Prostate Cancer Metastasis Associated DNA Methylation Regulated Candidate Genes And Clinical Values Evaluations

Part â…  Comparison of migration and invasion capability in prostate cancer cell lines PC-3 and LNCaPPurpose:Aim to find the most invasive prostate cancer cell line by testing the invasion and migration capability and make a foundation for searching metastasis tumor markers. Materials and Methods:Scratch healing assay and Boyden Chamber Transwell assay were conducted in cultured prostate cancer cell line LNCaP,22RV1, PC-3 and DU145 in vitro. The migration and invasion capability of the four cell lines were compared. Gelatin zymography was used to compare the secretion of MMPs. Results:Through Screening, we found the most invasive PC-3 cells and least invasive LNCaP cells. In scratch healing assay, PC-3 migrated more quickly than LNCaP (7.31 times, p<0.05). In Transwell assay, PC-3 had 3.38 times more invasive cells than LNCaP at the same seeding concentration and same time (p<0.05). And the MMPs were significantly higher in PC-3 than LNCaP in gelatin zymography. Conclusions: PC-3 had more invasiveness capability and higher MMPs level than LNCaP in vitro.Part â…¡ MBD-seq and RNA seq screening of diversity genes regulated by DNA methylation and validationPurpose:Aim to find the methylation regulated genes through diversities in DNA methylome and transcriptome of LNCaP and PC-3. Materials and Methods:By second generation sequencing, we examined the MBD-seq and RNA-seq of PC-3 and LNCaP cells. We further analyzed the data and found out methylation regulated and differently expressed genes in PC-3 and LNCaP. Results:Though high input screening, we found 14 genes differently expression between LNCaP and PC-3 (CAV1, CAV2, MET, PPARG, COL6A1, ALDH6A1, ALDH2, SMAD3, ITGA6, ITGB8, MCCC1, CAPN2, JAK1, LAMB1) and regulated by DNA methylation. They probably caused the invasiveness diversity in LNCaP and PC-3. We validated nine gene of them through qRT-PCR and Western blot. Results:By high input screening, we could easily find out the different expression genes regulated by DNA methylation.Part â…¢ Candidate gene COL6A1 and functional study in vitroPurpose:Further validation functions in vitro of methylation regulated COL6A1 gene. Material and methods:Construct overexpression and shRNA knock down lentivirus vectors of COL6A1. After packing of the lentiviruses, high COL6A1 expresser PC-3 cells were knocking down and low expresser LNCaP were overexpressed. Tranwell assay and wound healing assay were conducted to examine the functional change after altered COL6A1 expression level. We further analyzed the downstream signal pathway by western blot. Results:LNCaP cells with Overexpressed COL6A1 gained 50% additional migration rate (p<0.05), and 78.1% more invasive cell numbers (p<0.05) with higher level of MMP9 and CXCR4 expression. In COL6A1 knock down PC-3 cells, migration rate was 59.4% decreased than control group and invasive cells was 66.2%(p<0.05) relative to control. Conclusions:COL6A1 could promote the invasiveness in LNCaP and PC-3 cells in vitro.Part â…£ Clinical value of candidate gene COL6A1Purpose:To examine the clinical value of COL6A1 in prostate cancer specimen. Materials and Methods:We retrospectively collected 223 prostate specimens from prostate cancer patients treated in Fudan University Shanghai Cancer Center from 2007 to 2011 and 40 non-cancerous prostate controls from the same period of time. Clinical data were retrospectively collected from electrical database. COL6A1 expression level was examined by immunohistological chemistry and the clinical relevance of COL6A1 was analyzed. Results:In multivariate regression model, high Gleason score (>=8) (OR=4.204,95%CI=1.360 to 12.999, p= 0.013), high T stage (>=3)(OR=43.912,95% CI=12.340 to 156.263, p...