| Non-coding RNAs(nc RNAs)have been reported to be involved in numerous physiological and pathological process through regulating gene expression at the transcriptional and posttranscriptional levels.Ossification of ligamentum flavum(OLF)and osteoporosis(OP)are common chronic orthopedic disease.If unresolved,the OLF and OP would affect the quality of life of patients seriously.Up to now,very few nc RNAs have been identified to be correlated with OLF or OP.In this study,we identified the deregulated m RNA,lnc RNA,mi RNA and circ RNA in OLF by gene chip or sequencing.We also confirmed the function and mechanism of mi R-342-3p on osteogenic differentiation.This study provides a systematic perspective on the potential function of nc RNAs in the pathogenesis of OLF,and also suggests that mi R-342-3p may serves as a potential marker for diagnosis and treatment of OLF and OP.Methods:1.Sample collection: Ossified and normal LF tissues were obtained from OLF or sponal trauma patients who underwent posterior decompression laminectomy.The total RNA was isolated from the tissue by liquid nitrogen milling and detected by RNA electrophoresis and 2100 mass detection.Further experiments were performed on RNA from 4 qualified normal LF and 4 qualified OLF tissues.2.Identification of differentially expressed m RNA in OLF: we identified the deregulated m RNAs(fold change > 2,p < 0.05)in OLF with microarray analysis.Ten m RNAs were randomly selected to verify the microarray data in extension samples by q PCR.The GO and KEGG pathway analyses were conducted to confirm whether these deregulated m RNAs were involved in the ossification.3.Identification of differentially expressed lnc RNA in OLF: we identified the deregulated lnc RNAs(fold change > 2,p < 0.05)in OLF with microarray analysis.Ten lnc RNAs were randomly selected to verify the microarray data in extension samples by q PCR.During the process of h MSCs differentiated into osteoblasts,knockdown of two selected lnc RNAs with the respective si RNA examine the changes of the expressions of the pro-osteogenic differentiation-associated genes and the ALP staining.Ten selected m RNAs together with co-expressed lnc RNAs based on the degree of correlation were used to construct a CNC network.4.Identification of differentially expressed mi RNA in OLF: we identified the deregulated mi RNAs(fold change > 2,p < 0.05)in OLF with mi RNA sequencing analysis.Ten mi RNAs were randomly selected to verify the differential expression in extension samples by q PCR.During the process of h MSCs differentiated into osteoblasts,over-expression of one selected mi RNA with mi RNA mimics examine the changes of the expressions of the pro-osteogenic differentiation-associated genes and the ALP staining.The Target Scan,mi RBase,and mi Randa databases were used to predict mi RNA targets.We used the deregulated m RNAs to overlap with the mi RNA targets,and only genes that negatively correlated with their corresponding mi RNAs were included,to construct the mi RNA-m RNA network.5.Identification of differentially expressed circ RNA in OLF: we identified the deregulated circ RNAs(fold change>2,p< 0.05)in OLF with microarray analysis.Ten circ RNAs were randomly selected to verify the microarray data in extension samples by q PCR.During the process of h MSCs differentiated into osteoblasts,knockdown of one selected circ RNA with the si RNA examine the changes of the expressions of the pro-osteogenic differentiation-associated genes and the ALP staining.6.The level of mi R-342-3p in osteogenic differentiation: we examine the expression of ALP,OCN and mi R-342-3p by q PCR in OLF,plasma of patients,femurs of mice and the differentiated osteoblasts.7.The function of mi R-342-3p on osteogenic differentiation: examine the changes of the pro-osteogenic differentiation associated genes,the ALP staining and the activity,and the Alizarin red staining during the process of h MSCs and MC3T3-E1 differentiated into the osteoblasts after knockdown or overexpression of mi R-342-3p by transfection with antagomir or agomir.8.The mechanism of mi R-342-3p on osteogenic differentiation: after knockdown or overexpression of mi R-342-3p in h MSCs we examined the expression of ATF3 by q PCR and Western Blot.Examine the changes of the pro-osteogenic differentiation associated genes,the ALP staining and the activity,and the Alizarin red staining during the process of h MSCs differentiated into the osteoblasts after knockdown or overexpression of ATF3 by infected with lentivirus.Ch IP assay was performed to investigate the mechanism of ATF3 on the targets.9.The mechanism of the deregulation of mi R-342-3p: examine the expression of EVL and mi R-342-3p by q PCR during the process of osteoblast differentiation.We evaluated the methylation of the Cp G island of the EVL gene in h MSCs and h FOB1.19 cells using methylation-specific PCR(MSP).Results:1.Throuth m RNA,lnc RNA and circ RNA microarrays and mi RNA sequencing,a total of 2054 m RNAs,2567 lnc RNAs,627 circ RNAs and 28 mi RNAs were altered during the process of OLF.2.lnc RNA-m RNA co-expression network,mi RNA-m RNA target prediction network,and competing endogenous RNA(ce RNA)network of circ RNA-mi RNA-m RNA were constructed based on a correlation analysis of the differentially expressed RNA transcripts.3.A deregulated mi RNA-19b-3p-based mi RNA-circ RNA-lnc RNA-m RNA network was confirmed by gain-of-function and loss-of-function experiments in the process of osteoblast differentiation.4.mi R-342-3p was found to be deregulated with ossification or osteoporosis.5.mi R-342-3p promotes osteoblast activity and matrix mineralization.6.mi R-342-3p directly targets ATF3,which inhibits transcription of pro-osteogenic differentiation-associated genes.7.Demethylation of the EVL Cp G island induces overexpression of mi R-342-3p during osteogenic differentiation.Conclusion:In the present study,we identified the deregulated m RNAs,lnc RNAs,mi RNAs and circ RNAs in the process of OLF.The lnc RNA-m RNA co-expression network,mi RNA-m RNA target prediction network,and competing endogenous RNA(ce RNA)network of circ RNA-mi RNA-m RNA were constructed based on the correlation analysis of the differentially expressed RNA transcripts.mi R-342-3p was found to be deregulated with ossification or osteoporosis.We found that mi R-342-3p directly targets ATF3,which inhibits transcription of pro-osteogenic differentiation-associated genes,to promote osteoblast activity and matrix mineralization.In addition,it is the demethylation of the EVL Cp G island that induced overexpression of mi R-342-3p during osteogenic differentiation.Taken together,this study provides a systematic perspective on the potential function of nc RNAs in the pathogenesis of OLF,and suggests that mi R-342-3p may serves as a potential marker for diagnosis and treatment of OLF and osteoporosis. |
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