Volume 24, Issue 10, Pages 1726-1733 (October 2016) The TMSB4 Pseudogene LncRNA Functions as a Competing Endogenous RNA to Promote Cartilage Degradation in Human Osteoarthritis  Qiang Liu, Xiaoqing Hu, Xin Zhang, Linghui Dai, Xiaoning Duan, Chunyan Zhou, Yingfang Ao  Molecular Therapy  Volume 24, Issue 10, Pages 1726-1733 (October 2016) DOI: 10.1038/mt.2016.151 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 The differentially expressed genes in the different cartilage regions. (a) The expression of lncRNA-MSR was analyzed using quantitative polymerase chain reaction (qPCR). The ΔCt values were used to measure gene expression, which was normalized to the expression level of GAPDH. (Intact region group = 50, damaged region group = 50, *P < 0.05, **P < 0.01). (b) The expression of TMSB4, COL2A1, ACAN, MMP13, and ADAMTS5 were analyzed using qPCR. The results are shown as the mean ± standard error of the mean of at least three independent experiments (*P < 0.05, **P < 0.01). Molecular Therapy 2016 24, 1726-1733DOI: (10.1038/mt.2016.151) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 The newly identified lncRNA-MSR in chondrocytes is stimulated by mechanical stress. (a) The expression of lncRNA-MSR in chondrocytes by cyclic tensile strain (CTS) at a magnitude of 5% and 10% was examined using Northern blotting. ACTB was used as an internal control. (b) The chondrocytes were stimulated with mechanical stress for the indicated times. The expression of COL2A1, ACAN, TMSB4, and MMP13 were analyzed using qPCR at 0, 4, 12, and 24 hours (the 0 hour timepoint was used for comparisons). (c) The expression of TMSB4 and lncRNA-MSR in chondrocytes were analyzed using qPCR at the various magnitudes of CTS. (d) The expression of TMSB4 and lncRNA-MSR in chondrocytes were analyzed using qPCR for the various durations of CTS. The results are shown as the mean ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01). Molecular Therapy 2016 24, 1726-1733DOI: (10.1038/mt.2016.151) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The effects of lncRNA-MSR on TMSB4 expression and ECM in chondrocytes. (a, b) The mRNA expression of TMSB4, COL2A1, ACAN, MMP13, and ADAMTS5 were determined after overexpression of lncRNA-MSR or siRNA knockdown of lncRNA-MSR. (c) The chondrocytes were transfected with p-lncMSR or si-MSR, and the protein expression of COL2A1 and MMP-13 was analyzed using Western blotting. GAPDH was used as loading control. (d, e) The chondrocytes were transfected with p-lncMSR or si-MSR, and the cells were fixed and stained with rhodamine phalloidin and Hoechst 33342. Bar = 100 μm. The results are shown as the mean ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01 p-lncMSR versus control (treated with empty plasmid) and si-MSR versus the negative control (NC) siR-Ribo). Molecular Therapy 2016 24, 1726-1733DOI: (10.1038/mt.2016.151) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 LncRNA-MSR is targeted by TMSB4-targeting miR-152. (a) TMSB4 and lncRNA-MSR 3'-UTRs contain a highly conserved domain. Targeted miRNA seed matches within the high homology region are conserved between TMSB4 and lncRNA-MSR. The miRNAs binding to TMSB4 and lncRNA-MSR are matched by solid lines. (b, c) The luciferase reporter analysis of either the wild-type or mutant TMSB4 and lncRNA-MSR 3'-UTR activity. The miRNAs were cotransfected with the wild-type or mutant vector, and scrambled miRNA was used as a negative control. (d) The primary chondrocytes were transfected with the miR-152 mimic or inhibitor. Scrambled miRNA was used as a negative control. The expression of lncRNA-MSR and TMSB4 was analyzed using qPCR. (e) The protein expression of TMSB4 was analyzed using western blotting with GAPDH as loading control. The results are shown as the mean ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01). Molecular Therapy 2016 24, 1726-1733DOI: (10.1038/mt.2016.151) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 The effects of lncRNA-MSR on TMSB4 expression as a ceRNA in chondrocytes. (a) The chondrocytes were transfected with p-lncMSR or cotransfected with p-lncMSR and the miR-152 mimics. The expression of TMSB4 was determined using qPCR. (b) The chondrocytes were transfected with si-MSR or cotransfected with si-MSR and the miR-152 inhibitor. The expression of TMSB4 was determined using qPCR. (c, d) The protein expression of TMSB4 was analyzed using western blotting. GAPDH was used as loading control. The results are shown as the mean ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01). Molecular Therapy 2016 24, 1726-1733DOI: (10.1038/mt.2016.151) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 The mechanism of lncRNA-MSR regulation of ECM expression by functioning as a ceRNA. (a) The chondrocytes were cotransfected with the p-lncMSR and miR-152 mimics or cotransfected with p-lncMSR and si-TMSB4. The expression of ECM was analyzed using qPCR. (b) The protein expression of COL2A1 and MMP-13 was analyzed using Western blotting. (c) The chondrocytes were cotransfected with p-lncMSR and miR-152 inhibitor or cotransfected with si-MSR and p-TMSB4. The expression of ECM was analyzed using qPCR. (d) The protein expression of COL2A1 and MMP-13 was analyzed using western blotting. The findings in B and D were quantitatively analyzed using western blot images. The results are shown as the mean ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01). (e) The chondrocytes were cotransfected with the p-lncMSR and miR-152 mimics or cotransfected with p-lncMSR and si-TMSB4, and the cells were stained with rhodamine phalloidin. Bar = 50 μm. (f) The chondrocytes were co-transfected with si-MSR and miR-152 inhibitor or cotransfected with si-MSR and p-TMSB4, and cells were stained with rhodamine phalloidin. Bar = 50 μm. (g) A schematic model showing that lncRNA-MSR was stimulated by mechanical stress and regulated TMSB4 expression through the binding of miR-152, which induced cytoskeleton disassembly and ECM degradation. Molecular Therapy 2016 24, 1726-1733DOI: (10.1038/mt.2016.151) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions