Genome-wide Association Analysis Reveals Regulation Of At-risk Loci By DNA Methylation And The Function And Mechanism Of Claudin 3/4 In Prostate Cancer Metastasis

PART ?Genome-wide association analysis reveals regulation of at-risk loci by DNA methylation in prostate cancerObjectiveProstate cancer(PCa)is characterized by variable clinical manifestations and frequently unpredictable outcomes.Germline determinants of gene expression in tumorsare not studied frequently because of the complexity of transcript regulation due to somatically acquired alterations.In this study,based on the GWAS,we are aiming to find the regulation of at-risk loci by DNA methylation in prostate cancer,and to identify a new therapeutic strategy for the patients with prostate cancer.MethodsBased on the GWAS,we performed an expression quantitative trait locus(e QTL)analysis to identify genomic regions that regulate gene expression in PCa and identify a relationship between DNA methylation and clinical information.Using multi-level information published in The Cancer Genome Atlas,we performed e QTL-based analyses on DNA methylation and gene expression.To better interpret these data,we correlated Cp G loci and clinical indexes to identify the important loci for both PCa development and progression.In order to provide the mechanistic insights of the prostate cancer,an epigenetic association study of clinical indexes also detected risk loci and pyrosequencing was used with clinical samples for site validation.Results1,Based on the GWAS,methylated me QTLs and single nucleotide polymorphism analysis identified the locations of Cp G sites and genes within at-risk loci,including19q13.2-q13.43 and 16q22.2-q23.1.2,Based on the GWAS,we performed a correlation analysis between Cp G sites,gene expression levels and clinical indexes.We further performed a similar analysis to compare methylation sites and clinical information and identified high-risk Cp G sites that were significantly correlated with the aforementioned clinical information.3,These genes were further correlated with clinical information and the CpG sites were also correlated with the same clinical indexes.We identified 92 paired methylated QTLs and CASKIN1 that fulfilled the aforementioned criteria.4,We selected high-confidence Cp G sites reported to associate with PCa-related genes in other studies and evaluated methylation at related Cp G sites by using pyrosequencing clinical samples for site validation.The results was consisted with the results of me QTL anaylsis.ConclusionOur results demonstrated that although only a small proportion of genes are regulated via DNA methylation in PCa,these genes are enriched in important cancer-related groups.Based on the GWAS,we performed a correlation analysis between Cp G sites,gene expression levels and clinical indexes.Further we usd the pyrosequencing clinical samples for site validation.Finally we got several at-risk loci and genes regulated by DNA methylation in prostate cancer and could identify a new therapeutic strategy for the patients with prostate cancer.PART ?The function and mechanism of Claudin 3 and Claudin 4 in prostate cancer metastasisObjectiveSome research has showed the correlation between the epigenetic changes of prostate cancer and the expression level of Claudin 3 and Claudin 4.The abnormal expression of Claudins protein that belongs to tight junction proteins can lead to cancer.Based on the studies of epigenetic,we aim to further explore the function and invasive mechanism of Claudin 3 and Claudin 4 in prostate cancer and to provide new targets for the treatment of prostate cancer.MethodsIn human prostate cancer cells PC3 and LNCa P,we used the Si RNA to knock out Claudin 3 and Claudin 4,investigating their role in tumor metastasis.By using the WST-1 cell proliferation and cell toxicity assay and tumor cell growth inhibition tests,we evaluated the tumor cell proliferation and growth viability.Combined with the clonal formation assay and wound scratch assay,we further evaluated the tumor cell invasion ability.In order to verify the abnormal expression of Claudin 3 and Claudin4 impacts on the downstream signaling pathways,we tested the activation of signaling pathways and the expression levels of related proteins.Based on the results of the experiments,we revealed the function of Claudin 3 and Claudin 4 in metastasis of prostate cancer.Results1,By knockouting Claudin 3 and Claudin 4,we could obviously inhibited the tumor cell proliferation and growth viability,showing that it could restrict the metastasis of tumor cells.2,By knockouting Claudin 3 and Claudin 4,we could obviously inhibited the tumor cell clonal cluster formation and migration activity,showing that it could inhibit the metastasis of tumor cells.3,In the short term,by knockouting Claudin 3 and Claudin 4,we could inhibited the expression of the Bcl-xl and VEGFR2 in signaling pathways,showing that it could inhibit the metastasis by promoting tumor cell apoptosis and anti-tumor angiogenesis.4,In the long term,by knockouting Claudin 3 and Claudin 4,we could inhibited the expression of the GP130 and VEGFR2 in signaling pathways,showing that it could inhibit the metastasis by inhibiting tumor cell proliferation and anti-tumor angiogenesis.ConclusionOur results showed that the abnormal expression of Claudin 3 and Claudin 4 in prostate cancer can lead to the occurrence of tumor metastasis.By knockouting Claudin 3 and Claudin 4,we could inhibited the tumor cell growth viability,proliferation and invasive ability,and affected the expression of proteins in downstream signaling pathways,showing it could inhibit the tumor metastasis and providing therapeutic targets for the treatment of prostate cancer.