A Study of Molecular Signals Deregulating Mismatch Repair Genes in Prostate Cancer Compared to Benign Prostatic Hyperplasia

<div><p>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 <i>hMLH1</i>, <i>hMSH6</i> and <i>hMSH2</i> (34-85%, P<0.05) was observed in prostate cancer tissues. <i>hMSH6</i> polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of <i>hMLH1</i> 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 <i>hMLH1</i> and <i>hMSH6</i> 3’UTRs respectively. Relatively higher expression of DNA methyl-transferases (<i>DNMT1</i> and <i>DNMT3b</i>) and <i>HIF-1α</i> 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 <i>hMLH1</i> 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.</p></div

Clinical and demographic characteristics of the study participants

<p>*P value significant at 0.05.</p><p>Clinical and demographic characteristics of the study participants</p

Promoter hypermethylation of MMR genes.

<p>(a) Diagram showing the <i>in silico</i> mapping of putative CpG islands in the promoter region of <i>hMLH1</i>, <i>hMSH6</i> and <i>hMSH2</i> genes with position of forward and reverse primers demarcated as half arrows which were used in methylation assays. TSS indicates the transcriptional start site.(b) Gel photograph illustrating the methylation status of <i>hMLH1</i> and <i>hMSH2</i> promoter region CpG islands in prostate cancer and BPH tissues as determined by methylation-specific PCR in the left and right panels respectively. Primer sets for amplification were designated as unmethylated (U) and methylated (M).The presence of PCR product in lanes marked U indicates unmethylated <i>hMLH1</i> and <i>hMSH2</i>; product in lanes marked M indicates hypermethylated <i>hMLH1</i> and <i>hMSH2</i>. L indicates 100 bp plus ladder. (c) Distribution of methylation quotients [MQ = (logMLH1/ACTB) X1000] obtained from analyzing prostate cancer and BPH tissue DNAs using qMSP. The calibration curve was generated to determine quantitative accuracy of qMSP with five different dilutions of in vitro fully methylated DNA from normal healthy human lymphocytes.(d) Plot showing correlation between transcript levels of <i>hMLH1</i> (∆C_q) in prostate cancer tissue samples with methylation quotients corresponding <i>hMLH1</i> promoter. Pearson’s correlation coefficient (r) and P values are indicated for each test. * indicates P <0.05.</p

MMR gene expression in cancer and benign hyperplasia of prostate glands.

<p>(a) Bar diagram showing decreased expression of transcripts of <i>hMLH1</i>, <i>hMSH6</i> and <i>hMSH2</i> genes in prostate cancer tissues compared to that of BPH patients. 18S rRNA was used as endogenous control. * indicates P <0.05. (b) Fold differences of <i>hMLH1</i>, <i>hMSH6</i> and <i>hMSH2</i> expression in each of 15 cancer tissue specimens with respect to population mean of ∆C_q estimate of the said gene in BPH tissues. (c) Upper panel: Plots showing correlation between transcript levels of <i>hMSH6</i> and <i>hMSH2</i>, <i>hMSH6</i> and <i>hMLH1</i> and <i>hMSH2</i> and <i>hMLH1</i> in prostate cancer. Lower panel: Plots showing correlation between transcript levels of <i>hMSH6</i> and <i>hMSH2</i>, <i>hMSH6</i> and <i>hMLH1</i> and <i>hMSH2</i> and <i>hMLH1</i> in BPH. Pearson’s correlation coefficient (r) and P values are indicated for each test. * indicates P <0.05. (d) Upper panel: Representative result of Western blot for MMR proteins in cancer and benign tissue of prostate. β-Actin acted as endogenous control. Lower panel: Bar diagram showing difference in expression of <i>hMLH1</i>, <i>hMSH6</i> and <i>hMSH2</i> at the protein level between BPH and prostate cancer tissue samples. Fold changes and statistical significance are indicated. * indicates P <0.05 and n indicates the number of samples. (e) Photomicrograph of representative prostate archival specimens immune-stained with antibodies against hMLH1, hMSH6 and hMSH2 at 400x magnification showing decreased expression of the MMR genes in prostate cancer tissues compared to benign hyperplastic tissues.</p

Interaction of MMR gene 3'UTRs with microRNAs.

<p>(a) Bar diagram illustrating positive 3'UTR activity of <i>hMLH1</i>, <i>hMSH6</i> and <i>hMSH2</i> genes in HEPG2 cells following reporter gene assay. Fold changes were appended in the diagram. (*) indicates statistical significance of functionality of 3'UTR regions measured in terms of P value by t-test. (b) Entire 3'UTR region of <i>hMLH1</i>, <i>hMSH2</i> and <i>hMSH6</i> were mapped for putative microRNA binding sites. The highlighted and boldfaced segments within the 3'UTR sequences represent the seed positions for the microRNAs. (c) Multiple alignments indicated that miR-155, miR-21 and miR-141 seed sequences are evolutionarily conserved across mammalian and non-mammalian species. (d) Normalized relative light units (RLU) in HEPG2 and PC3 cells were measured for <i>hMLH1</i> and <i>hMSH6</i> 3'-UTR constructs in pSiCHECK2 with (+) and without (-) the effect of microRNAs. Co-transfection of the pRNAU6.1 empty vector (+) with the 3'UTR-pSiCHECK2 constructs of <i>hMLH1</i> and <i>hMSH6</i> were set as 100% and percentage reduction in luciferase activity mediated by the three microRNAs were measured in relation to this was shown in the bar diagram. Statistical significance in terms of P values was measured with t-test. (*) indicates the statistical significance P<0.05. (e) Bar diagram showing upregulation of hsa-miR-21, hsa-miR-155 and hsa-miR-141transcripts in prostate cancer tissues compared to that of BPH patients. * indicates P<0.05. (f) Heat map showing the combination of two dendrograms displayed above and to the right. The rows represent genes on the right of the figure. Individual patient sample is shown as columns. Color represents expression level of the gene. Red represents low expression, while green represents high expression. The expression levels are continuously mapped on the color scale provided at the bottom of the figure. The dendrograms show stratification of samples into two groups: BPH (B) and prostate cancer (P), based on gene expression.</p