGL3-PDLIM4-970bp表达活性最弱,说明在启动子-1275bp至-970bp区域内可能存在一个正调控元件。前列腺癌细胞株转染PDLIM4启动子,经5-aza-dc处理后,与未处理的细胞比较启动子活性变化。结果显示,LNCaP经5-aza-dc处理后PDLIM4的启动子活性明显上调(P0.05),而PC-3经5-aza-dc处理后启动子活性发生下调,RWPE1未发生明显改变。经5-aza-dc处理后启动子活性的变化与蛋白表达水平的变化相一致。说明了甲基化在激素依赖的LNCaP是主要的调控机制,而在激素非依赖的PC-3细胞中可能存在非甲基化调控机制。综上所述,抑癌基因PDLIM4在中国汉族人群前列腺癌组织和激素依赖的前列腺癌细胞中沉默,其启动子区域的甲基化对基因的沉默起着重要作用,去甲基化后可使PDLIM4在转录和蛋白翻译水平上恢复表达。而在激素非依赖的前列腺癌细胞中,其表达可能存在其他的调控机制,需要进一步的研究探索。【英文摘要】Prostate cancer (prostate cancer, PCa) is the most common malignant cancer in Europe and America countries, and is the second leading cause of death to lung cancer in men. Although its incidence in China is lower than Europe countries, prostate cancer has become more important cause of death in the old men as the average lifespan increasing and the living standards improving in our country. Compelling evidence in biochemistry, genetics and epidemiology has shown that the many oncogenes and tumor suppressor genes were involved in the pathogenesis and progression of prostate cancer in addition to environment, food and race, which have a strong associations with the development and progression of prostate cancer.It has been revealed that PDLIM4 is silenced in prostate cancer according to oligonucleotide microarrays results between prostate cancer and benign tissues. PDLIM4, also called RIL (reversion-induced LIM gene), maps to chromosome 5q31.1, and is identified as a candidate tumor suppressor. The PDLIM4 gene was firstly identified in experiments on elucidating genes whose expression was critical for neoplastic transformation in rat fibroblasts. PDLIM4 expression is ubiquitous in normal tissues, but lost in cancer cell lines. PDLIM4 reexpression leads to a suppression of cancer cell growth and clonogenicity in soft agar and sensitizes cancer cells to apoptosis. Several studies has demonstrated that PDLIM4 is frequently methylated in cancer, and that methylation is associated with loss of expression and can predict the prognosis of prostate cancer. As the important part of the emerging epigenomics, methylation of CpG island results in the inhibited expression of cancer suppressor genes, and following impact on pathogenesis and progression of tumors. Methylation of DNA is different from mutation and absence, without altering the DNA sequence, therefore, methylation of DNA is a reversible change. Hypermethylation of PDLIM4 correlates with loss of gene expression, which could be restored in methylated cell lines by 5-aza-dc, an inhibitor of DNA methyltransferases. Our work focus on the regulatory mechanisms underlying promoter CpG islands methylation on PDLIM4 gene in prostate cancer.In attempt to examine whether the expression of PDLIM4 was also down-regulated in Chinese Han population, immunohistochemistry was used to detect the expressions of PDLIM4, DNA methyltransferases DNMT3A and DNMT3B in patients with prostate cancer and BPH. Consistent to observations in Western populations, the expression of PDLIM4 in prostate carcinoma tissues was significantly lower than that in BPH and cancer adjacent tissues in Chinese Han patients, While no significant alterations in the expression of DNMT3A and DNMT3B were observed in tumor tissues and BPH, suggesting that the expression of PDLIM4 was decreased in prostate carcinoma tissues in Chinese Han. We utilized western blotting to study the expression level of PDLIM4 in prostate cancer cell lines. The results showed that PDLIM4 expressed in RWPE1 cell lines and was lost in PC-3 and LNCaP cells. Pretreatment with 5-aza-dc significantly upregulated the expression of PDLIM4 protein in LNCaP cells at the concentration of 10mol/L, and did not affect the expression levels in RWPE1 and PC-3 cells.To further investigate regulatory mechanisms of PDLIM4 gene in prostate cancer, PCR assay was carried out to clone PDLIM4 promoter fragment into pGL3-basic vector to construct pGL3-promoter recombinant report plasmid, resulted in construct containing 3kb and 1.2 kb fragment of its 5flanking region sequence (pGL3-PDLIM4-3kb) and 1.2kb sequence (pGL3-PDLIM4-1.2kb), respectively. Then we utilized EXOIII to truncate the promoter to construct an array of truncated promoter plasmids, using pGL3-PDLIM4-1.2kb plasmid as template, and producing pGL3-PDLIM4-970bp, pGL3-PDLIM4-803bp, pGL3-PDLIM4-653bp, pGL3-PDLIM4-645bp, pGL3- PDLIM4-627bp plasmid. Array of above promoter plasmids were transfected into different tissue cell lines and prostate cell lines to test the promoter activity. Transient transfection results showed that the promoter activities of pGL3-PDLIM4-3kb and pGL3-PDLIM4-1.2kb were evident in tested cancer cell lines, and lower signals were observed in prostate cancer LNCaP and PC-3 cells when compared with others. Furthermore, luciferase activities of 5-end delete mutants bearing variable sequence regions in PDLIM4 promoter were detected in prostate cancer cells, and the 0.97kb promoter displayed lowest activity in cells when compared to other promoter reporters tested, suggesting that transcript factors bound to cis-elements in this region could be important for regulation of PDLIM4 promoter activity.Treatment of prostate cancer cells with 5-aza-dc resulted in pronounced elevation of promoter activity in LNCaP after transfection. However, downregulation of luciferase activity was observed in PC-3 and DU145 after transfection. No detectable change was shown inRWPE1 cells under same conditions. These results indicated that methylation is the prominent regulatory mechanism in LNCaP, but not in PC-3. There may be other regulatory mechanisms in PC3 to induce gene silence.In short, tumor suppressed g