Effect Of CCN3 On Survival Of MSCs And Its Mechanism

Nephroblastoma overexpressed（Nov,CCN3）is an exocrine protein expressed in many tissues and organs of the human body,which is involved in regulating cell adhesion,mitosis and other biological processes^[1-3].Recent studies have found that the expression of CCN3 is closely related to bone formation and development^[3-5].Studies have shown that CCN3 can be used as a secretory ligand to bind Notch1 and participate in signal transduction.Our previous study found that CCN3 is involved in regulating Notch,thereby inhibiting BMPS-induced osteoblastic differentiation of mesenchymal stem cells^[6-8].Because cell differentiation and cell proliferation are closely related in the process of cell development,CCN3inhibit the differentiation of mesenchymal stem cells.Is its function mainly to promote cell proliferation?However,there are few studies on the regulation of CCN3 on cell proliferation,and the existing research conclusions are inconsistent and the mechanism is not clear^[9-11].Mouse embryonic fibroblasts（MEFs）,as a research model of MSCS,have been increasingly used in MSCS related studies^[12-14].This study MEFs as MSCs research model,the use of recombinant adenovirus expression and knock CCN3 reduction,understand CCN3 influence on MEFs alive,to explore the role of CCN3 in MSCs proliferation process and mechanism,this study will enrich regulating MSCs growth mechanism is the basis of research results,and to solve the problem of source of seed cells in bone tissue engineering provides some new ideas.Part Ⅰ:Objective:To investigate the effect of CCN3 on the survival（proliferation,apoptosis and migration）of MSCs.Methods:Mice fibroblast MEFs were used as the model of MSCs,and CCN3 was overexpressed and down-regulated by recombinant adenovirus.Flow cytometry,CCK-8 and Hoechst were used to detect the effects of CCN3 on proliferation and apoptosis.The effect of CCN3 on cell migration was detected by Trans Well assay.The expression of proliferation marker protein PCNA,Cyclin E,Cyclin B1,pro-apoptotic protein Bax,anti-apoptotic protein Bcl-2,cell migration marker protein Snail,Vimentin and Twist were detected by Western Blot.The effect of q RT-PCR on BMPs mRNA expression was investigated.Results:The results of CCK-8,flow cytometry,Hoechst and Trans Well experiments showed that,compared with the GFP control group,overexpression of CCR3 could promote the proliferation and migration of MEFs,and inhibit their apoptosis.WB results showed that overexpression of CCN3 could significantly promote the expression of PCNA,Cyclin E,Bcl-2,Vimentin,Snail and Twist proteins,and inhibit the expression of Cyclin B1 and Bax proteins.QRT-PCR indicated that overexpression of CCN3 could inhibit BMP2,4,6,7 and 9 mRNA levels,while interference with CCN3 could significantly promote the expression of BMP9 mRNA.Conclusion:CCN3 can positively regulate cell proliferation and migration,and inhibit cell apoptosis.CCN3 is a potent inhibitor of BMP9.Part Ⅱ:Objective:To explore the possible mechanism of CCN3regulation on cell proliferation from three aspects:CCN3 concentration,MSCs differentiation degree and MSCs cell density.Methods:The expression of CCN3 was detected by ELISA,and the effect of different concentrations of CCN3 on cell proliferation was detected by CCK-8.Two MSCs cell lines with different degrees of differentiation were selected as the research objects,and the effect of CCN3 on their proliferation was detected by CCK-8.The cell density of MEFs was controlled,and the influence of CCN3 on low,medium and high density MEFs was investigated by CCK-8 assay.Results:ELISA results showed that the concentration of CCN3 in low density（30%）MEFS cells was about 0.27ng/m L,the concentration of CCN3 in medium density（60%）was about0.97-1.05 ng/m L,and the concentration of CCN3 in high density（90%）was about 16.04-27.71 ng/m L.The concentration of CCN3 in Ad-CCN3group increased to 849.62-1629.48ng/m L,while the concentration of CCN3 in sic CCN3 group was between 9.09-22.5ng/m L.The results of CCK-8 showed that low concentration of CCN3 promoted the proliferation of intermediate density cells better,and CCN3 promoted the proliferation of intermediate density cells more strongly.Conclusion:CCN3 regulation of MSCs proliferation is mainly related to the density of MSCs.Part Ⅲ:Objective:To explore the mechanism of CCN3 regulating the proliferation of MSCs through Notch.Methods:Ad-CCN3 and ad-SICCN3 were used to up-regulate and down-regulate CCN3,Ad-DNNotch1 to down-regulate Notch1,MAPK inhibitor to down-regulate MAPK.CCK-8 was used to detect cell proliferation,q PCR was used to detect Notch downstream gene expression,and WB was used to detect H3K9 and MAPK pathway protein levels.Results:CCN3 can promote the level of Notch1 mRNA,and inhibit the level of DLL1 mRNA,but interfere the expression of CCN3.DAPT and Ad-DNNotch1 could inhibit the pro-proliferation effect of CCN3.Overexpression of CCN3 inhibited the mRNA expression of Notch downstream classical target genes RBPJ,HEY1,p300,p53 and p21,as well as the expression of H3K9 protein.PD98059 significantly affected the pro-proliferation effect of CCN3 on MEFs,and overexpression of CCN3 promoted the protein expression of Erk1+2,p-Erk1+2,JNK,p-JNK and p38,but inhibited the protein expression of p-p38.Conclusion:CCN3 can promote the proliferation and migration of MEFs,and inhibit the apoptosis of MEFs mainly by inhibiting the classical Notch signal and activating the MAPK signal pathway.Part Ⅳ:Objective:To analyze the effect of CCN3 on tumor development by using database.Methods:The expression level of CCN3in tumors and the relationship between CCN3 and the development,metastasis and prognosis of breast cancer were analyzed by TCGA database,GEO database,Oncomine database,GEPIA2 website and Timer website for gene expression profile interactive analysis.Results:Data analysis of multiple databases showed that compared with adjacent normal tissues,CCN3 expression level was decreased in breast cancer,especially in luminal B type.Survival analysis results show that the high expression of CCN3 poor survival associated with breast cancer,and mainly with three negative breast cancer and the tube cavity of patients with breast cancer development,which associated with increased survival rate,high CCN3expression CCN3 expression levels and breast cancer patients age,stage of development,and negatively correlated with tumor size,information was positively associated with lymph node metastasis.Finally,the GEO database analysis showed that the distant metastasis of breast cancer patients,especially bone metastasis,was closely related to CCN3-Notch1,which may play a regulatory role by affecting the expression of Post N and other proteins.Conclusion:CCN3 is not supported as an oncopromoter gene by biocredit analysis.