MicroRNA-205 Regulates ECFC By Targeting NOTCH2 To Modulate Distraction Osteogenesis (DO) Angiogenesis

Objective Inflammation,trauma,and tumors of the oral and maxillofacial region can contribute to the various defects of osseous tissues in the oral and maxillofacial region.Distraction osteogenesis(DO)provides a novel strategy for the treatment of bone defects.However,the long treatment duration is accompanied by different complications,which has significantly limited its wide clinical application.Thus,exploring the osteogenesis mechanism of DO and promoting rapid mineralization can aid to shorten the stable stage of DO.Angiogenesis has been reported to complement osteogenesis in DO consolidation phase and play a key role in osteogenesis as well as bone remodeling.Therefore,it is important to explore the molecular mechanisms that can facilitate the interaction between DO osteogenesis and angiogenesis.A number of previous studies have revealed that endothelial colony forming cell(ECFC)possesses a close association with DO angiogenesis.Micro RNAs(miRNAs)have been found to play important role in regulating osteogenesis,angiogenesis and other biological processes.Nevertheless,the underlying mechanism through which miRNAs can regulate ECFC to participate in DO angiogenesis is not well understood.This study aimed to identify the key genes which can regulate early angiogenesis of DO by setting up animal model of canine mandibular DO,comparing the differential miRNAs in the tissues at early DO stage,and exploring the potential mechanisms by which they can modulate DO angiogenesis.Method1.Differential miRNAs screening in the regenerative tissues during the course of canine mandibular DO: DO animal models were first established.Their mandibular regenerative tissues were obtained at 14 and 28 days after operation.Immunohistochemistry was performed using CD31 and VEGF antibodies to observe angiogenesis.MiRNAs sequencing was performed on the regenerative tissue at the same time points as described above to screen for the various differentially expressed miRNAs,which were validated by PCR.To further validate the candidate miRNAs,canine DO animal models were set up.The samples from this model were harvested 10,14,21,28,and 42 days after operation,respectively.Thereafter,PCR was used to observe the changes in the levels of differentially expressed miRNAs in the context of DO.For the animals that were sacrificed 42 days after operation,their peripheral blood was collected to obtain serum and ECFC at 7,14,21,28,35 and 42 days after operation,respectively.The validation of candidate miRNAs in the serum and ECFC was performed using q RT-PCR to explore the potential changes of candidate miRNAs during early osteogenesis and angiogenesis in DO.2.Role of miR-205 in regulating ECFC angiogenesis: Canine peripheral blood derived ECFC was isolated and cultured for identification through morphological observation,flow cytometry,Dil-Ac-LDL and FITC-UEA-1double staining experiments.Lentiviral transfection was used to silence or overexpress miR-205 in ECFCs.MiR-205 expression in ECFC was confirmed by q RT-PCR.The potential effect of miR-205 on the ability of angiogenesis in ECFC was examined by CCK8,Transwell,wound healing assays,as well as Matrigel tube formation and chick chorioallantoic membrane(CAM)angiogenesis experiments.Using bioinformatics,the downstream target genes of miR-205,NOTCH2 and FOXF1,were screened.They were validated by the dual luciferase reporter assay.m RNA and protein levels of the downstream genes and angiogenesis factors in ECFC with miR-205 silenced/overexpressed were further examined by PCR and WB,and the secretion of VEGF by ECFC was analyzed by ELISA.3.(1)Regulation of ECFC angiogenesis by NOTCH2: Lentiviral transfection was employed to silence NOTCH2 in ECFC.The transfection efficiency of NOTCH2 was examined by q RT-PCR and WB.The effects of NOTCH2 on the proliferation and migration of ECFC,as well as angiogenesis under in vitro and in vivo settings were examined.The possible changes in the m RNA and protein levels of downstream angiogenesis factors were further examined by PCR and WB,and the secretion of VEGF by ECFC was analyzed by ELISA.(2)MiR-205 targets NOTCH2 to regulate ECFC angiogenesis: Double transfection of ECFC was performed with miR-205 silencing and NOTCH2 silencing lentiviruses.They were divided into three different groups,including NOTCH2 silencing negative + miR-205 silencing negative(NOTCH2i-NC +miR-205i-NC),NOTCH2 silencing negative + miR-205 silencing(NOTCH2i NC + miR-205i),NOTCH2 silencing + miR-205 silencing(NOTCH2i +miR-205i).The changes in the levels of NOTCH2,VEGF and b FGF were detected by q RT-PCR and Western blot analyses.The secretion of VEGF by ECFC was measured by ELISA.In addition,CCK8,Transwell,wound healing assays as well as Matrigel tube formation,and CAM angiogenesis experiments were used to detect the different functional changes.4.Role of mir-205-modified ECFC in DO: 27 beagle dogs were selected to establish the canine DO animal models.Animals were divided into three different groups: normal DO group,group injected with miR-205 silencing negative lentivirus modified ECFCs(negative group,NC group)and group injected with miR-205 silencing lentivirus modified ECFCs(the miR-205 i group).Each group contained nine dogs in,among which three were randomly selected for the sample collection 14,28 and 42 days after operation,respectively.The