The Significance And Pathomechanism Of PRMT2and Its Variants In Breast Carcinomas

Breast cancer is one of the most common cancers in the women,and the mechanismof which is not yet fully understood. Estrogen receptor α (ERα) is a transcription factor andinvolved in a broad range of biological processes, including cell proliferation,differentiation, morphogenesis and apoptosis, as well as the development and progressionof breast cancer. Protein arginine N-methyltransferases (PRMTs) are eukaryotic enzymeswhich are common in mammals and have11members today. Their activities influence awide range of cellular processes, including cell growth, nuclear/cytoplasmic proteinshuttling, differentiation and embryogenesis, RNA splicing and transport, andposttranscriptional regulation. Subsequent research indicated PRMT2acts as an Estrogenreceptor α (ERα) coactivator and enhances estrogen-related transcription. Reports haveshown that it is clearly involved in lung function or the inflammatory response, promotesapoptosis, as well as leptin signaling and Wnt signaling regulation. However, themechanism of PRMT2in breast cancer is not yet fully understood, and no study has yetaddressed the alternative splicing events in the processing of the PRMT2transcript and nospliced PRMT2variants have been reported.We found and verified that the expression of PRMT2in breast carcinoma wassignificantly higher than that in normal breast tissues, and it was significantly higher inERα-positive breast carcinomas than that in ERα-negative carcinomas. To obtain andcharacterized of four novel PRMT2splice variants by using SMART RACE and RT-PCRtechnology. To analysis the expression of them in various of tissues and cancer cells byNorthern blot and Western blot. The exprssion of PRMT2and its variants was examed byreal-time PCR in breast cancer cell lines. The eukaryotic expression plasmidpcDNA3.1/NT-GFP-PRMT2/α/β/γ/L2were constructed and transfected into breastcancer cells. The expression and subcellular localization of GFP-fusion exogenous proteinin the transfected breast cancer cells were observed under laser confocal scanningmicroscope. To test the effection on the ERα transcription activation of PRMT2and its variants interact with ERα by using the dual luciferase reporter assays. To test the domainof PRMT2and its variants interact with ERα. in vitro and in mammalian cells withGST-pull down and immunoprecipitation. To construct PRMT2microRNA expressingeukaryotic recombinant, and identify biological activity of recombinant in breast cancerMCF7cells after transfection, and to investigate its effects on the cell growth properties ofMCF7cells and nude mice xenograft tumors.The results showed that PRMT2and its splice variants protein expressed differently inhuman cancer cell lines and normal human fetal tissues, and is significantly associated withERα positivity status. Confocal microscopy scanning revealed a distinct intra-cellularlocalization of PRMT2variants. PRMT2and its variants bound to the ERα both in vitroand in mammalian cells in a ligand-independent fashion, but enhanced the ERα-mediatedtransactivation activity of ERE-Luc in a dosage-dependant of estrogen. MicroRNA inducedPRMT2knockdown enhances the estrogen-mediated proliferation response and colonyformation to MCF-7cells, promotes E2-stimulated expression of ERα target genesCyclinD1and c-myc, the cell cycle phase, and promote the growth of nude mice xenografttumor. These results suggest that PRMT2and its splice variants may play a role in theformation and development of breast cancer by modulating the estrogen-ERα signalingpathway. Part1Significances of PRMT2and ERα expression in breastcarcinomaObjective: To investigate the expression and clinical significance of PRMT2and ERαinthe breast carcinoma. Methods: The expression of PRMT2and ERα were detected byimmunohistochemistry in198cases of breast tumor (56carcinomas and15adenomas) and20of normal thyroid. Results: overall, PRMT2protein immunocytochemical positivitywas found in4of20(20.0%) normal specimens, but158of198(79.8%) breast carcinomaspecimens were positive for PRMT2protein, significantly different from normal specimens(P<0.05, χ2test). Furthermore,126of142(88.7%) ERα-positive specimens were positivelystained, whereas low level staining for PRMT2was observed in ERα negative breast tumortissues, the cases of positive staining was32of56(57.1%), significantly different from theERα-positive carcinomas (P<0.05, χ2test). The expression of PRMT2is ralated to thehistologic grade in ERα-positive carcinomas (P=0.013). Conclusion: The expression ofPRMT2in breast carcinoma was significantly higher than that in normal breast tissues, andit was significantly higher in ERα-positive breast carcinomas than that in ERα-negativecarcinomas. The percentage of PRMT2expression may be correlated with the histologicgrade.Part2Identification and expression analysis of novel spliced variantsof PRMT2in breast cancerObjective: To identify and expression analysis of four novel spliced variants of PRMT2inbreast cancer. Methods:To obtain and characterized of four novel PRMT2splice variantsby using SMART RACE and RT-PCR technology. To analysis the expression of them invarious of tissues and cancer cells by Northern blot and Western blot. The exprssion ofPRMT2and its variants was examed by real-time PCR in breast cancer cell lines. Theeukaryotic expression plasmid pcDNA3.1/NT-GFP-PRMT2/α/β/γ/L2were constructedand transfected into breast cancer cells. The expression and subcellular localization ofGFP-fusion exogenous protein in the transfected breast cancer cells were observed under laser confocal scanning microscope. Results: Identification and characterization of fournovel PRMT2splice variants, and the mRNA expression profile overlap with thedistribution of ERα, with the strongest abundance in estrogen target