Role Of Long Non-coding RNA LncAPP In Prostate Cancer Progression

Background:Prostate cancer is second leading in male cancer rank and sixth in cancer mortality.The incidence in China has been rising rapidly during last two decades.And when prostate cancer diagnosed in China,more than half of the patients' clinical stage are advanced or metastases.Different from western countries,most of prostate cancer confirmed are at early stage.This is partly due to lack of PSA screening in Chinese population,and genetic vairance among races may also play a vital role in it.According to National Cancer Center SEER(Surveillance Epidemiology and End Results)Database,five-year survival rate of localized prostate cancer is nearly 100%,compared to only 29.3% of metastatic prostate cancer.Therefore,it is urgent and of great meaning to explore early prostate cancer diagnosis biomarker for Chinese population.Researches on human genomics and trancriptomics have revealed that only 19,000 genes with protein coding ability accounting just 1-2% of the whole genome,while rest 98-99% are dark matters-non-coding transcripts.With great success application of next generation sequening(NGS),long non-coding RNAs which were regarded as transcript noise now show an important role in biological process.LncRNAs could regulate gene expression at epigenetic,transcriptional and post-transcriptional levels,bind proteins to affect its function,and excert oncogenic or tumor suppressive function during tumor development,progression and metastasis.Moreover,large numbers of lncRNAs are found to be a biomarker for prostate cancer diagnosis and molecular typing,and few of them are related to prostate cancer metastasis.Preliminary research laid a solid foundation of prostate cancer tissues NGS in Chinese population.Hence,we sought to elucidate molecular mechanisms of prostate cancer progression and metastasis.Purpose:To identify novel prostate cancer associated lncRNAs based on NGS transcriptomics data of 65 paired tumor tissues and adjacent normal prostate tissues,explore diagnostic value of lncRNAs,and investigate its function and molecular mechanism in tumor progression and metastasis.Methods:PCa associated lncRNA transcripts from RNA-seq data were identified and screened via bioinformatics analysis,ncbi annotations and literature review.LncRNAs were selected and validated in clinical urine samples.A total of 291 patients with suspected prostate cancer were enrolled in Changhai hospital.The predictive values of lncAPP score and PCA3 score were evaluated by receiver operation curve,diagnostic model construction via logistic regression,decision cure analysis and a biopsy-specific nomogram.Nuclear and cytoplasmic RNA separation assays,RNA FISH were performed to explore subcellular location of lncAPP.CCK-8proliferation assay,transwell migration and invasion assays,flow cytometry cell cycle assays and cell apoptosis assays,and colony formation assays were detected with ectopic overexpression and knocking down of lncAPP.In vivo subcutaneous tumor xenograft models and tail intravenously injection metastatic models were constructed to assess biological function of lnxLOX5-1.Using miRDB and Target Scan database to predict possible miRNAs and their binding sites to lncAPP,MS2-RIP,Ago2-RIP and dual luciferase reporter gene system were conducted to validate lncAPP competitively binding to miRNAs.Target genes of miRNAs were also verified by lncAPP/miRNA regulation.Micro-array chips were used to analyze changes of transcripts via exogenous over-expression and knocking down of lncAPP in PC3 cell lines,and differentially expressed genes and key signal pathways were screened out.Results:Herein,we identify lncAPP from differently expressed lncRNAs according to classification of Gleason score,clinical stage and metastasis.The diagnostic accuracy of lncAPP as potential urinary biomarker was evaluated,and lncAPP score were sufficient to discriminate positive and negative biopsies.Comprehensive model based on clinical parameters plus lncAPP score could significantly improve diagnostic performance of PCa.Urinary based lncAPP is a promising novel biomarker for early detection of PCa,especially in PSA 4-10 ng/mL cohort.Gain and loss of function assay in prostate cancer cells demonstrated that lncAPP involved in maintaining prostate cancer growth,a feature specific to cancer cells.In vitro study of CCK8assays,transwell migration and invasion assays verified that lncAPP enhanced proliferation ability and promoted cell migration and invasion.In vivo study of subcutaneous tumor xenograft model and tail intravenously injection metastatic model validated that lncAPP could enhance tumor cell proliferation and metastases,and siRNA target lncAPP could abolish tumor growth and metastases.Through miRDB and Target Scan database,potential miRNAs whose sequence could match to lncAPP were predicted.Expression levels of lncAPP and predicted miRNAs were detected with ectopic overexpression or knockdown of lncAPP expression levels.MS2-RIP,Ago2-RIP and dual luciferase reporter systems were constructed to confirm that lncAPP could bind miR218/miR646 to exert biological functions.Rescue experiments proved that lncAPP promote cell migration and invasion via competitively binding miR218/miR-646 to facilitate down-stream genes expression in prostate cancer cells.Conclusion:Urinary based lncAPP score may serve as a prostate cancer diagnosis biomarker and were sufficed to discriminate positive from negative biopsies.LncAPP is essential for maintaining tumor growth through inhibiting miRNA218/646 mediated downstream genes expression levels changes.In vivo study suggested that lncAPP could be a potential therapeutic target for cancer progression and metastases.