MiR-144-3p Regulate The Proliferation And Differentiation Of Osteoclast By Targeting RANK

Part I:miR144-3p expression is decreased in patients with osteoporosisObjective:Osteoporosis is a common anaphylactic disease of bone homeostasis.Recent studies have also found that miRNAs also play an important role in bone development,bone homeostasis and bone remodeling.In order to further investigate the role of miRNAs in osteoporosis,we selected 10 miRNAs which have proved are bone metabolism-related miRNAs in previous studies for quantification.Methods:10(miR-7-5p,miR-24-3p,miR-27a-3p,miR-100,miR-125b,miR-128,miR-145-5p,miR-211-5p,miR-144-3p,miR-122a)miRNAs was choose.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to investigated the expression of these 10 miRNAs in serum and bone tissue in osteoporotic and non-osteoporotic patients.Results:In our study,we found that the expression of miR-24-3p,miR-27a-3p,miR-100,miR-125b and miR-122a was significantly increased in the osteoporosis group in both serum and bone tissue samples,and miR-144-3p,in contrast,was reduced in serum and bone tissue samples in the osteoporosis group relative to the normal group.Conclusion:The expression of miR-24-3p,miR-27a-3p,miR-100,miR-125b,miR-122a and miR-144-3p in serum and bone tissue of patients with osteoporosis is consistent,those 5 miRNAs maybe as endogenous diagnostic biomarkers in osteoporotic patients.To investigate the underlying mechanisms by which miRNAs interact with bone metabolism,we chose miR-144-3p as our target miRNA for further investigation.Part? RANK is target gene of miR-144-3pObjective:The mechanism of action for traditional miRNAs is that miRNAs bind to the 3'UTR of the target gene mRNA,thereby affecting the stability of the mRNA and inhibiting the translation of the protein.In this chapter we study the target gene of miR-144-3p in bone and its mechanism of action.Methods:In order to further study the target genes for miR-144-3p,we first used online miRNAs prediction software to analyze the target genes of miR-144-3p.Then we quantified the expression of these target genes and then constructed the 3' UTR expression vector to studies the mechanism of action in vitro and in vivo.Results:Using online miRNAs target gene prediction software,we found that BMPR1A,COL11A,SMAD4,ESPRG and RANK are related to bone metabolism and remodeling.qRT-PCR results showed that the expression of SMAD4 and RANK was significantly increased in patients with osteoporosis.And the content of serum RANK mRNA also showed a significant increase.Bioinformatics analysis found that 9 bases(seed sequence)in the sequence of RANK 3'UTR were perfectly matched with miR-144-3p.And this seed sequence is completely conserved among humans,chimpanzees,mice,dogs and lizards.Expression of miR-144-3p in CD 14+ PBMCs cells was significantly down-regulated relative to undifferentiated cells(Day 0)after induction by M-CSF and RANKL.In vitro dual reporter luciferase experiments also demonstrated that miR-144-3p can target inhibit the expression of RANK.Conclusions:Based on our experimental results,the evidence revealed that miR-144-3p directly affects RANK mRNA and protein expression at the transcriptional and translational level.It is demonstrated that RANK is the target gene of miR-144-3p..However,whether miR-144-3p can participate in osteoclast differentiation by regulating the expression of RANK remains to be further studied.Part III mi-144-3p regulates osteoclast differentiation by target RANKObjective:A recent study found that OPG/RANKL/RANK system is directly related to osteoclast differentiation.In the present study,it has been identified that miR-144-3p can specifically regulate the expression of RANK,and whether miR-144-3p can also regulate osteoclast differentiation by regulating RANK expression.we designed the next experiment to in order to further study.Methods:In this study,TRAP staining was used to quantitative osteoclast number.qRT-PCR was performed to revealed expression level of osteoblast formation and osteoblast-specific in CD1414+PBMCs induced by M-CSF and RANKL.We then performed Flow Cytometry and CCK-8 experiments to check the proliferation and apoptosis of CD 14+PBMCs induced by M-CSF and RANKL.Results:Mimics and inhibitors of miR-144-3p significantly reduce or increase the expression of both MFATC and CTSK genes.The number of TRAP-positive cells in the miR-144-3p mimic-treated group was significantly reduced,and the number of TRAP-positive cells in the miR-144-3p inhibitor-treated group was significantly increased.Mimics and inhibitors of miR-144-3p reduced and increased the proliferation of CD14 ?PBMCs,respectively,relative to the negative control.Flow Cytometry results showed that miR-144-3p mimics significantly increased apoptosis in CD14 + PBMCs compared to the negative control.Conclusions:Mimics and inhibitors of miR-144-3p significantly reduce or increase the expression of both MFATC and CTSK genes.And subsequent experiments of cell proliferation and apoptosis also demonstrated that the effect of miR-144-3p on osteoclasts,strongly suggests that miR-144-3p can influence osteoclast formation by targeting RANK.