Molecular Mechanism Of MiR-204-5p Overexpression On Regulating The Growth Characteristics And Differentiation Potential Of Adipose Derived Stem Cells

In recent years,adipose derived stem cells(ADSCs)have attracted extensive attention because of their therapeutic potential in different fields of applied science.Among them,ADSCs derived from livestock has significant pluripotency,plasticity,differentiation potential and immunomodulatory characteristics,with levels comparable to human ADSCs.Using livestock ADSCs as drug delivery target can provide reference for determining disease mechanism and the effectiveness and safety of disease model.In addition,livestock ADSCs can be applied to somatic nuclear transplantation to improve cloning efficiency and gene transfection efficiency in gene-edited animals.With the increasing application of livestock ADSCs in the field of human medicine and animal husbandry,it is particularly necessary to explore its biological characteristics and molecular regulation mechanism.As an important part of epigenetic modification,miRNAs play an important role in regulating the proliferation and multidirectional differentiation of ADSCs in vitro,but little is known about their molecular regulation mechanism in livestock ADSCs.In this study,cashmere goat adipose mesenchymal stem cells(gADSCs)were used as the research object to explore the effects of miR-204-5p overexpression on the growth characteristics and differentiation potential of gADSCs.The molecular mechanism of miR-204-5p overexpression regulating the growth characteristics and differentiation potential of gADSCs was explored by combined transcriptome and proteome analysis and molecular biology.A comprehensive understanding of the regulatory role of miRNAs on the growth characteristics and differentiation potential of gADSCs at the cellular and molecular levels.The results are not only important to improve the stem cell characteristics of gADSCs and thus its application in somatic cell cloning,but also provide a theoretical basis for the application of ADSCs in stem cell therapy and regenerative medicine.1.Effects of miR-204-5p overexpression on the growth characteristics and differentiation potential of gADSCsAfter overexpression of miR-204-5p into gADSCs in a lentivirus mediated manner,it was found that the cell morphology did not change,but the growth was slow.Through the detection of CCK-8 and Ed U proliferation activity,it was found that miR-204-5p overexpression decreased the proliferation activity of gADSCs.Through cell cycle detection and apoptosis detection,it was found that the cells in G0/G1 phase decreased when gADSCs of miR-204-5p overexpression were cultured for 72 hours in vitro,but the cells in G2/M phase did not increase,the cells in S phase increased.In addition,the number of apoptotic cells of gADSCs of miR-204-5p overexpression decreased at 24 h,48h and 72 h.Through oil red O staining and lipid droplet quantitative detection,it was found that the content of lipid droplets in differentiated adipocytes of gADSCs of miR-204-5p overexpression was low.The results of real-time quantitative PCR showed that the transcriptional levels of PPARG,IRS1 and PERILIPIN in differentiated adipocytes of gADSCs of miR-204-5p overexpression decreased significantly at 15 days.The results of ELISA showed that NGF secreted by differentiated neurons of gADSCs of miR-204-5p overexpression increased significantly,TAU,MAP2 and RBFOX3 were highly expressed at the protein level,and ENO2,TAU and RBFOX3 were highly expressed at the m RNA level.By detecting the content of ALB and urea and the expression of marker genes in differentiated hepatocytes of gADSCs of miR-204-5p overexpression,it was found that the content of ALB and urea increased slightly,and AFP,ALB,HNF4 A and KRT18 were highly expressed at the m RNA level,but only AFP and HNF4 A were highly expressed at the protein level.The results showed that overexpression of miR-204-5p inhibited gADSCs growth,proliferation,and apoptosis,triggered cell cycle arrest,and inhibited gADSCs differentiation into adipocytes,but promoted its differentiation into neurons and hepatocytes.2.Transcriptome and proteome were used to analyze the regulation of miR-204-5p overexpression on the growth characteristics and differentiation potential of gADSCsWe performed differential expression analysis of transcriptome and proteomic on uninfected gADSCs(NC),g ADSC infected with lentivirus empty vector(LV?Con)and miR-204-5p up lentivirus vector(LV?miR),and focused on the differential genes and proteins in LV?Con?vs?LV?miR.According to the functional notes,we found13 differential genes and 10 differential proteins were screened in LV?Con?vs?LV?miR,which were related to cell proliferation inhibition.Among them,JAG1 is the ligand of NOTCH3.Studies have shown that NOTCH3 plays an important role in inhibiting cell proliferation.Real-time quantitative PCR and western blot confirmed the upregulation of JAG1 and NOTCH3 in the LV?miR.In addition,PKA pathway can down regulate CREB1 expression and inhibit cell proliferation.The results of real-time quantitative PCR and western blot showed that the expression of PKA was upregulated in LV?miR,while the expression of its downstream CREB1 was downregulated.Among the 19 differential genes and 16 differential proteins related to cell cycle,KEGG enrichment analysis