CircPGLYRP4 Promotes Osteoblast Differentiation And Mineralization By Binding HuR And Enhancing ZEB1 MRNA Stability

Research backgroundOsteoporosis is a systemic bone disease that is prone to fractures due to the loss of bone density and bone mass caused by a variety of reasons,the destruction of bone microstructure,resulting in increased bone fragility.The prevalence is 14.4%in men over 50 years of age and 20.7%in women over 50 years of age[1],especially in women over 60 years of age.Osteoporosis is divided into two categories:primary and secondary.Primary osteoporosis is divided into three types:postmenopausal osteoporosis(type Ⅰ.),senile osteoporosis(type Ⅱ)and idiopathic osteoporosis(including juvenile type).The dynamic balance of osteoblastic osteogenesis and osteoclast bone resorption is disrupted,so osteoporosis manifests as an endocrine metabolic imbalance[2].Osteoporosis is characterized by osteopenia,microstructural destruction,and fragility fractures[3].Osteoblasts contribute less bone mass to explain the reduced bone thickness,which is characteristic of osteoporosis[4].Osteoporosis has been found to be triggered by interference with various targets in the pathway of osteoblast proliferation,differentiation,and activation[5].Understanding the molecular mechanisms that mediate osteoblastic and osteoclast differentiation will contribute to a comprehensive understanding of osteoporosis.CircRNAs are a new set of noncoding transcripts that do not have 3’ and 5’ ends,but instead form a closed loop that differs from linear RNA.The study found that circRNA has at least three major capabilities:regulating the biological function of miRNA,participating in transcriptional regulation and self-translation.In particular,it acts as a sponge to adhere to miRNA molecules and thus control protein expression.Recently,some reports have shown abnormal expression of circRNA in osteoporosis patients and regulates the process of bone production and absorption.For example,knocking out circCDR1as in patients with hormonal osteonecrosis of the femoral head promotes the differentiation of human bone marrow stromal stem cells(BMSCs)into osteogenesis while inhibiting the adipogenic differentiation of BMSCs[11].In addition,tumor necrosis factor-α(TNF-α)promotes bone resorption by promoting osteoclast differentiation and inhibits osteoblastic differentiation,and CircHmboxl was found to be involved in inhibiting this process and inhibiting osteoclast differentiation[12].Therefore,mining the key circRNAs in the process of osteoporosis progression can provide a reference and basis for finding early diagnostic markers of osteoporosis and developing new therapeutic targets.The RNA-binding protein HuR(human antigen R)binds to many transcripts,coding and noncoding and controls their splicing,localization,stability,and translation.Through regulation of target transcripts,HuR is associated with cellular events including proliferation,aging,differentiation,apoptosis,and stress and immune responses.HuR,in turn,affects processes such as cancer and inflammation.ZEB1 Zinc finger E-box combined with cognate box 1(ZEB1)is most commonly characterized as a zinc finger transcription factor triggering the epithelial-mesenchymal conversion(EMT)program critical for development as well as a range of pathological states,including cancer10-13.Previous studies have shown that global deletion of ZEB1 in mice causes perinatal lethality due to severe bone defects14.However,primary cell lineages are affected in these mice and their molecular mechanisms remain undefined.In addition,it is unclear whether ZEB1 is a postnatal bone formation or whether ZEB1 loss is associated with certain cell lineages associated with osteoporosis in humans and mice.Recent studies have found that circRNAs can regulate the translation of endogenous competitive mRNAs by spongesizing microRNAs(miRNAs).Interestingly,recent evidence suggests that circRNAs can play an important role in regulating the pathogenesis of disease by interacting with RNA-binding proteins(RBPs).For example,a recent study showed that circPDE4B prevents articular cartilage degeneration by interacting with RIC8A and MID1,suggesting that circRNA plays a key role in regulating chondrogenesis by interacting with RBPs.Some of these circRNAs have been identified as regulators of BMSC proliferation or differentiation via sponge miRNA25.However,these previous studies