Mechanism Of MiR-140-5p Targeting HDAC4 To Regulate Growth And Differentiation Of Growth Plate Chondrocytes And The Effect Of Total Flavonoids Of Rhizoma Drynariae

Tibial Dyschondroplasia(TD)is a metabolically disturbed skeletal disease that often occurs in broilers with short growth cycles.The clinical manifestations of TD in broilers are characterized by difficulty in standing,dyskinesia,decreased growth performance,and skeletal deformities.Its typical pathological characteristics are the appearance of non-vascularized and non-calcified hyaline cartilage plugs in the proximal metaphyseal growth plate of the tibia and the accumulation of chondrocytes in the growth plate with abnormal growth and differentiation.A questionnaire survey by our research group showed that the incidence of TD with clinical and subclinical symptoms in the Chinese poultry breeding industry is about 5%in recent years.Due to the complex and diverse pathogenesis,it is often manifested as subclinical symptoms,coupled with the serious contamination of broiler feed.It has not only caused problems for the front-line researchers in the diagnosis and treatment of TD,but also caused huge economic losses to the broiler breeding industry.Previous researches have confirmed that there are many differentially expressed miRNAs and their target genes in the tibial growth plate after the occurrence of TD.In order to further explore the regulatory role of differentially expressed miRNAs in the pathogenesis of TD,primary growth plate chondrocytes were cultured as the experimental model to reveal the effect of miR-140-5p and its target genes on the growth and differentiation of growth plate chondrocytes in-vitro.Also,an in-vivo experiments was conducted to evaluate the prevention and treatment of the total flavonoids of Rhizoma drynariae on TD and its impact on miRNA expression.The current study lay a foundation for the pathogenesis of TD and the evaluation of the prevention and treatment effect of traditional Chinese medicine.1.Establishment and evaluation of primary growth plate chondrocytes;an isolation and culture method(mechanical double-enzyme method).The growth of poultry bones is accomplished by a highly organized growth plate,having a lot number of chondrocytes located at the proximal end of the tibia.Therefore,the precise isolation and culture of chondrocytes from the tibial growth plate can provided sufficient cell samples for this study and provided necessary conditions for further study of the growth and differentiation of chondrocytes.In this study,the chondrocytes were obtained from the growth plate by hyaluronidase combined with type IV collagenase.The survival rate of cells was detected by trypan blue staining.While the chondrocytes were identified by toluidine blue staining,cell immunofluorescence(Col-Ⅱ,Aggrecan)and PCR,the growth of chondrocytes was detected by blood cell count and CCK-8 method,and the growth curve was drawn.Different chondrocytes’ isolation and culture methods were evaluated by cell purity,number of living cells,and a total number of cells.The results confirmed that the cells isolated by the mechanical double-enzyme method were growth plates’ chondrocytes.The cell survival rate was higher than 98%,and the chondrocytes were in the proliferation stage and did not differentiate.Comparing and analyzing different methods of separating and culturing chondrocytes,the chondrocytes isolated by the mechanical double-enzyme method have the advantages of large number,high purity and good cell condition.Moreover,the chondrocytes were successfully passed to the third generation.In conclusion,the mechanical double-enzyme method was relatively a mature method for the isolation and culture of primary growth plates’ chondrocytes,which provided sufficient experimental samples for current study and accumulated a practical experience for the isolation and culture of other primary cells.2.Functional study of miR-140-5p in growth plate chondrocytesmiRNAs are involved in regulating biological development,cell growth,differentiation,apoptosis and metabolism.The detection of whole-genome miRNA levels showed significant differences in the expression of miRNAs between normal and abnormal development of animals.Previous high-throughput sequencing results showed that the expression of mir-140-5p decreased significantly in the tibial growth plate of TD broilers,but the function of miR-140-5p in the growth plate chondrocytes was unknow.Here,the expression profile of miR-140-5p in the growth plate and different tissues were first verified by RT-qPCR in the animal model of broiler TD induced by thiram.LV3-gga-miR-140-5p mimics and LV3-gga-miR-140-5p inhibitors were simultaneously transfected into growth plate chondrocytes.The function of miR-140-5p in growth plate chondrocytes was detected by the changes of external cell morphology and the levels of growth plate chondrocytes markers related to growth and differentiation.The results showed that the expression of miR-140-5p was significantly reduced in the TD group as compared with the normal group(p<0.05 or p<0.01),which was consistent with the miRNA sequencing results.Also,the expression level of miR-140-5p in the broiler tibial growth plate was significantly higher than that in the internal organs,skin and muscle(p<0.01),with tissue expression specificity.After 72 hours of transfection,the external morphology of