Regulation Of Parent Genes By BCL2L2-PABPN1 Readthrough And Its Roles In Prostate Cancer

Background and objective:Prostate cancer is one of the most common malignant tumors in the male population.According to the statistics of International Agency for Research on Cancer(IARC),there were more than 1.4 million new cases and estimated 375,000 deaths worldwide in 2020.The incidence and detection rate of prostate cancer are increasing in China.Although most patients can be relieved by treatment with drugs or surgery,disease progression into castration-resistant prostate cancer results in poor prognosis.Studies on molecular mechanisms of prostate cancer tumorigenesis and progression are important for improving diagnosis and treatment.Transcriptional readthrough results from transcription beyond stop signal of one gene and continued transcription of the downstream gene,which yields chimeric transcripts(also called conjoined genes).The parent genes are usually adjacent genes located on the same chromosome in the same transcription direction.Some readthrough transcripts can encode new proteins.However,little is known about the regulatory mechanisms,biological functions and the relationships of these readthrough transcripts with disease processes,especially in tumors.High-throughput gene sequencing has shown that there are many readthrough transcripts in prostate cancer,but their expression and roles remain unclear.We have identified the overexpression of BCL2L2-PABPN1 readthrough transcripts in prostate cancer tissues by RNA sequencing.BCL2L2-PABPN1(BP)readthrough transcripts are produced by transcriptional readthrough of BCL2L2(B cell lymphoma-2 like 2)gene and its downstream adjacent gene PABPN1(poly(A)binding protein nuclear 1).The parent gene BCL2L2,a member of the BCL2 family,is expressed in various tissues and is involved in cell proliferation,differentiation,and apoptosis.PABPN1 encodes PABPN1 protein which is expressed in the nucleus and has a variety of biological functions.Abnormal expression of PABPN1 has been associated with various human diseases.However,the expression,localization and function of BP readthrough transcripts in prostate cancer have not been reported.In the present study,we aim to(1)identify and analyze the BP readthrough transcripts in prostate cancer;(2)explore the regulatory mechanism of BP readthrough transcripts on parent genes;(3)investigate the effects of BP readthrough transcripts on the biological behavior of prostate cancer cells;and(4)analyze the expression of BP readthrough transcripts and clinical significance in prostate cancer.Materials and Methods:1.Bioinformatics analysis,reverse transcription polymerase chain reaction(RTPCR),Sanger sequencing and rapid amplification of cDNA ends(RACE)were used to identify and characterize the BP readthrough transcripts in prostate cancer cells.2.Flag sequence was added to the downstream of open reading frame(ORF)of BP readthrough transcripts.Expression plasmids,including pc-Flag,pc-BP-Flag and pc-BPmut-Flag(in which the start codon of ORF was mutated)were constructed.3.Nuclear/cytoplasmic separation,Western blot,immunocytochemistry and immunofluorescence were used to examine the expression and subcellular localization of BP readthrough proteins in prostate cancer cells.4.RT-PCR and quantitative PCR(Q-PCR)were used to examine the expression of BP readthrough transcripts and their parent genes,BCL2L2 and PABPN1,in fresh prostate cancer(n=14)and benign prostate hyperplasia(n=8)tissue specimens.5.Small interfering RNA(siRNA)was designed according to the junction sequence of BP readthrought transcripts,and artificial overexpression plasmids of BP readthrough transcripts were constructed.Expression of parent genes were analyzed by PCR and Western blot,with or without siRNA treatment.6.MicroRNAs(miRNAs)that may potentially interact with both BP readthrough transcripts and BCL2L2 were analyzed by bioinformatics tools.7.Luciferase reporter gene plasmids that contained the miRNA binding sequences or their mutated sequences in BCL2L2-3’ UTR or BP readthrough transcripts were constructed.8.Biotin-labeled oligonucleotide probes were designed according to the junction sequence of BP readthrought transcripts.9.Dual luciferase reporter gene assays,RNA dot hybridization,and RNA immunoprecipitation were used to analyze effects of BP readthrough transcripts on parent genes,to elucidate the competitive endogenous RNA(ceRNA)mechanisms by which BP transcripts inhibit miRNA and promote expression of BCL2L2.10.CCK-8,EdU assays,clone formation assay,wound scratch assays,transwell cell migration and invasion assays were used to examine the effects of BP readthrough transcripts on the proliferation,invasion and migration of prostate cancer cell lines.11.Archived tissue samples of prostate cancer(n=60)and benign prostate hyperplasia cases(n=25)diagnosed in our hospital in 2017 were collected,and the expression of BP readthrough transcripts were examined by in situ hybridization.12.Relationship between BP readthrough transcripts expression level and patient age,PSA level,Gleason