Atractylodes Lancea Volatile Oils Target ADAR2-miR-181a-5p Signaling to Mesenchymal Stem Cells Chondrogenic Differentiation

6 Background ： The Rhizoma Atractylodis has long been recommended for the treatment 7 of different diseases in traditional Chinese medicine. The main component of Rhizoma 8 Atractylodis is Atractylodes lancea volatile oils which possess anti-microorganism, 9 anti-tumour, cognitive protection and immunoregulation. The study aimed to elucidate 10 the mechanism of Atractylodes lancea volatile oils promoting mesenchymal stem cells 11 (MSCs) chondrogenic differentiation. 12 Method ： lancea volatile oils were extracted from Chinese medicine Cangzhu by volatile oil extractor. MSCs culture were treated with Atractylodes lancea volatile oils medium . Real-time reverse transcription PCR was conducted to verify the 16 candidate microRNAs discovered by microarray analysis. Western-blot analyzed the 17 expressions of mark genes. Sanger sequences identified the changes of the base pairs, 18 which would be edited by ADAR2 enzyme. Toluidine blue staining identified the 19 changes in cells chondrogenic differentiation. chondrogenic differentiation of MSCs. Atractylodes lancea volatile oils promoted the expression of 23 ADAR2 enzyme, which may edit the precursor of miR-181a-5p. A dual-luciferase 24 reporter system assay verified that transcription factors yingyang1(YY1) was targeted 25 by miR-181a-5p which was downregulated in MSCs chondrogenic differentiation. 26 27 Conclusion ： This work demonstrates the mechanism of Atractylodes lancea volatile 28 oils, promoting MSCs to chondrogenic differentiation. It may provide an alternative 29 strategy for treatment purposes and diagnosis in the clinic. replaced in the pLUc-YY1 MUT plasmid. (D) Western blotting identified the most 498 effective fragment of YY1 in silence. The grouping of gels cropped from different parts 499 of the same gel with target gene and β-actin. * P <0.05 vs. nc group (E)Cells were treated with NC, siADAR2, siYY1, siADAR2 and siADAR2+siYYY1 group. The chondrocytes genes were analyzed by q-pcr to determine the differential expression in siADAR2 and siYY1. *** P <0.05 vs. NC group, **** P <0.01 vs. NC group (F) The 503 toluidine blue identified the MSCs chondrogenic differentiation in treatment with siADAR2 and siYY1.

differentiation of MSCs. Atractylodes lancea volatile oils promoted the expression of 23 ADAR2 enzyme, which may edit the precursor of miR-181a-5p. A dual-luciferase 24 reporter system assay verified that transcription factors yingyang1(YY1) was targeted 25 by miR-181a-5p which was downregulated in MSCs chondrogenic differentiation.  alterations, which mainly concern an imbalance in tissue remodelling due to defection 53 in chondrocyte behaviour [5]. Moreover, the previous study has reported that an          Reverse TGGTGGTGGTGGTGGTGATGG.

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The expression of mature miRNAs was determined using the 2 -△△Ct method, using 168 U6 as a reference gene. To identify A-to-I changes in the precursor sequences, PCR 169 products from the analysis of pri-and pre-miRNA-181a-5p were tested by Sanger    MSCs aggregates were stained with toluidine blue. Compared with the control group 242 and 0.3μg/ml treated group, the 3μg/ml treated group had significantly increased 243 toluidine blue staining (Fig.2D).

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The Atractylodes lancea volatile oils impact the expression of ADAR enzyme. 245 There are three subtypes of ADAR enzymes existing, ADAR1, ADAR2 and ADAR3.

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The fold change of miR-181a-5p mimic was measured by q-PCR. The expression 290 of miR-181a-5p inhibitor was upregulated comparing with the control group (Fig.5A).

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The relative expression of miR-181-5p was measured by western blotting. The for 24h was the most effective segment to silence the expression of YY1 (Fig.6D).

