The Study On The Biological Function Of Long Noncoding RNA Linc00844 In Prostate Cancer

BackgroundProstate cancer(PCA)is the one of the most common cancers and the second leading cause of cancer-related deaths in men in western countries.The pathogenesis of PCA has obvious geographical and ethnic differences and the incidence rate in China was significantly lower than that in western countries.But with the aging of our population and the improvement of living conditions,the incidence rate of PCA was significantly increased in China.PCA is an androgen dependent malignant tumor.Androgen plays an important role in PCA growth,metastasis and differentiation.The androgen receptor(AR)signaling pathway has greatly improved our understanding of PCA,which led to the successful development of androgen deprivation therapy in the treatment of PCA.However,after a period time of androgen deprivation therapy,patients with PCA usually become less sensitive to the treatment,and then transformed into androgen independent PCA,eventually leading to metastasis and death.Therefore,the mechanism of AR signaling pathway can't fully explain the occurrence and progression of PCA,which indicates that there are other mechanisms led to the development of PCA.Therefore,the research on pathogenesis and treatment of PCA remains to be further studied.Since the development of androgen deprivation therapy,the study of prostate cancer has entered a bottleneck.Studies have shown that,in addition to the androgen receptor signaling pathway,PI3K/Akt and other signaling cascades are also involved in prostate cancer formation,but effective therapies have not been found in these studies.As the discovery of catalytic RNA in the early 80s,researchers realize that RNAs is not only the intermediate between DNA and protein molecules,but a dynamic,multifunction molecule.RNAs regulate a variety of cellular processes,including cancer.The findings eventually led to the emergence of RNA based therapies,which greatly broadened the scope of drug development.At present,our understanding on the occurrence and development of PCA is mainly based on the research of protein encoding gene.Long noncoding RNA(lncRNA)is a kind of noncoding RNA with a length of more than 200 nucleotides,lncRNAs are from genomic DNA by transcription and splicing,but their functions are poorly understood.In recent years,more and more studies show that lncRNAs are involved in many cellular activities,including embryonic development,cell growth,differentiation and metabolism.In addition,the study found that a variety of IncRNAs in patients with PCA showed that the abnormal expression of lncRNAs may be involved in the occurrence and development of PCA,such as lncRNA PCA3(prostate cancer antigen 3),which can be detected in benign and malignant PCA in urine,is significantly increased in about 95%of patients with PCA.In addition,PCAT-1 is highly expressed in metastatic prostate cancer and has a high degree of tissue-specific carcinoma.PCGEM1(prostate cancer gene expression marker 1)is increased in PCA and can promote the proliferation of tumor cells and the formation of community.More and more studies show that lncRNAs play a very important role in the occurrence and development of cancer.In order to further clarify the regulatory mechanism of lncRNA in PCA and find the effective clinical diagnostic markers and potential therapeutic target in PCA,we performed the high-throughput sequencing of lncRNA in PCA.The results showed that lncRNA linc00844 had an abnormal expression in PCA.However,the relationship between linc00844 and the development of PCA and its potential regulatory mechanism is still not clear.In order to elucidate the relationship between linc00844 and PCA and its potential biological function,we detected the clinical specimens and performed the cell biological function study on linc00844.ObjectiveThe study of the pathogenesis and clinical diagnosis and treatment of prostate cancer is a hot topic in cancer research.In the early stage,we found that the expression of IncRNA linc00844 in prostate cancer tissues was significantly lower,suggesting that linc00844 is associated with prostate cancer,but the specific correlation and regulatory mechanism are unknown.The purpose of this study is to elucidate the biological function of linc00844 and its clinical correlation with prostate cancer and expression regulation mechanism and to look for the prostate cancer clinical diagnosis and prognosis of effective markers and therapeutic targets,providing a new theoretical basis and the development of prostate cancer drugs.Methods1.real-time quantitative PCR analysis to detect the expression of linc00844 andmRNA in prostate cancer tissues and cells;2.to analyze the relationship between the expression of linc00844 and the clinicopathological parameters of prostate cancer patients by TCGA and Taylor prostate cancer database analysis and gene cluster enrichment analysis;3.molecular cloning method was used to construct linc00844 overexpression vector to transfect prostate cancer cells.The effect of linc00844 on invasion and migration of prostate cancer cells was observed by transwell assay;4.using TCGA prostate cancer database to analyze the survival curve,and to observe the correlation between the expression level of linc00844 and the prognosis of patients with prostate cancer;5.androgen was used to stimulate prostate cancer cells and real-time quantitative PCR was used to detect whether androgen signaling pathway can regulate the expression of linc00844.Results(1)the expression of linc00844 in PCA:real-time fluorescence quantitative PCR(qRT-PCR)to detect the expression of linc00844 in PCA tissues and normal tissues.The results showed that compared with normal tissues,the expression of linc00844 in PCA tissue was significantly lower.Furthermore,the use of high-throughput sequencing data of TCGA and Taylor database also showed that linc00844 levels were significantly lower in PCA tissue,suggesting that linc00844 may be a potential PCA suppressor gene.(2)the relationship between linc00844 and PCA in clinical pathology:the clinical specimens of PCA were divided into two groups based on Gleason scores:Gleason>7 group and Gleason?7.Then the expression of linc00844 was detected by qRT-PCR in different groups.The results showed that the expression level of linc00844 in Gleason>7 group was significantly lower than that in Gleason?7 group.In addition,the results of high-throughput sequencing data in TCGA and Taylor database also showed a lower expression of linc00844 in patients with a higher Gleason score in PCA.(3)Using an alternate way to further characterize the clinical associations of linc00844:we performed Gene Set Enrichment Analysis(GSEA)using 6 datasets generated from 3 independent high-throughput sequencing studies.As expected,genes down-regulated in metastatic PCA samples,in TMPRSS2-ERG fusion positive tumors,and in PCA samples were significantly enriched in the linc00844 high expression group of patients,indicating a potential tumor suppressive role of linc00844.(4)the relationship between linc00844 expression and survival:the PCA patients were divided into two groups,high linc00844 expression and low linc00844 expression groups.Then the patients were followed up.The results showed that the relapse free survival and overall survival in PCA patients with high linc00844 expression group were significantly higher than those in patients with low linc00844 expression group.(5)Role of linc00844 on invasion and metastasis of PCA cells:we transfected the PCA cell line PC3 and DU 145 cells with a linc00844 overexpression vector.A significant decrease in cell migration and invasion was noted in both PC3 and DU 145 cells when linc00844 was overexpressed.(6)Regulation of the expression of linc00844 by the AR signaling:To query the effect of AR signaling on linc00844 expression,we first utilize the AR ChIP-sequencing data in VCaP cells,and found a significant AR peak in the linc00844 locus upon treatment with dihydrotestosterone(DHT).And more importantly,the peak decreased upon the treatment with AR antagonist bioclutamide,JQ1 or MDV3100.We further performed DHT stimulation experiment in VCaP cells,the results showed that linc00844 is significantly up-regulated in a time-dependent manner and this up-regulation could be reversed by MDV3100 treatment.Conclusion1.IncRNA linc00844 is a potential prostate cancer suppressor gene that inhibits the invasion and migration of prostate cancer cells;2.The expression of linc00844 is associated with the prognosis of patients with prostate cancer,and has the potential to be a molecular marker and therapeutic target for prostate cancer patients.