Exploring The Mechanism Of Action Of M6A Methyltransferase WTAP In Castration-resistant Prostate Cance

Objective(s)Castration-resistant prostate cancer(CRPC)is the most malignant and prognostic prostate cancer disease.N6 methyladenine(m6A)modification is one of the most common RNA modifications,which is involved in the occurrence and development of a variety of malignant diseases.The main objective of this study was to prepare androgen-resistant cell lines(Lncap AI)based on androgen-dependent cell lines(Lncap AD),to simulate clinical conditions,and to explore biological behavioral differences between these two cell lines.Find out the key m6 A that plays a regulatory role between androgen dependent cell line(Lncap AD)and androgen resistant cell line(Lncap AI).Method(s)The Lncap AD cell line was cultured and passaged 60 times among ten months.The morphology of the Lncap AI cell line was observed by light microscopy and HE staining.AR levels identification were detected in q RT-PCR and Western Blot assay.Cell counting kit8(CCK-8),5-ethynyl-2’-deoxyuridine(Ed U)assay,wound healing assay and cell adhesion assays were used to observe the ability of proliferation,migration and adhesion.Scanning Electron Microscopy(SEM)and Transition Electron Microscopy(TEM)were used to observe microculture structure.The PSA secrete ability was evaluated by Elisa assay.At last,the CCLE database combined with q RT-PCR was used to detect the changes of adhesion pathway related markers.CCLE database combined with q RT-PCR assay and Western blot assay were used to find out the difference of m6 A methylated methyltransferases(METTL3,METTL14,WTAP),demethylase(FTO,ALKBH5)and methylated reading protein(YTHDF1,YTHDF2,YTHDF3,YTHDC1,YTHDC2)between Lncap androgen dependent cell line(Lncap AD)and Lncap androgen resistant cell line(Lncap AI).Dot blot was used to detect the changes of total m6 A methylation modification level after Lncap AD,knockdown of WTAP(Lncap ADsi WTAP)and Lncap AI.TCGA data was used to analysis the differences of WTAP expression value,TNM stage,Gleason score,survival analysis and immune infiltration score between prostate cancer disease and normal group.Finally,the proliferation of prostate cancer cells was observed by CCK-8 assay,Edu assay,cell clone assay and drug resistance of prostate cancer cells was observed by IC-50 value.Me RIP-seq technology was used to detect the methylation level of Lncap androgen dependent cell line(Lncap AD),Lncap androgen resistant cell line(Lncap AI)and Lncap androgen dependent cell line silenced WTAP(Lncap ADsi WTAP).Detect the sequencing sequence statistics and quality control,reference genome alignment reads statistics,reference genome alignment regional distribution,whole genome peak scanning,and the gene distribution of reads.Take Lncap AD cell line as a reference,compare the differences between Lncap AI and Lncap ASsi WTAP by peak analysis,motif analysis gene/ transcript overall result analysis and differential gene and differential peak association analysis.The methylation levels of LNCa P androgen dependent cell line(LNCa P AD),LNCa P androgen resistant cell line(Lncap AI),LNCa P androgen dependent cell line silencing WTAP(Lncap ADsi WTAP)were detected by m6 A SEQ method.With LNCa P ad as the reference,the RNA SEQ and m6 A SEQ differential genes between Lncap AI vs Lncap AD,Lncap ADsi WTAP vs Lncap AD were analyzed by four quadrant diagram,Further,under the condition of | logfc | > 1,p.adjust < 0.05,the differential genes were screened,and the AR(androgen resistant)related gene set was searched by Genecards database.Wayne diagram intersected the AR gene set with Lncap AI vs Lncap AD,Lncap ADsi WTAP vs Lncap AD,looking for the candidate target genes related to AR receptor regulated by WTAP from androgen sensitivity to androgen resistance,QPCR and Western blot experiments were used to jointly verify the expression at the m RNA and protein levels.Explore the GGAC region of NR3C1,design primers respectively,and verify it with me RIP-q PCR to find the sequence with potential binding between the target gene and WTAP.RIP-q PCR was further used to verify the binding ability of Lncap AD and Lncap AI cell lines stably overexpressing WTAP and WTAP in empty group to target genes.Then,the stability of WTAP protein was analyzed by protease inhibitor CHX.Finally,the effects of WTAP/target gene/reading protein on the cell biological behavior(proliferation and drug resistance)of prostate cancer cell lines were analyzed to explore whether the expression changes between WTAP/target gene/reading protein axes are the key factors affecting the cell biological behavior(proliferation and drug resistance).Result(s)The Lncap AD cell line was cultured and passaged 60 times among 16 months.The Lncap AI cell line showed a morphologic change at the end stage of culture,the cells turned slender and cell space turned separated compared to the Lncap AD cell line.Compared with the Lncap AD cell line,the Lncap AI cell line can observe obvious morphological changes in culture,such as elongated cell shape profile,cytoplasmic richness,good cell light transmittance,larger cell-to-cell gap,colony-like growth,and obvious inter-colony space.The natural androgen-resistant cell line PC3 grows in a tile-like pattern,with vigorous cell division and proliferation,strong vitality and less cytoplasm.The m RNA and protein levels of the Lncap AI cell line AR are higher than those of the Lncap AD cell line.Compared with Lncap AD cell lines,Lncap AI cell lines showed strong proliferation,migration and adhesion capabilities in CCK-8 experiments,Edu experiments,scratch healing experiments and adhesion experiments.The cell membrane surface structure of the scanning electron microscope(SEM)shows the presence of many cilia in the Lncap AI cell line and PC3 cell line,while the smooth surface of the Lncap AD cell line is not.Transmission electron microscopy(TEM)can observe pseudopodoid structures.The Elisa method detected the ability of prostate cancer cell lines to secrete PSA,and there was no significant difference(p>0.05)between