Purification Of MEG3 LncRNA Assembled In Vivo And Its Downstream Target Genes Screening

Backgrounds: Breast cancer is the main malignant tumor that causes death in women,due to the lack of targeted and effective therapy targets.Therefore,finding a specific key therapeutic target is a key problem to be solved in breast cancer research.lnc RNAs doesn't encode proteins,the length of them are more than 200 nucleotides,studies show that lnc RNA play an important role in cell cycle and differentiation and other life activities,such as the transcription and post transcription regulation of cell structural integrity,the determination of subcellular localization and controlling epigenetic modification.The function of long noncoding RNA is usually dependent on their interacting proteins,such as lnc RNA TERRA,which is mediated by the interaction with hn RNPA1 to achieve cell immortalization.In recent years,many researches have been working on the research of the mechanism of lnc RNA and tumor occurrence and development by the way of study the interaction proteins of lnc RNA,and study the key proteins or transcription factors of signaling pathways.Lnc RNA MEG3(Maternally Expressed Gene3)is a maternally inherited imprinting gene,which is located in human chromosome 14q32 and MEG3 is expressed in many normal tissues of human cells.It is highly expressed in brain and pituitary gland.MEG3 is not expressed or low-expressed in many human tumor cell lines,including non small cell lung cancer,and breast cancer.The re expression of MEG3 in cancer cell lines such as He La,H4 and CH157-MN can prohibit cell proliferation.In addition,the study showed that MEG3 could promote the expression of tumor suppressor gene p53 and enhance the combination of p53 and its target gene promoter.MEG3 can induce apoptosis by stimulating p53 dependent transcription in vitro and in vivo.In recent years,in the lung cancer cells,Ch IRP has been applied to detect the interactions of MEG3 with JARID2 and its target genes that can recruit PRC2 complex,and form a complex of DNA-RNA to achieve H3K27 methylation,thereby promoting EMT process.In this paper,we established and optimized the affinity and purification technology of lnc RNA MEG3 intracellular binding protein in breast cancer,combined with mass spectrometry(MS-LC) technology to screen the interaction proteins of lnc RNA MEG3.Moreover,in breast cancer cells,the changes of downstream target genes were identified through RNA-seq technology,and after the clustering analysis,the target genes related to phenotypes were selected and further studied.By detecting MEG3 binding protein and its downstream target genes,to further clarify MEG3 have an inhibition effect in cell proliferation,migration and clone formation is of BC is of great significance,and our research provide reference in the development of lnc RNA in cancer.Methods: 1.Using RT-PCR method,designing the near 3 'terminal primer of MEG3,the parts of MEG3 were detected in normal human embryo lung fibroblasts MRC5 and different breast cancer cell lines(T47D).HCC1954;MDA-MB-231;MCF7)Expression in 2.Using lentivirus transfection--System Lenti-x Tet-On 3G Inducible Expression System construct Plvx-TRE3G-MEG3 and Plvx-TET3 G plasmide,then transfecting them into breast cancer cell lines MDA-MB-231 and MCF7 cells.Using puromycin to selecting the MEG3 stable Expression single clones.3.MEG3 knockdown by RNAi and oligonucleotide(ASO)have little effect,in this paper,through CRISPRi technology,we design the sg RNA targeting the MEG3 promoter region,the plasmid of sg RNA has a KRAB region,when d CSA9 combining with the target area,KRAB plays a role of inhibiting the transcription of MEG3,thus stable knockdown MEG3 at the transcription level in normal mammary epithelial cells.4.Using MTT to detect the influence of stable overexpression and downregulated MEG3 on cell proliferation;The effects of stable overexpression and downregulated MEG3 on cell migration were detected by scratches.The effects of stable MEG3 overexpression and knockdown on cell cloning ability were detected by using the clone formation assay.5.RNAseq was used to detect the change of target gene after MEG3 overexpression or knockdown.6.Using the optimized in vivo lnc RNA binding protein affinity purification technology,screened MEG3 upstream binding protein: run silver staining gel,compared with the blank control and reverse the sense one have obvious different band,after sending the band of each sample respectively to the company,the different band was separately cut out and send to the company,after the cross analysis of the lnc RNPs we get the key protein.7.Through the cross analysis of overexpression and knockdown of MEG3 cells of downstream target genes,screened the target genes that in two overexpressing cell lines were up-regulated or down regulated commonly and the genes in overexpression cell line and knockdown cells were up-regulated or down regulated differently.Then pick out key genes and study the genes effect on cell migration.Results: Firstly,In breast cancer We establish stable cell line MCF7 and MDA-MB-231 that overexpressed MEG3 and in mammary epithelial cells MCF10 A we knockdown MEG3 by CRISPR-d CAS9 technology.Cell function test showed that overexpression of MEG3 can inhibit cell proliferation,migration and clone formation ability,but knockdown MEG3 can obviously promote cell migration.Then we established and optimized the affinity and purification technology of lnc RNA MEG3 intracellular binding protein in breast cancer,combined with mass spectrometry(MS-LC)technology to screen the interaction proteins of lnc RNA MEG3,we got 12 candidate RNP of MEG3.Finally,the changes of downstream target genes were identified by RNA-seq technique,and after the clustering analysis,8 genes related to migration phenotype was selected.Conclusion: The results of this study show that has been initially confirmed that overexpression of MEG3 can inhibit cell proliferation,migration and colony formation ability,knockdown of MEG3 can significantly promote cell migration.The downstream target genes of MEG3 were screened by RNA-seq technology,and 8 possible target genes related to migration phenotype were screened out after cluster analysis.The affinity purification technology of MEG3 binding proteins was optimized,and 12 key proteins that could bind to MEG3 were screened,establishing a foundation for research mechanism of lnc RNA MEG3 in the development of breast cancer...