showed that THBS1,CDKN1 A and CCND1 were enriched in PI3K-AKT signaling pathway.The results of real-time quantitative PCR and western blot showed that THBS1,CDKN1 A and CCND1 were upregulated in LV?miR,and two key factors PIK3 CA and AKT1 in this pathway were also highly expressed in LV?miR.Among the 3 differential genes and 4differential proteins related to apoptosis,NRAS can regulate the expression of anti apoptotic factor BCL2 by acting on ERK1/2.Through real-time quantitative PCR and western blot,we found that the expression of NRAS,ERK1/2,p ERK1/2 and BCL2 were upregulated in LV?miR.Among the 8 differential genes and 2 differential proteins related to adipocyte differentiation,ADIPOQ,LEPTIN and PPARG were enriched in AMPK signaling pathway.Through real-time quantitative PCR and Western blot,we found that expression of ADIPOQ,LEPTIN and AMPK were upregulated in LV?miR,while PPARG was downregulated.In addition,JAG1 negatively regulates adipocyte differentiation,and its expression was upregulated in LV?miR.Among the 8 differential genes and 7 differential proteins related to neuron differentiation,two key proteins in NOTCH signaling pathways,NOTCH3 and JAG1,have been identified again,and these two proteins have been confirmed were significantly highly expressed in LV?miR.It has been shown that NOTCH3 expression promoted neuron differentiation.We found only one differential gene associated with hepatocyte differentiation in LV?Con?vs?LV?miR,namely E2F8.Real-time quantitative PCR and western blot showed that E2F8 was highly expressed in LV?miR.The results suggest that the inhibitory effect of miR-204-5p overexpression on gADSCs proliferation may be achieved by activation of JAG1/NOTCH3 signaling or by promoting the expression of PKA and down regulating the expression of CREB1.miR-204-5p overexpression may also upregulate the expression of CDKN1 A and CCND1 by acting on the THBS1/PIK3CA/AKT1 axis in the PI3K-AKT signaling pathway,which in turn triggered cell cycle arrest.The anti-apoptotic effect of miR-204-5p overexpression on gADSCs is likely to promote BCL2 expression by activation of NRAS/ERK1/2 signaling.miR-204-5p overexpression may inhibit PPARG expression by AMPK signaling,and then inhibit the differentiation of gADSCs into adipocytes,and also to inhibit the adipocyte differentiation process by acting on the JAG1/NOTCH3 axis.Furthermore,the promoting effect of miR-204-5p overexpression on gADSCs differentiation into neurons may be achieved by acting on the JAG1/NOTCH3 axis.Moreover,miR-204-5p overexpression may also promote the differentiation of gADSCs into hepatocytes through E2F8.3.Molecular mechanism of miR-204-5p overexpression regulating the growth characteristics and differentiation potential of gADSCsThrough the detection of CCK8 cell proliferation activity,the cell proliferative activity was decreased after gADSCs treated with the NOTCH activator Valproic acid(VPA),and the expression of JAG1 and NOTCH3 increased,but the inhibitor LY411575 promoted the proliferation of gADSCs.After gADSCs treated with PKA activator 8-Bromo-c AMP,PKA was upregulated,CREB1 was downregulated,and cell proliferation activity was reduced.The effects of inhibitor H89 2HCl was opposite.The results of cell cycle detection showed that the gADSCs treated with PIK3 CA activator 740 Y-P would prevent the cells entering the G2/M phase and cause cell cycle arrest,and the expressions of PIK3 CA,AKT1,CDKN1 A and CCND1 were upegulated.The gADSCs treated with inhibitor HS-173 would promote the cells to enter the G2/M phase.Apoptosis detection showed that the number of apoptotic cells of gADSCs treated with NRAS inhibitor Lonafarnib(SCH66336)were increased and the expression of NRAS,ERK1/2,p ERK1/2 and BCL2 downregulated.AMPK activator AICAR can inhibit the differentiation of gADSCs into adipocytes,upregulates the expression of AMPK and downregulates the expression of PPARG,ADIPOQ and LEPTIN,while the gADSCs treated with inhibitor Dorsomorphin(compound C)2HCl has the opposite effects.Further analysis showed that the gADSCs treated with NOTCH activator Valproic acid(VPA)also inhibited adipocyte differentiation of gADSCs.In addition,treatment of gADSCs with Valproic acid(VPA)upregulated the expression of the neuron marker genes ENO2,MAP2,RBFOX3 and TAU,which in turn promoted gADSCs differentiated into neurons.After gADSCs treated with LY411575,although only RBFOX3 was downregulated at the m RNA level after differentiation,the marker genes ENO2,MAP2 and TAU were low expressed at the protein level.In conclusion,miR-204-5p overexpression inhibited the proliferation of gADSCs by acting on JAG1/NOTCH3 signaling pathway and PKA/CREB1 signaling pathway,triggered cell cycle arrest by acting on the THBS1/PIK3CA/AKT1/CDKN1 A axis in PI3K-AKT signaling pathway,and achieved anti-apoptotic effects by promoting BCL2 expression through activation of NRAS/ERK1/2 signaling pathway,inhibited PPARG expression by promoting the expression of ADIPOQ and LEPTIN and acting on the AMPK signaling pathway,and then inhibited gADSCs differentiated into adipocytes.miR-204-5p overexpression can also promote gADSCs differentiated into neurons and inhibit gADSCs differentiated into adipocytes by acting on JAG1/NOTCH3 signaling pathway.Furthermore,miR-204-5p overexpression may also promote the differentiation of gADSCs into hepatocytes through E2F8.