the three prostate cancer cell lines.The CCLE database combines q RT-PCR to verify adhesion signaling pathway-related molecules(L1CAM,ICAM1).The CCLE database was used to compare the expression patterns of 10m6A-related enzymes in prostate cancer cell lines(DU-145,PC-3)with those in the normal prostate epithelial cell line(RWPE-1),and four WREs(WTAP,FTO,ALKBH5,YTHDF2)were downregulated.Among them,the m RNA and protein levels of WTAP were different in the three CRPC cell lines,and the trend was the same in Lncap AI and PC3 cell lines,which was opposite to that in Lncap AD.Compared with normal prostate tissue,the overall m6 A level of prostate cancer samples of TRAMP mice were higher,and the overall m6 A level increased with the increase of weeks of age of TRAMP mice.The same results were obtained in the observation of overall m6 A level in dot blot assay,and the overall m6 A level decreased significantly after knocking down WTAP in Lncap AD cells.WTAP can play an important role in clinical staging of prostate cancer;WTAP also had significant expression differences in Gleason score,and the survival rate of patients with high Gleason score was lower by TCGA database.Overexpression of WTAP resulted in higher proliferation than the control in three CRPC cell lines(Lncap AD,Lncap AI and PC3),while silencing of WTAP showed the opposite results by CCK8,colony formation,and Ed U assays.In the analysis of WTAP and cell drug resistance,silencing WTAP led to the enhancement of cell drug resistance,while overexpression of WTAP led to the weakening of drug resistance.The regional distribution of reference genome alignment showed that the percentage of sequencing sequence in exon region was the highest.Motif analysis showed that GGAC was m6 A modified RRAC region.Compared with androgen sensitive cell line Lncap AD,the distribution of motif decreased significantly in androgen resistant cell line Lncap AI by silence WTAP cell line Lncap ADsi WTAP group.The overall transcript expression density map of gene/transcript overall result analysis shows that the overall transcript expression sites of the three groups of samples are consistent,and the overall transcript expression box diagram shows that the median of the three groups of samples is in a straight line.RNA-seq and me RIP-seq analysis of differential genes and differential peak results showed that in Lncap AI vs Lncap AD group,a total of 2702 genes m6 A peak and m RNA expression were up-regulated in hyper up;There were 3298 genes in hyper down,m6 A peak was up-regulated and m RNA expression was down regulated;There were 3093 genes in hypo up,m6 A peak was down-regulated and m RNA expression was up-regulated;There were2590 genes in hypo down,m6 A peak and m RNA expression were down regulated.In Lncap ADsi WTAP vs Lncap AD group,a total of 2450 genes m6 A peak and m RNA expression were up-regulated in hyper up;There were 5188 genes in hyper down,m6 A peak was up-regulated and m RNA expression was down regulated;There were 610 genes in hypo up,m6 A peak was down-regulated and m RNA expression was up-regulated;There were 1060 genes in hypo down,m6 A peak and m RNA expression were down regulated.The differential genes of Lncap ADsi WTAP vs Lncap AD and Lncap AI vs Lncap AD are associated with differential peak.GO and KEGG enrichment analysis.The common GO enrichment includes 17 Biological process(BP),12 Cellular component(CC)and 7 Molecular function(MF)and 2 KEGG signal pathway.Using the androgen related dataset from Genecards database,the intersection with Lncap AI vs Lncap AD,Lncap ADsi WTAP vs Lncap AD was taken,and a total of 16 differential intersection genes were obtained.Six genes with consistent expression in the three groups of biological repeats(adra2c,NR3C1,CD83,gatad2 b,PDP1 and Rac3)were selected for verification.The NR3C1 gene was finally determined by q PCR combined with Western blot,which changed with the expression of WTAP.The m6 A abundance map of NR3C1 was drawn by IGV software,seven GGAC sites on the sense chain were found,and primers containing only one GGAC region were designed respectively.Using the me RIP-q PCR method,it was found that the fourth pair of primers in GGAC had significant differences between IP group and input group.Rip q PCR experiment confirmed that WTAP could bind to nrc3c1.In Lncap AD and Lncap AI cell lines,control cells or cells overexpressing WTAP were treated with cycloheximide(CHX),and the expression of WTAP decreased with the increase of CHX treatment time.Another important difference gene found in me RIP-seq and RNA-seq analysis in CRPC cell line is YTHDF2.CCK8,clone formation and drug resistance analysis showed that YTHDF2 affected the progress of CRPC through cell proliferation and drug resistance.QRT-PCR experiments were used to detect the m RNA level of NR3C1 in two cell lines,and in both strains,overexpressed WTAP and NR3C1 expression increased,while after stable overexpression of WTAP,silencing NR3C1 could reverse the expression of the above three genes and return to near the original level.This shows that NR3C1 is indeed a target gene for WTAP modifications to play a role.Drug resistance experiments confirmed that WTAP regulates the resistance of CRPC cells by regulating NR3C1.WTAP affects the disease progression of CRPC through AR signaling pathway.HPA database found that the protein expression of WTAP,NR3C1 and ythdf2 in low stage group was lower,while the protein expression of WTAP,NR3C1 and ythdf2 in high stage group was higher.This indicates the co carcinogenic effect of WTAP,NR3C1 and YTHDF2 in prostate cancer samples.Conclusion(s)Simulation of CRPC progression,Lncap AD cell line turned to Lncap AI cell line with androgen deprivation therapy(ADT)and sustained Flutamide stimulation.M6 A methyltransferase WTAP may be involved in the occurrence and development in the process from androgen sensitivity to androgen resistance.Our findings enrich the landscape of m6A-modulated tumor malignancy and provide new insights into potential biomarkers and therapeutic targets of Lncap AI treatment.