chondrocytes in the control group was oval,with clear cell outline and tight arrangement.The external morphology of chondrocytes in the mimics group was"paving stone" like,and the cells were uniformly distributed.While the external morphology of chondrocytes in the inhibitor group was irregular,blurred outline with disordered distribution.Compared with the control group,the expression levels of the chondrocyte-specific secretion markers Col-II and Sox9 in the overexpression miR-140-5p group were significantly up-regulated(p<0.05 or p<0.01)in the NC and the inhibitor groups.The expression of chondrocyte differentiation-promoting transcription factors MEF2C,Runx2 and chondrocyte hypertrophy markers Col-X,COMP,IHH,MMP-13,and VEGFA were significantly up-regulated(p<0.05 or p<0.01),but the expression of miR-140-5p potential target gene HDAC4 was decreased(p<0.05 or p<0.01).In conclusion,overexpression of miR-140-5p promoted the growth and differentiation of growth plate chondrocytes,while the silencing of miR-140-5p resulted in chondrocyte growth arrest with an unable capability to differentiate into mature chondrocytes.3.Study on the mechanism of miR-140-5p in growth plate chondrocytesIn all organisms,miRNAs play an important regulatory role by incompletely pairing bases with target mRNAs,leading the silencing complex to inhibit the translation of target mRNAs or promote their incomplete degradation.Histone deacetylase 4(HDAC4)is a potential target gene of miR-140-5p.Studies have found that HDAC4 regulates chondrocyte hypertrophy and endochondral osteogenesis by binding to pro-chondrocyte differentiation factor and inhibiting its activity.However,the direct targeting relationship between miR-140-5p and HDAC4 and its underlying mechanism by which HDAC4 affects chondrocyte growth and differentiation is unknown.To this end,in this study,the associative relationship between miR-140-5p and HDAC4 was evaluated by dual-luciferase reporter gene;the interacting protein with HDAC4 was screened by co-immunoprecipitation assay.The chondrocytes were treated with specific inhibitors and agonists of HDAC4,respectively to examine the regulatory relationship between HDAC4 and its interacting proteins.The dynamic expression changes of miR-140-5p,HDAC4 and their interacting proteins were detected in chondrocytes throughout the culture cycle by promoting chondrocyte differentiation.The results showed that the combination of miR-140-5p mimic and HDAC4-WT significantly reduced the fluorescence activity(p<0.01);HDAC4 interacted with Col-II,Col-X and COMP;compared with other groups,the expressions of chondrocyte differentiation promoting factors Runx2,MEF2C and chondrocyte hypertrophy markers Col-X,COMP,IHH,MMP-13 and VEGFA were significantly up-regulated in HDAC4 inhibitor group(p<0.05 or p<0.01).Under the condition of promoting differentiation culture,the expressions of Runx2,MEF2C,col-x,comp,IHH,MMP-13 and VEGFA were up-regulated in varying degrees,and the expression of HDAC4 was down-regulated(p<0.05 or p<0.01).In conclusion,HDAC4 is a target gene of miR-140-5p,inhibiting the expression of HDAC4.HDAC4 interacts with Col-II,Col-X and COMP.It inhibits chondrocyte differentiation,inhibits the expression of intramembranous osteogenic target protein BMP2,endochondral ossification signaling pathway IHH/PTHrP,and VEGFA following Sox9,BMP2,Runx2,MEF2C,Col-X,MMP-13,COMP and VEGFA regulation that play a key positive regulatory role in chondrocyte growth and differentiation.4.Study on the effect of the total flavonoids of Rhizoma drynariae on Tibial Dyschondroplasia in broilersThe total flavonoids of Rhizoma drynariae is a Chinese traditional medicine,which is often used to prevent and cure osteoporosis,fractures and other bone diseases.However,whether Rhizoma drynariae with therapeutic effect against TD has not been reported.Therefore,in order to explore the therapeutic effect of Rhizoma drynariae on broiler TD,a total of 320 one-day-old AA chickens were randomly divided into control group,TD group(supplemented with 50 mg/kg of thiram in the diet),TDR group(self-recovery group)and Rhizoma drynariae group(20 mg/kg/day).The therapeutic effect of total flavonoids of Rhizoma drynariae was evaluated by clinical manifestations,growth performance,tibial parameters,growth plate chondrocyte development and angiogenesis.RT-qPCR and Western-blot were used to detect the expression of BMP2/Runx2,IHH/PTHrP,miR-140-5p and their target genes.The results showed that the total flavonoids of Rhizoma drynariae has a good effect on the growth performance,clinical manifestations,tibial parameters,chondrocytes and blood vessels in the growth plate of TD broilers.The expression levels of BMP2/Runx2,IHH/PTHrP,miR-140-5p and chondrocyte hypertrophy markers(Col-X,COMP,MMP-13 and VEGFA)were up-regulated in different degrees in Rhizoma drynariae group(p<0.05 or p<0.01),but the expression of HDAC4 was down-regulated all the time(p<0.05 or p<0.01).The total flavonoids of Rhizoma drynariae positively regulate the signal axis of intramembrane osteogenesis and endochondral osteogenesis,up regulate the expression of miR-140-5p and down regulate the expression of HDAC4.It can promote the growth and transition of growth plate chondrocytes and the development and invasion of growth plate vessels.In conclusion,total flavonoids of Rhizoma drynariae can be used as a potential medicine for the treatment of TD.