Primary Study On Biological Functions Of BRM-b Isoforms Coded By Smarca2-b Transcriptive Variants

Chromatin-Remodeling-Factor BRM participates chromatin remodeling and gene expression regulation as a vital component of SWI/SNF(Mating Type Switch/Sucrose Non-Fermenting)complex,which uses either BRM or its counterpart BRG1 as the ATPase.BRM is translated by Smarca2,which is considered as a tumor suppressor gene.Our previous study found that small protein isoforms(BRM-b)can be coded by short Smarca2 mRNA transcriptive variants(Smarca2-b)which transcriptively initiated within intron 27 of Smarca2.Total four kinds of BRM isoforms(BRM-b)can be coded by these Smarca2-b variants,and these vatiants are significantly expressed in most human and mouse cells detcted.The expression of Smarca2-b transcriptive variants are also sensitive to serum starvation and complex regulated by CCND1 and CDK4.Conclusively,biological signification of existing Smarca2-b transcriptive variants and coded BRM-b protein isoforms in tumor cells is necessary to be explored.Bioinformatics analysis indidcates that all BRM-b protein isoforms coded by Smarca2-b transcriptive variants harbor two intact structures,Bromodomain and AT-Hook motif,which “reads” acetylated-lysine code and attachs to minor groove of DNA helix respectively,prompts that BRM-b may have important regulatory feature via unrevealed mechanism,which is worth to be explored.Our study preliminarily discussed the biological characters of Smarca2-b variants,as well as their coded BRM-b protein isoforms which harbor Bromodomain and AT-Hook motif,for epigenetic regulation in tumor and their potential value in clinical tumor diagnosis & treatment.ObjectivePreliminary investigation of the biological character of Smarca2-b transcriptive variants and their coded BRM-b proteins in three aspects: First,to investigate the existence of Smarca2-b transcriptive variants’ expression pattern within different mouse tissues at different growth stages,to get some clue in whether the variants participate in or are involved in the regulation of tissue growth and development;Second,to explore the localization of BRM-b isoform inside the cells;And third,to preliminarily study the effect of Smarca2-b transcriptive variants and their coded BRM-b proteins in neoplastic cells.Materials and Methods1.Use RT-PCR to detect the levels of mSmarca2-a/b variants within tissues of 3-day,7-day and mature(>2 months)C57BL/6 mouse;2.Construct the over-expressing vectors by cloning the whole Open Reading Frame(ORF)together with 5’-Untraslational Region(5’-UTR)of hSmarca2-b variants into pEGFP-N1 plasmid,which translate hBRM-b-EGFP fusion proteins in cells;3.Transfect the hBRM-b-EGFP over-expressing vectors into hSmarca2-b null Hep-3B cells with Liposome;the transcription of mRNA variants and the translation of hBRM-b-EGFP fusion protein isoforms are dected and verified by RT-PCR and Western Blot respectively after transfection;Localize the fusion proteins in Hep-3B cells followed by nuclear staining and fluorescent imaging;4.Detect the apoptosis of Hep-3B cells after transfected with over-expressing vectors by Flowcytometer;5.Scratch test to identify the alteration of cell migrative ability caused by transfected over-expressing vectors.6.Use CCK-8 to identify the alteration of Hep-3B cell proliferation caused by transfected over-expressing vector.Results1.The moderated expression of mSmarc2-a/b variants was observed in spleen,heart,kidney,lung and liver obtained from postnatal day 3 normal C57BL/6 mouse,The expression of mSmarc2-a in most postnatal day 7 mouse tissues was high,while low in several tissues,mSmarc2-b showed significant expression levels but detected null in liver,kidney and cerebellum;The expression of mSmarca2-a/b was observed in all adult C57BL/6 mouse tissues detected,although there were not much in heart and pancreas;2.The expression of hSmarca2-b in most collected human-derived cell lines were significant but depleted in heptacelluarcarcinoma Hep-3B cells and nasopharyngeal carcinoma 5-8F cells;3.Five over-expressing vectors(Named as N2,N4,N6,N8 and N10,respectively)containing different partial cDNAs of hSmarca2-b variants were constructed with pEGFP-N1 plasmid,and their sequencing results were consistent with expectations;The bioinformatics analysis of these vectors indicates that three defferent hBRM-b protein isoforms(b2,b3 and b4)could be coded,all of which contain intact Bromodomain and AT-Hook motif;In fluorescent imaging,the whole body of transfected cells in Vector Control emitted green fluorescent light whereas the cells transfected with constructed over-expressing vectors emmited bright green fluorescent light only from nucleus.4.FCS detection result indicated that as the hBRM-b-EGFP over-expressing vectors transfected into Hep-3B cells 48 h later,all vectors but N8 significantly elevated apoptosis rate statistically contrast to vector control;5.Scratch test indicated that the transfection of any over-expressing vector didn’t change the migration distance of Hep-3B cells contrast to vector control;6.Cell proliferation detection with CCK-8 indicated that all hBRM-b-EGFP over-expressing vectors but N8 inhibited cell proliferation: Staistical difference of cell number in N2,N4,N6 and N10 were observed 96 h after tranfection contrast to vector control.Conclusion1.The expression of mSmarc2-b in 7d C57BL/6 mouse cerebellum,liver and kidney are depleted,they may be involved in their developmental regulation.2.Five hBRM-b-EGFP over-expressing vectors are constructed successfully (Named as N2,N4,N6,N8 and N10 respectively).In transfectd Hep-3B cells, hSmarca2-b variants can be detected by RT-PCR,while hBRM-b-EGFP fusion protein isoforms can be detected by Western Blot with anti-EGFP antibody;3.Contrast to the distribution all over the transfected Hep-3B cell of EGFP expressed by pEGFP-N1,all hBRM-b-EGFP isoforms are resident in Hep-3B nucleus alone,further bioinformatics indicated that hBRM-b contain potential Nuclear Localization Signal sites;4.Except for N8,the expression of the other four hBRM-b-EGFP over-expressing vectors in Hep-3B promoted cell apoptosis significantly,which seems related to their common exon 2 as well;5.The migration ability of Hep-3B cell is not altered by the transfection of hSmarca2-b over-expressing vectors;6.Except for N8,the expression of the other four h BRM-b-EGFP over-expressing vectors in Hep-3B inhibited cell proliferation significantly,which seems related to their common exon 2 as well.