The Initial Confirmation Of LncRNAs Contributing To Proliferation And Osteogenic Differentiation Of Human Periodontal Ligament Stem Cell Inducing By Icariin

Objective: Icaiin,an active flavonoid extracted from Chinese medicinal herb Epimedii, with clear chemical structure, which the role of clinical efficacy have been verified. Icaiin confirmed promote bone marrow mesenchymal stem cells into osteoblasts. Long non-coding RNA is a kind of non-coding RNA which is longer than 200 nucleotides with regulating fuction. Recent evidence suggests that a complex network of transcription factors, chromatin regulators, and noncoding RNAs exist in pluripotent cells to regulate the balance between self-renewal and multilineage differentiation. Lnc RNA play a pivotal role in the regulation of stem cell differentiation and proliferation.This study was designed to screen out the optimal lnc RNAs which may play a vital role in the osteogenic differentiation process of h PDLSCs inducing by Icariin and study these lnc RNAs’ mechanism of action.Materials and Methods:The primary h PDLSCs were identified using flow cytometry analysis; on this basis, using Alizarin Red staining confirmed that h PDLSCs have the ability to differentiate multiple. Alizarin Red staining and q RT-PCR was used to confirmed that icariin has osteoinduction activity specific to h PDLSCs. In the end, we achived the expression profiles of icariin treated cells at day 0 and day 21 and performed a fold change of more than two times screening between the two groups obtained in this experiment. The differently expressed lnc RNAs’ structure, function and regulatory mode was combined with microarray analysis as well as literatures to locate targets at 2 lnc RNAs. Meanwhile, using the method of bioinformatics analysis, we get these 2 lnc RNAs’ target gene. The result of microarray was validated by q RT-PCR tests.Result: Calcium nodules were observed under an optical microscope with Alizarin Red; Lnc RNA microarray and m RNA microarray was carried out to detect the difference of expression profile of m RNA and lnc RNAs between two groups of human periodontal ligament stem cells respectively be treated with icariin at the concentration of 10-7mol/L at day 0 and day 21. Then we performed a fold change screening between the m RNAs and lnc RNAs at different groups. Fold Change≥2.0, P-value≤0.05 or Fold Change≤2.0, P-value≤0.05 is the threshold we used to screen up or down regulated lnc RNAs and m RNA. By comparing differentially expressed m RNA level of day 0 group and day 21 group, 117 m RNAs disordered, 78 up-regulated while 39 down-regulated by more than 2 times. By comparing lnc RNA, 48 disordered, 39 up-regulated while 9 down-regulated. lnc-COL14A1-1, lnc-USP25-2 were selected as the study objects. Bioinformatics analysis was employed to predict the target genes of two selected lnc RNAs. Real-time quantitative reverse transcriptase-polymerase chain reaction was used to validated the expression levels of m RNA and lnc RNA were agreed with gene chips.Conclusion:1×10-7mol/L of Icaiin may has excellent osteoinduction activity specific to h PDLSCs. According to the Co-expression network and lnc RNA target predition results,two lnc RNAs predicted related to osteogenic genes may play a key role in the osteogenic process of h PDLSCs. Icariin promoted the proliferation and differentiation of h DPSCs through alteration of gene expression profiles.