score,WHO/ISUP prognostic grade group,tumor stage and prognosis was analyzed.Results:1.Identification and characterization of BCL2L2-PABPN1 readthrough transcripts devived from BCL2L2 and PABPN1,and pBP readthrough proteins.1)Three BCL2L2-PABPN1 readthrough transcripts were identified in prostate tumor cells.BCL2L2-PABPN1-201/202(BP1)contained the same ORF(1002bp).BCL2L2-PABPN1-203(BP2,1098bp)contained an extra new exon(96bp)in ORF.2)BP1 and BP2 readthrough transcripts were mainly located in cytoplasm.3)Predicted pBPl and pBP2 readthrough proteins,about 37 kDa and 40 kDa,were encoded by BP1 and BP2 readthrough transcripts,respectively.4)pBPl/pBP2 readthrough proteins were mainly located in the nucleus.2.Regulation of parent genes by BP readthrough transcripts.1)Expression of BP1/BP2 readthrough transcripts was positively correlated with expression of the parent genes BCL2L2 and PABPN1(P<0.05).2)BP1/BP2 readthrough transcripts promoted parent gene expression.Expression of BCL2L2 and PABPN1 decreased with the knock-down of BP1/BP2 readthrough transcripts,whereas artificial overexpression of BP1/BP2 readthrough transcripts by pc-BP1/pc-BP2 plasmid restored the expression of BCL2L2 and PABPN1.3)Bioinformatics analysis showed that both BP1/BP2 readthrough transcripts and BCL2L2 could bind to miR-1306-5p.4)Expression of miR-1306-5p was negatively correlated with expression of BP1/BP2 readthrough transcripts and BCL2L2(P<0.05).5)The expression of BCL2L2 was significantly down-regulated by artificial overexpression of miR-1306-5p.Dual luciferase reporter gene assays and RNA dot hybridization showed that miR-1306-5p interacted with BCL2L2 expression and led to decrease of BCL2L2 levels.6)Dual luciferase reporter gene assays and RNA dot hybridization showed the interaction between BP1/BP2 readthrough transcripts and miR-1306-5p.7)MiR-1306-5p was associated with the RNA-induced silencing complex(RISC),and the binding of miR-1306-5p to RISC could be inhibited by BP1/BP2 readthrough transcripts,which supressed the negative post-transcriptional regulation of BCL2L2 by miR-1306-5p.These experiments indicated that BP1/BP2 readthrough transcripts may function as ceRNA and competitively bind to miR-1306-5p to promote expression level of BCL2L2.3.BCL2L2-PABPN1 readthrough transcripts promoted proliferation,clone formation,invasion and migration of prostate cancer cells.1)Proliferation,clone formation,migration and invasion of prostate cancer cells were inhibited by the knock-down of BP1/BP2 readthrough transcripts.2)Proliferation,clone formation,migration and invasion of prostate cancer cells were promoted by the artificial overexpression of BP1/BP2 readthrough transcripts.3)Artificial overexpression of BP1/BP2 readthrough transcripts reversed the inhibitory effects of si-BP1/si-BP2 on prostate cancer cells.4.BCL2L2-PABPN1 readthrough transcripts overexpression was a poor prognostic factor in prostate cancer.1)Expression of BP1/BP2 readthrough transcripts in prostate cancer tissue samples was significantly higher than that in benign prostate hyperplasia tissue samples(P<0.05).2)Expression of BP1 readthrough transcript was positively correlated with baseline PSA level,Gleason score,WHO/ISUP prognostic grade group,and clinical T stage of prostate cancer,and BP2 readthrough expression was positively correlated with Gleason score and WHO/ISUP grade group(P<0.01).3)Overexpression of BP1/BP2 readthrough transcripts was an effective molecular mark of prostate cancer progression(P<0.05).4)Overexpression of BP1/BP2 readthrough transcripts was an adverse prognostic factor for progression-free survival(PFS)and disease-specific survival(DSS)for prostate cancer patients(P<0.05).Conclusion:1.We identified three BCL2L2-PABPN1 readthrough transcripts derived from transcriptional readthrough of BCL2L2 and PABPN1 genes,which were mainly located in cytoplasm.BCL2L2-PABPN1-201 and BCL2L2-PABPN1-202(collectivelly named as BP1)had the same ORF sequence(1002bp).The ORF of BCL2L2-PABPN1-203(BP2,1098bp)had an extra exon of 96bp.The ORFs encoded pBPl and pBP2 readthrough proteins,which were mainly located in the nucleus.2.BP1/BP2 readthrough transcripts were overexpressed in both prostate cancer cells and tissues,and the expression level was positively correlated with the parent genes BCL2L2 and PABPN1.BP1/BP2 readthrough transcripts served as ceRNA for miR-1306-5p,and promoted BCL2L2 expression by protecting BCL2L2 from miR-1306-5p inhibition(ceRNA mechanism).3.Proliferation,clone formation,invasion and migration of prostate cancer cells were promoted by BP1/BP2 readthrough transcripts.4.Expression of BP1/BP2 readthrough transcripts in clinical prostate cancer tissue samples were significantly higher than that of benign prostate hyperplasia tissue samples.BP1/BP2 expression levels were positively correlated with baseline PSA level,Gleason score,WHO/ISUP prognostic grade group,and clinical T stage.Overexpression of BP1/BP2 readthrough transcripts was an adverse prognostic factor for progression-free survival(PFS)and disease-specific survival(DSS)in prostate cancer patients.