have limited to exploring the role of osteoporosis-associated circRNAs in sponge miRNAs.It is unclear whether circRNA can modulate bone metabolism through other mechanisms,for example by interacting with RBP.Here,we show that circPGLYRP4 is a circRNA expression downregulated in BMSC in osteoporosis rats and bone tissue in postmenopausal osteoporosis patients.We further demonstrated that circPGLYRP4 interacts with HuR proteins to facilitate HuR translocation into the cytoplasm.Then,the function of cytoplasmic HuR is to stabilize and enhance ZEB1 mRNA expression.Thus,circPGLYRP4 has been identified as a key regulator of bone metabolism as a candidate therapeutic target for the prevention of postmenopausal osteoporosis.objectiveThis study mainly investigated the expression of circPGLYRP4 in osteoporosis tissues and cells,clarified the effect of HuR on osteogenic differentiation and mineralization,explored the effect of circPGLYRP4 on HuR,and further studied the effect of the combination of circPGLYRP4 and HuR on downstream ZEB1,so as to determine whether circPGLYRP4 plays the role of osteogenic differentiation and mineralization through the HuR/ZEB 1 pathway.methodThe serum of 40 osteoporosis patients and non-osteoporosis patients was selected clinically,and the expression of circPGLYRP4 and HuR and ZEB1 mRNA in the above samples was detected by qRT-PCR method.The effect of dexamethasone on osteogenic differentiation of BMSCs was observed by using dexamethasone at a concentration of 200 μM.The western blot method was used to determine the generation of bone alkaline phosphatase BALP and bone morphogenetic protein BMP,the marker protein of osteogenic conversion process,to detect the process of osteogenic differentiation.Alizarin red staining was then used to determine the extent of mineralization of BMSCs induced by the dexamethasone group compared to the control group(con).RIP and RNA pulldown experiments were used to confirm whether circPGLYRP4 was bound to HuR,nucleoplasmic isolation assays were used to separate the components of the nucleus from the cytoplasm,and then the regions where HuR was affected by circPGLYRP4 were verified by westernblot,and the cell sublocalization of circPGLYRP4 and HuR and the subcellular localization of ZEB1mRNA were confirmed by FISH binding IF staining.The effect of circPGLYRP4 on HuR and ZEB1 was demonstrated by function loss and acquisition,and finally the effect on osteogenic differentiation and mineralization was verified by PCR,ALP,ARS and westernblot.outcomeThrough the analysis of online data,circPGLYRP4 showed low expression in osteoporosis samples,and at the same time,5 pairs of clinical sample data were detected by qRT-PCR,and the results showed that circPGLYRP4 showed low expression in serum of osteoporosis patients with consistent consistency.Through the online prediction website RBPsuite,circPGLYRP4 can be combined and play a role in HuR,and the designed RIP and RNA pulldown experiments prove the accuracy of the above predictions.At the same time,HuR was expressed low in osteoporosis samples,and the osteogenic differentiation and mineralization ability of BMSCs was enhanced after overexpression of HuR.Overexpression of circPGLYRP4,it was proved by nucleoplasm isolation test that circPGLYRP4 made HuR converge in the cytoplasm,and the HuR content of the nucleus decreased significantly,and the differential expression was confirmed by synchronous qRT-PCR and westernblot.Through online data analysis,HuR binds to and functions with ZEB1 mRNA,and the prediction of binding is confirmed by FISH and IF staining localization.Through qRT-PCR and transfection,functional acquisition and deletion experiments,circPGLYRP4 overexpression can enhance the expression of HuR and ZEB1 mRNA,and enhance the osteogenic differentiation and mineralization of BMSCs.conclusion1.circPGLYRP4 is low in osteolian tissues and cells;2.Overexpression of circPGLYRP4 can promote osteogenic differentiation and mineralization of BMSCs,and inhibition of circPGLYRP4 expression can inhibit osteogenic differentiation and mineralization.3.circPGLYRP4 binds to and promotes the translocation of HuR from the nucleus to the cytoplasm;4.HuR can bind to ZEB1 mRNA and overexpression of HuR can affect the expression content of ZEB1 mRNA;5.circPGLYRP4 promotes the stability of ZEB1 mRNA by binding to HuR,thereby promoting osteogenic differentiation and mineralization.