Target Gene Sequence Of Prostate Cancer Samples In Chinese Popolations And The Mechanism Of Recurrent Mutation Gene KMT2D In Prostate Cancer Progression

Background and objectiveProstate cancer(PCa)is the second malignancies in men worldwide,threating the health if older men.The gene sequencing researchs found that there were lots of gene mutations and copy number varients in prostate caner samples.These indicate that genome alternations are important mechanism in the development of prostate cancer.Despite the recent generation of massive amount of sequencing data on prostate cancer,most of them are restricted in Western populations and few functional analyses by cell culture or animal models are performed,which are the main limitations and chanlleges in prostate cancer genomic studies.The histone lysine(K)methylation transferase KMT2D(also known as MLL2/ALR/MLL4)is a part of the KMT2 family,acting as catalytic subunits of mammalian COMPASS-like complexes to catalyze the formation of H3K4mel.The H3K4mel is found enrichment at enhancers and considered a principal mark of active enhancers.Through these activated enhancers,cells can dynamically regulate their gene expression responding to developmental,hormonal and other signals based on transcription factor availability.Recent cancer genetic researches have uncovered frequent somatic mutation in KMT2D in variety of cancer types.The role of KMT2D in cnacers have shown to be cancer type-dependment.The KMT2D has been shown to exhibit tumor suppressor as well as oncogenic properties.In prostate cancer,KMT2D is one of recurrent mutation genes and mutation in KMT2D is only found in prostate cancer,not in high grade prostatic intraepithelial neoplasia(HGPIN).For the high mutation rate and "PCa-specific",KMT2D is suggested as potential "driver" alteration,highlighting an urgent need to understand its mechanism in the progression of PCa.1.Target gene sequence of prostate cancer samples in Chinese populationsTo understand the mutation profile in Chinese prostate cancer patients,we performed the target next generation gene sequencing with a panel of 32 high mutation genes in prostate cancer,along with the expression of 25 prostate cancer related genes detected by qRT-PCR.The results showed the distinct mutation spectrum in Chinese prostate cancer patients,while KMT2D,AKAP9,GLI1 was the significant recurrent mutation genes in our sequencing study.Among them,KMT2D was the most mutated gene and the mutation of KMT2D associated with high mRNA level due to the linkage disequilibrium.In addition,combining with qRT-PCR analysis,KMT2D expression correlated with AR signaling and cell cycle progression.2.The expression level and clinical correlation of KMT2D in prostate cancerBesides gene mutation,the expression level is also an essential aspect to explore the relationship between KMT2D and cancer.Higher protein expression level in tumor is one of characteristics of oncogenes.Therefore,we used another cohort of 51 prostate cancer and 3 BPH samples to verify its protein level by IHC.The results showed that the expression of KMT2D was significantly higher in prostate cancer than BPH cases and high level of KMT2D indicated poor prognosis.Moreover,the expression level and clinical correlation of KMT2D also were analyzed by oncomine and cBioportal online platform.The results indicated that KMT2D mRNA level was higher in prostate cancer tissues and positively correlated with progression-free survival and disease aggression.3.The biological relevance analysis in prostate cancer cell lines with KMT2D knockdownTo investigate the mechanism of KMT2D in prostate cancer,we used siRNA to build a KMT2D-silenced cell model for analyzing the function changes in prostate cancer cells when KMT2D deficiency.The results of MTT assay and EdU cell proliferation detectation showed cell proliferation was reduced after KMT2D silenced.The cell cycle and apoptosis analysis by flow cytometer reveled G2/M block and significantly higher percentage of apoptosis cells with KMT2D deficiency.These indicate KMT2D play an important role in the maintance of cell proliferation,while the loss of it would cause cell cycle block and apoptosis.The enhanced migration and invasion capacity promote tumor metastasis.We found the reducation of cell migration and invasion by wound healing test and Transwell assay in KMT2D-silenced group.The immunofluorescence staining also revealed the high expression of E-cadherin and low expression of N-cadherin in KMT2D silenced cells.Because of these,KMT2D contributes to the process of tumor metastasis.The loss of it attenuates the abilities of migration and invasion in tumor cells.DNA instability is one of reasons caused genomic mutation and causes cancer.Single cell gel electrophoresis,immunofluorescence staining and western blot were combined to detect DNA damage level after KMT2D silencing.The results provided compelling evidence that KMT2D deficiency results in DNA damage.Recent study suggest that DNA damage is one of the important triggers of cellular senescence.We found loss of KMT2D resulted in cell senescence in prostate cancer cells by SA-β-Gal assays.So the deficiency of KMT2D induces DNA danmage and promotes cell senescence.Above all,KMT2D play important role in the maintaining of tumor cell proliferation,migration,and invasion.It also participate in the process of anti-apoptosis and DNA damage repair.KMT2D may act as an oncogene to promote prostate cancer progression.4.The biological functional analysis in vivo with KMT2D knockdownTo explore the effect of tumor growth in vivo after KMT2D silencing,we used xenograft tumor model.As expected,the tumor groth were slower in KMT2D silenced group.Therefore,KMT2D is a critical gene associated with tumor groth.For the deficiency of KMT2D significantly inhibits tumor groth in vivo,it may be a potential target for prostate cancer therapy.5.The downstream target of KMT2D in prostate cancerThe immunofluorescence staining of H3K4mel revealed that the the loss of KMT2D decreased the total level of H3K4mel.As H3K4mel is marker of enhancer and could regulate the transcriptional activity,we performed RNA-seq to analyze the transcriptional changes after KMT2D silencing.The results showed significantly clustered of PI3K-Akt pathway by KEGG.The phosphorylation of Akt was reduced with KMT2D deficiency.In addition,the ChIP-seq of H3K4mel was performed to explore the direct target of KMT2D.Two siginifant genes LIFR,upstream regulater of PI3K-Akt and KLF4,transcription factor controls EMT related genes were selected.The downregulation of gene expression of LIFR and KLF4 was also confirmed by qRT-PCR,indicating the LIFR and KLF4 were direct target of KMT2D.Futhermore KMT2D mRNA level was positively correlated with that of LIFR and KLF4.Together,we conlunde KMT2D can regulate the level of H3K4mel of LIFR and KLF4,modulating their gene expression,which finally influence the PI3K-Akt pathway and EMT related genes and contributes to tumor growth and metastasis.