dynamic changes in morphological osteogenesis were observed using HE staining.The expression of osteogenic protein OCN was observed at 42 days after operation by immunohistochemistry.For the animals that were sacrificed 42 days after operation,they were imaged 14,28,35,and 42 respectively days after operation,respectively,using CBCT.Result1.By analyzing the miRNA-seq data of DO14 and DO28,it was observed that the top ten miRNAs with differential expression were miR-141,miR-205,miR-196 b,miR-33 a,miR-206,miR-133 c,miR-147,miR-503,miR-153 and miR-219-5p.Thereafter,we used q RT-PCR to validate the expressions in regenerative tissues of DO14 and DO28.The expression trends of miR-141,miR-33 a,mir-205 and miR-206 were consistent with the sequencing results,with miR-205 exhibiting the largest difference and the most significant statistical difference(P < 0.001).The immunohistochemical results also demonstrated that the levels of CD31 and VEGF were upregulated at DO28,thereby indicating occurrence of abundant early angiogenesis.The expression of miR-205 in the distraction tissues,peripheral blood serum and peripheral blood ECFC continuously decreased over time,and the expression was lowest at 28 days after operation and then leveled off.In summary,miR-205 played an important role in regulating DO angiogenesis process.2.The cells cultured in vitro displayed colony growth and proliferated rapidly.Flow cytometry analysis indicated that these cells expressed CD31,CD34,as well as CD105 but did not express CD133 and CD45.In addition,the positive dual staining experiment with Dil and UEA suggested endocytic function,thus demonstrating that cultured canine peripheral blood derived ECFCs were isolated and can be used in subsequent experiments.q RT-PCR results revealed that miR-205 was successfully transfected,and its expression was correspondingly elevated and decreased after over-expression or silencing.Dual-luciferase results demonstrated that FOXF1 did not directly bind to miR-205,while miR-205 could target NOTCH2.PCR and WB results demonstrated that the m RNA and protein levels of NOTCH2,VEGF and b FGF decreased significantly after over-expression of miR-205.Functional experimental results indicated that the proliferation,migration,Matrigel tube formation,and CAM angiogenesis of ECFCs were markedly inhibited after overexpression of miR-205.However,upon silencing mir-205 in ECFC,all these functions were enhanced.3.(1)PCR and WB results demonstrated that NOTCH2 expression in ECFC was successfully silenced,and the m RNA as well as protein levels of the downstream angiogenesis related factors VEGF and b FGF decreased substantially.The results of ELISA assay revealed that the levels of VEGF released by ECFC was markedly decreased.Functional experiments found that silencing NOTCH2 significantly inhibited the proliferation,migration,tube formation,and CAM angiogenesis of ECFC.(2)PCR and WB results demonstrated that ECFC with only miR-205 silenced(NOTCH2i-NC + miR-205i)displayed elevated expression of NOTCH2,VEGF and b FGF at both RNA and protein levels.ELISA results indicated that silencing mir-205 alone could significantly promote VEGF release;The proliferation,migration,tube formation,and CAM angiogenesis.After silencing NOTCH2 in ECFC with low expression of miR-205(NOTCH2i + mir-205i),expression of the various factors and the cellular biological functions were restored to the normal levels,thereby clearly suggesting that miR-205 may target NOTCH2 to regulate ECFC angiogenesis in vitro as well as in vivo.4.Gross view of DO regenerative tissue showed marked differences in the osteogenesis effects among the three different groups,with the best bone formation effect in the ECFC group injected with silenced miR-205(miR-205i).CBCT results showed that the bone density(HU value)increased gradually at each time point,and the bone density of the miR-205 i group was at the highest level at all the time points.The results of 6 weeks after operation(DO42)indicated that trabeculae filled in the distraction area in all groups.There was a broken end found between the osseous tissues of the normal DO group and the NC group while the callus tissue of the miR-205 i group was observed to be completely connected.HE staining results demonstrated that during the early stage of distraction(DO14 and DO28),the time of trabecular bone formation in the miR-205 i group was relatively earlier than that in the control group.HE and immunohistochemical results of DO42 indicated that mature bone could be observed in the distraction space of the miR-205 i group,whereas the expression of osteogenesis protein OCN was found to be high.In other groups,still some fibers or immature osseous tissues were detected.Conclusion1.MiR-205 was identified as the major micro RNA that displayed the most significant difference during the early stage of DO,which was closely related to early angiogenesis.2.Overexpression of miR-205 can significantly inhibit the proliferation,migration,angiogenesis both under in vivo and in vitro settings and VEGF release of ECFC.Conversely,silencing miR-205 can effectively promote these functions of ECFC.3.Inhibition of NOTCH2 can suppress angiogenesis of ECFC under in vivo and in vitro conditions.MiR-205 can regulate the angiogenesis function of ECFC by targeting NOTCH2.4.MiR-205-modified ECFC can promote the new bone formation of DO and significantly shorten the consolidation phase.