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Compared with the control group, the expressions of SOX9, COLLAGEN2 and were 330 significantly decreased in the siRNA-ADAR2 and siRNA-YY1 group, and the 331 expression of miR-181a-5p increased significantly in the siRNA-ADAR2 and siRNA-332 YY1 group (Fig.6E). These results indicated that miR-181a-5p might promote MSCs 333 chondrogenic differentiation with silencing ADAR2 and YY1 expression. Moreover, 334 the stain of toluidine blue showed that the degree dyeing of MSCs with siADAR2, 335 siYY1 and siADAR2+siYY1 were all lighter than negative control groups. (Fig.6F). 336 These results showed that ADAR2 and YY1 were upregulated factors in MSCs 337 chondrogenic differentiation.       The MSCs were seeded at a density of 1.210 6 in 6-well plates for 7days.The toluidine 460 blue staining was used for chondrogenic differentiation, see the higher panel.       Atractylodes lancea volatile oils enhance MSCs chondrogenic differentiation. (A)The MSCs were seeded at a density of 4×104 cells in 96-well plates. Cells were treated as control, DMSO, 0.3μg/ml,3μg/ml,15μg/ml,30μg/ml groups. The CCK8 assay analyzed the cytotoxicity of Atractylodes lancea volatile oils for MSCs chondrogenic differentiation. ****P<0.05 vs. control group (B) The MSCs were seeded at a density of 2.1×106 cells in 6mm dishes for 7days and treated with different concentrations of Atractylodes lancea volatile oils. Cells were then harvested, and the mRNA levels of SOX-9, type II collagen and Aggrecan were analyzed by q-PCR. *, **, *** P<0.05, ****P<0.001 vs. control group (C)The protein levels of SOX-9, type II collagen and Aggrecan were analyzed by Western blot. The grouping of gels cropped from different parts of the same gel with target gene and β-actin. *, ** P<0.05 vs. control group (D) The MSCs were seeded at a density of 1.2 × 106 in 6-well plates for 7days.The toluidine blue staining was used for chondrogenic differentiation, see the higher panel. Figure 3 ADAR2 enzymes express positively in Atractylodes lancea volatile oils(Atr). The MSCs were seeded at a density of 7×106 cells in 10mm dishes and treated with Atractylodes lancea volatile oils. (A) The protein levels of ADAR1, ADAR2 and ADAR3 were analyzed by Western blot. The grouping of gels cropped from different parts of the same gel with target gene and β-actin. * P<0.05 vs. control group (B) The mRNA levels of ADAR1, ADAR2 and ADAR3 were analyzed by q-pcr. **, *** P<0.05 vs. control group (C)Cells were treated with silent segments of ADAR2 and then analyzed by Western blot to identify the most effective segment. * P<0.05 vs. control group (D)Cells were treated with control, siADAR2, Atr and siADAR2+Atr group, and then analyzed by Western blot to identify the effect of siADAR2 in chondrogenic differentiation with Atractylodes lancea volatile oils. The grouping of gels cropped from different parts of the same gel with target gene and β-actin. *, ** P<0.05, ****P<0.001 vs. control group, #### P<0.001vs. Atr group (E) The toluidine blue staining identi ed the MSCs chondrogenic differentiation in treatment with siADAR2 and Atractylodes lancea volatile oils.  miR-181a-5p downregulates the MSCs chondrogenic differentiation. Cells were treated with mimic and inhibitor of miR-181a-5p. (A)The mRNA levels of SOX-9, type II collagen and Aggrecan were analyzed by q-pcr. *, *** P<0.05, ****P<0.001 vs. control group (B)The protein levels of SOX-9, type II collagen and Aggrecan were analyzed by Western blot. The grouping of gels cropped from different parts of the same gel with target gene and β-actin. *, ** P<0.05 vs. control group (C)The toluidine blue identi ed the MSCs chondrogenic differentiation in treatment with miR-181a-5p. (D) Cells were treated with control, Atr and siADAR2 group, and then analyzed by q-pcr and Sanger sequence. *, *** P<0.05 vs. control group Figure 6 The 3'UTR of YY1 was the speci c target of miR181a-5p. (A)The protein level of YY1 was analyzed by Western blot. The grouping of gels cropped from different parts of the same gel with target gene and βactin. *, *** P<0.05 vs. control group (B) miR-181a-5p reduced the relative luciferase activity of pLUc-YY1.