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N. Zhong, J. Sun, Z. Min, W. Zhao, R. Zhang, W. Wang, J. Tian, L

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Presentation on theme: "N. Zhong, J. Sun, Z. Min, W. Zhao, R. Zhang, W. Wang, J. Tian, L"— Presentation transcript:

1 MicroRNA-337 is associated with chondrogenesis through regulating TGFBR2 expression 
N. Zhong, J. Sun, Z. Min, W. Zhao, R. Zhang, W. Wang, J. Tian, L. Tian, J. Ma, D. Li, Y. Han, S. Lu  Osteoarthritis and Cartilage  Volume 20, Issue 6, Pages (June 2012) DOI: /j.joca Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

2 Fig. 1 Histology of left femoral head at different development stages and expression of six miRNAs in cartilage development. (A) Representative histological images of the left femoral heads collected from rats at postnatal day 0 (D0), day 21 (D21) and day 42 (D42) and stained with Safranin O. (B) The expression levels of six miRNAs detected by RT-qPCR in rat articular cartilage at postnatal day 0 (D0), day 21 (D21) and day 42 (D42). n = 10 for each time point. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

3 Fig. 2 Expression down-regulation of miR-337 during the cartilage development. (A) Agarose electropherogram of miR-337 expression in rat femoral heads at postnatal day 0 (D0), day 7 (D7), day 14 (D14), day 21 (D21), day 28 (D28), day 36 (D36) and day 42 (D42). Cartilage was collected from five female and five male DA rats for each development stage. Stem-loop RT-PCR was performed by the pooled RNA from each time point sample and U6 was used as a reference. (B). MiR-337 expression detected by RT-qPCR in rat femoral heads at postnatal day 0 (D0), day 7 (D7), day 14 (D14), day 21 (D21), day 28 (D28), day 36 (D36) and day 42 (D42). miR-337 relative expression was normalized to small RNA U6. n = 10 for each time point. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

4 Fig. 3 miR-337 up-regulation in endochondral ossification model induced by bone morphogenetic gelatin. (A) Representative histological images of endochondral ossification model induced with bone morphogenetic gelatin at 1 (1W), 2 (2W), 3 (3W), and 4 (4W) weeks and stained with HE. (B) MiR-337 expression detected by RT-qPCR in endochondral ossification model induced with bone morphogenetic gelatin at 1 (1W), 2 (2W), 3 (3W), and 4 (4W) weeks. miR-337 relative expression was normalized to snRNA U6. n = 9 for each group. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

5 Fig. 4 Different expression trends of miR-337 in rat cartilage of CIA and PIA. (A) Representative macroscopical and microscopical images of DIP joints of CIA rats. The arthritis was induced in DA rats with homologus collagen type II and IFA. Macroscopical (upper panel) and microscopical (below panel) images were taken at day 26 after arthritis induction. DIP joints from control (left panel) and arthritic rats (right panel) were sectioned and stained with HE. (B) MiR-337 expression detected by RT-qPCR in rat DIP joint cartilage of CIA. (C) Representative macroscopical and microscopical images of ankle joints of PIA rats. The arthritis was induced in DA rats with pristane. Macroscopical (upper panel) and microscopical (below panel) images were taken at day 26 after arthritis induction. Ankle joints from control (left panel) and arthritic rats (right panel) were sectioned and stained with HE. (D) Agarose electropherogram of miR-337 expression in rat ankle joints of PIA. (E) MiR-337 expression in rat ankle joints of PIA by semi-quantitative RT-PCR. U6 was used as reference. n = 10 for each arthritis group and n = 7 for control group. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

6 Fig. 5 Effect of miR-337 on TGFBR2 expression. (A) Schematic sequence match between miR-337 and 3′UTR of TGFBR2 gene. TGFBR2 gene was predicted as a target gene of miR-337 by using TargetScan softwares and 3′UTRs of human, rat and mouse TGFBR2 were well matched with miR-337. (B) Luciferase activities of TGFBR2 reporter gene were obtained in HEK-293A cells which were transfected with luciferase expression plasmids containing 3′UTRs of the predicted miR-337 target site. The cells were also treated with either miR-337 mimic/inhibitor or negative mimic/inhibitor. Twenty-four hours after transfection, the cells were assayed for firefly luciferase and Renilla luciferase test. (C) Three cell lines were transfected with luciferase expression plasmids TGFBR2/pMIR-report. (D) The expression of miR-337 when the C-28/I2 cells were treated with miR-337 mimic and negative mimic after transfected 24 h and 48 h. The mRNA level of TGFBR2 was detected by RT-qPCR at the same time. (E) The miR-337 expression when the cells were treated with miR-337 inhibitor and negative inhibitor after transfected 24 h and 48 h. The mRNA level of TGFBR2 was detected by RT-qPCR at the same time. (F) Western blots for TGFBR2 or β-actin from C-28/I2 cells transfected with 50 nM miR-337 mimic and miR-neg or mock (left panel) and relative band density (right panel). (G) Western blots for TGFBR2 or β-actin from C-28/I2 cells transfected with 200 nM miR-337 and miR-neg or mock (left panel) and relative band density (right panel). The results are shown from a representative of three different experiments. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

7 Fig. 6 Expression changes of cartilage-specific genes in C-28/I2 cells transfected with miR-337 mimic and inhibitor. (A) Cartilage-specific gene expression detected by RT-qPCR analysis C-28/I2 cells tranfected with miR-337 mimic for 24 h. (B) Cartilage-specific gene expression by RT-qPCR analysis C-28/I2 cells tranfected with miR-337 inhibitor for 24 h. The relative expression was normalized against β-actin expression. The experiment was performed at least in triplicate. The results are shown from a representative of three different experiments. Osteoarthritis and Cartilage  , DOI: ( /j.joca ) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

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