tissues. PRMT2and itssplice variants protein expressed differently in human cancer cell lines and normal humanfetal tissues. The results of real-time PCR showed that the expressions of PRMT2and itsvariants were significantly higher in ERα positive breast cancer cells than in ERα negatives.The results of laser confocal miccroscope scanning showed that the GFP-PRMT2α andGFP-PRMT2γ fusion protein expression were predominantly localized to the nuclearcompartment excluding the nucleoli, and a weak fluorescence was detected in the cytosol,as well as that of the wild type PRMT2. Whereas, cells expressing GFP-PRMT2β, showedan even distribution of the GFP fusion proteins between the nucleus including the nucleoliand the cytoplasm. the GFP-PRMT2L2fusion protein expression resulted in apredominantly cytoplasmic staining concentrated around the nuclear compartment.Conclusion: Our studies report that identification and characterization of four novelPRMT2splice variants and demonstrate different expression of them in normal humantissues and human cancer cell lines, and is significantly associated with ERα positivitystatus. Confocal microscopy scanning revealed a distinct intra-cellular localization ofPRMT2variants.Part3The interaction between PRMT2and its variants with ERαObjective: To investigate the effection on the ERα transcription activation of PRMT2andits variants interact with ERα. Methods: to test the effection on the ERα transcriptionactivation of PRMT2and its variants interact with ERα by using the dual luciferasereporter assays. To test the domain of PRMT2and its variants interact with ERα. in vitroand in mammalian cells with GST-pull down and immunoprecipitation. Results: PRMT2and its variants bound to the ERα both in vitro and in mammalian cells in aligand-independent fashion, but enhanced the ERα-mediated transactivation activity ofERE-Luc in a dosage-dependant of estrogen. N-termini of PRMT2and its variants isresponsible for the binding to ERα protein and transactivation activity. Conclusions: PRMT2and its variants are all the co-activators of ERα. PRMT2and its variants bound tothe ERα both in vitro and in vivo, and N-termini of PRMT2and its variants is responsiblefor the binding to ERα protein and transactivation activity.Part4MicroRNA induced PRMT2knockdown affect estrogen-mediated proliferation in breast cancer MCF-7cellsObjective: To construct PRMT2microRNA expressing eukaryotic recombinant, andidentify biological activity of recombinant in breast cancer MCF7cells after transfection,and to investigate its effects on the cell growth properties of MCF7cells with differenttreatments. To investigate the effect of PRMT2knockdown on the growth of nude micexenograft tumors, and to clarify the preliminary mechanism of the effect of PRMT2on thegrowth of breast cancer. Methods: According to sequence of PRMT2mRNA, the PRMT2pre-microRNA was designed and synthesized, then cloned into the GFP taggedpcDNATM6.2-GW/EmGFP-miR vector and transfected into breast cancer MCF7cells. Todetect integrity of inset fragment through sequencing analysis. The biological activity ofrecombinant through identify interference efficiency of PRMT2microRNA recombinant byway of indirect immunofluorescence and western blot. The proliferation and colonyformation of MCF7cells were measured by crystal violet assay and colony formation assay,and the cell cycle was observed by cell flow cytometry (FCM). The stable MCF7cell linewith over-expression of PRMT2-N-miRNA and PRMT2-C-miRNA and the MCF7celltransfected with the empty vector control-miRNA were cultured respectively. ThePRMT2-N-miRNA/PRMT2-C-miRNA/control-miRNA MCF7cell line was inoculatedsubcutaneously in nude mice. To estimate the the effect of PRMT2knockdown on thegrowth of nude mice xenograft tumors, the volume and weight of xenograft tumor wasmeasured and the expression of cyclin D1and c-myc protein was detected in nude micetransplantation tumor by using the immunohistochemical method. Results: Sequence ofinset fragment in microRNA expressing recombinat was correct, and PRMT2proteinexpression of microRNA recombinant was decreased after transfection. microRNA inducedPRMT2knockdown did not change the growth property of MCF7cells without any treatment. The microRNA recombinant-transfected MCF7cells treated with4-OHTproliferated at the same rate as the control cells, whereas a stronger promotion of theproliferation of the microRNA recombinant-transfected MCF7cells cultivated with E2wasobserved. The colony formation assay showed that microRNA induced PRMT2knockdownenhances the estrogen-mediated proliferation response to MCF-7cells, and FCM assayindicated that the percentages of MCF7cells in G2phase were increased (P<0.05). Thenude mice xenograft tumors models of PRMT2-N-miRNA/PRMT2-C-miRNA/control-miRNA MCF7were established successfully. Compared with the control-miRNA MCF7cell group, the nude mice xenograft tumor growth ability was greatly increased inPRMT2-N-miRNA and PRMT2-C-miRNA groups (P<0.05). Among the three groups, theweight of PRMT2-N-miRNA and PRMT2-C-miRNA groups xenograft tumor were heaverthan that of the control-miRNA group (P<0.05), and the expression of cyclin D1and c-mycprotein were up-regulated in PRMT2-N-miRNA and PRMT2-C-miRNA groups xenografttumor (P<0.05). Conclusion: The microRNA expressing plasmid which were successfullyconstructed and have biological activity in MCF7cells after transfection. microRNAinduced PRMT2knockdown enhances the estrogen-mediated proliferation response andcolony formation to MCF-7cells, promotes E2-stimulated expression of ERα target genesCyclinD1and c-myc, and the cell cycle phase. MicroRNA induced PRMT2knockdowncould promote the growth of nude mice xenograft tumor of breast cancer MCF7cell line,and enhance the expression of cyclin D1and c-myc protein in nude mice xenograft tumor.