Basu Sanmitra, Majumder Subhadipa, Bhowal Ankur, Ghosh Alip, Naskar Sukla, Nandy Sumit, Mukherjee Subhabrata, Sinha Rajan Kumar, Basu Keya, Karmakar Dilip, Banerjee Soma, Sengupta Sanghamitra
Department of Biochemistry, University of Calcutta, Kolkata, West Bengal, India.
Centre for Liver Research, Institute of Post-Graduate Medical Education & Research, Kolkata, West Bengal, India.
PLoS One. 2015 May 4;10(5):e0125560. doi: 10.1371/journal.pone.0125560. eCollection 2015.
Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, P<0.05) was observed in prostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of hMLH1 was inversely related (r = -0.59, P<0.05) with methylation quotient of its promoter which showed a significantly higher methylation density (P = 0.008, Z = -2.649) in cancer patients. hsa-miR-155, hsa-miR-141 and hsa-miR-21 gene expressions were significantly elevated (66-85%, P<0.05) in tumor specimens and negatively correlated (r = -0.602 to -0.527, P<0.05) with that of MMR genes. hsa-miR-155 & hsa-miR-141 and hsa-miR-155 & hsa-miR-21 were demonstrated to bind to their putative seed sequences in hMLH1 and hMSH6 3'UTRs respectively. Relatively higher expression of DNA methyl-transferases (DNMT1 and DNMT3b) and HIF-1α genes (34-50%, P<0.05) were also detected in tumor tissues. This study provides statistical evidence that MMR deficiency is correlated with hypermethylation of hMLH1 promoter and upregulation of hsa-miR-155, hsa-miR-141 and hsa-miR-21 in prostate cancer. This comparative study reflects that microRNA expression level, particularly hsa-miR-155, exhibits predictive signature of prostate adenocarcinoma.
前列腺癌是老年男性主要的致死原因之一。临床上对于早期检测前列腺癌的有用生物标志物存在未满足的需求，以减少过度治疗及其伴随的发病率。本项基于人群的研究调查了破坏前列腺癌患者DNA错配修复途径中多个功能相关基因表达的因素，以确定区分前列腺腺癌与良性增生的分子特征。使用实时PCR、蛋白质印迹和免疫组织化学比较前列腺癌组织样本与良性前列腺增生组织样本之间的基因表达。使用PCR偶联的限制性片段长度多态性和测序对三个错配修复（MMR）基因的七个单核苷酸多态性的基因型进行评估。通过甲基化特异性PCR和亚硫酸氢盐测序检测启动子甲基化。通过基于3'非翻译区（UTR）的双荧光素酶测定验证微小RNA与MMR基因之间的相互作用。在前列腺癌组织中观察到三个MMR基因hMLH1、hMSH6和hMSH2同时减少（34 - 85%，P<0.05）。hMSH6多态性rs1800932（Pro92Pro）在癌症患者中具有临界保护作用（比值比 = 0.33，95%置信区间 = 0.15 - 0.75）。hMLH1的相对转录水平与其启动子的甲基化商呈负相关（r = -0.59，P<0.05），在癌症患者中其启动子甲基化密度显著更高（P = 0.008，Z = -2.649）。hsa-miR-155、hsa-miR-141和hsa-miR-21基因表达在肿瘤标本中显著升高（66 - 85%，P<0.05），并且与MMR基因的表达呈负相关（r = -0.602至 -0.527，P<0.05）。已证明hsa-miR-155与hsa-miR-141以及hsa-miR-155与hsa-miR-21分别与hMLH1和hMSH6 3'UTR中的假定种子序列结合。在肿瘤组织中还检测到DNA甲基转移酶（DNMT1和DNMT3b）和缺氧诱导因子-1α（HIF-1α）基因相对较高的表达（34 - 50%，P<0.05）。本研究提供了统计学证据，表明错配修复缺陷与前列腺癌中hMLH1启动子的高甲基化以及hsa-miR-155、hsa-miR-141和hsa-miR-21的上调相关。这项比较研究表明，微小RNA表达水平，特别是hsa-miR-155，表现出前列腺腺癌的预测特征。
