Effects Of Hsa-let-7c/IGF-1R Axis On The Proliferation And Osteo/Oontogenetic Differentiation Of Human Dental Pulp Stem Cells Induced By IGF-1

Hsa-let-7c is one member of the miRNAs let-7 family and is a key regulatory factor in the proliferation and differentiation of stem cells,especially for the maintenance of "stemness".IGF-1 is referred to insulin-like growth factor-1.Previous studies have shown that IGF-1 can regulate the homing,proliferation and differentiation ability of mesenchymal stem cells.IGF-1 mainly plays a role in activation of the downstream signaling pathways through IGF-1R.Let-7c family is closely involved in cell proliferation,migration,and regulation in the post-transcription of IGF-1/MAPK signaling pathway.However,the effects of hsa-let-7c on IGF-1-mediated proliferation and osteo/odontogenetic differentiation of human dental pulp stem cells(human dental pulp stem cells,hDPSCs)remain unknown.Part ?.Relationship of hsa-let-7c and IGF-1R in hDPSCs Objective To investigate the relationship between hsa-let-7c and IGF-1R,hsa-let-7c was over/downexpressed in hDPSCs by lentiviral vector transfection.Methods The gene expression level of hsa-let-7c was detected by real time RT-PCR and the protein level of IGF-1R was tested by western blot after hsa-let-7c was over/downregulated in hDPSCs for 48 hours.Results: Real time RT-PCR showed that the expression level of hsa-let-7c gene was significantly upregulated(P<0.01)in let-7c(+)when compared to the control group,while the expression level of IGF-1R protein was significantly downregulated at the same time.In contrast,compared to let-7c(-)Con,the expression level of hsa-let-7c gene was significantly decreased(P<0.05)and the expression of IGF-1R protein was increased in let-7c(-)hDPSCs.Conclusion: Hsa-let-7c over/downexpression can significantly affect the expression level of IGF-1R in hDPSCs.The expression level of hsa-let-7c and IGF-1R exhibited an inversed relationship.Part ?.The effects of hsa-let-7c / IGF-1R axis on the IGF-1-mediatedactivity and proliferation of hDPSCs Objective: To determine the effects of hsa-let-7c/IGF-1R axis on the IGF-1-mediated viability and proliferation ability of hDPSCs.Methods: 100 ng/mL IGF-1 was used to treat hsa-let-7c over/downexpressed hDPSCs for 0,1,3,5,7,9 days,then the cell proliferation ability was detected by CCK-8 kit and the cell growth curve was drawed.Flow cytometry(FCM)was detected to investigate the cell cycle distribution and cell apoptosis.The proliferation index(PI= G2/M+S)was calculated to explore the impact of hsa-let-7c/IGF-1R axis on the proliferation of hDPSCs.Results: CCK-8 and FCM assays demonstrated that the proliferation ability of hDPSCs exhibited no significant differences between the experimental and control groups after hsa-let-7c were over/downregulated(P>0.05).These experimental groups and the control groups exhibited similar average rate of apoptosis by FCM assay(about 7%,P>0.05).Conclusion: Hsa-let-7c/IGF-1R axis had no significant regulatory ability on the viability and proliferation of IGF-1 treated hDPSCs.Part ?.The effects of hsa-let-7c/IGF-1R axis on IGF-1-mediateddifferentiation of hDPSCs and the MAPK signaling pathway involvement Objective: To explore the effects of hsa-let-7c/IGF-1R axis on the IGF-1-mediated differentiation of hDPSCs and the involvement of MAPK signaling pathway.Methods: Total RNA and total protein were collected after hsa-let-7c over/downexpressed hDPSCs were treated with 100 ng/mL IGF-1 for 0,3,7 days.Real time quantitative reverse transcriptase polymerase chain reaction(Real time RT-PCR)and western blot were conducted to detect the osteo/odontogenetic markers(ALP/ALP,RUNX2/RUNX2,OCN/OCN,OSX/OSX and DSPP/DSPP,etc).Alizarin red staining was performed after hsa-let-7c over/downexpressed hDPSCs were treated with 100 ng/mL IGF-1 or mineralization inducing media for 14 days.Total protein extraction from hDPSCs was used to detect MAPK signaling pathway related proteins(ERK/P-ERK,JNK/P-JNK,P38/P-P38).Results: Real time RT-PCR and western blot results showed that the osteo/odontogenetic markers(ALP/ALP,RUNX2/RUNX2,OCN/OCN,OSX/OSX and DSP/DSPP,etc)were significantly decreased(P<0.05)in let-7c(+)+IGF-1 group when compared with the let-7c(+)Con+IGF-1 group.Conversely,in comparison with let-7c(-)Con+IGF-1,the osteo/odontogenetic markers(ALP/ALP,RUNX2/RUNX2,OCN/OCN,OSX/OSX and DSP/DSPP,etc)were increased in let-7c(-)group+IGF-1 to some extent and the differences were statistically significant(P<0.05).Alizarin red staining displayed that MM can exacerbate the capacity of calcium nodule formation.In the medium of MM or MM+IGF-1,less calcium nodules were formed in let-7c(+)compared to let-7c(+)Con group while more calcium nodules were generated in let-7c(-)in comparing with let-7c(-)Con group(P<0.05).MAPK signaling pathway test results exhibited that ERK,P-ERK,P-JNK,P38 and P-P38 were significantly downregulated in let-7c(+)group while JNK had no significant difference between the groups(P<0.05).The ratios of P-ERK/ERK,P-JNK/JNK,P-P38/P38 were significantly downregulated(P<0.05).In contrast,ERK,P-ERK,P-JNK,P38 and P-P38 were significantly upregulated in let-7c(-)group while JNK was declined.The ratios of P-JNK/JNK,P-P38/P38 were significantly upregulated while the ratio of P-ERK/ERK remains raised(P<0.01).Conclusion: Hsa-let-7c / IGF-1R axis can significantly affect the IGF-1-mediated differentiation of hDPSCs.Hsa-let-7c overexpression significantly reduced the osteo/odontogenic differentiation capacity of hDPSCs,while hsa-let-7c downexpression dramatically upregulated the osteo/odontogenic differentiation capacity of hDPSCs inversely.MAPK signaling pathway was closely involved in the regulation of hsa-let-7c/IGF-1R axis on IGF-1-mediated differentiation of hDPSCs.