LncRNA MALAT1 promotes osteoarthritis by modulating miR-150-5p/AKT3 axis

Abstract

Background: Many studies have reported that long noncoding RNAs (lncRNAs) could act as sponges for microRNAs (miRNAs) and play important roles in the regulation of osteoarthritis (OA). Yet, the underlying mechanisms of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in OA are still unclear. Therefore, we aimed to explore the regulation mechanisms of MALAT1 in OA procession.

Methods: IL-1β treatment in chondrocyte was used to mimic OA in vitro. MALAT1, miR-150-5p and AKT3 expression levels were detected via qRT-PCR. The protein levels of AKT3, MMP-13, ADAMTS-5, Bax, Bcl-2, cleaved-PARP, collagen II and aggracan were measured by western blot. MTT assay was performed to detect cell proliferation ability. The apoptosis of chondrocytes was determined using flow cytometry and western blot. Luciferase assay and RNA immunoprecipitation (RIP) assays were used to confirm the relationship among MALAT1, miR-150-5p and AKT3.

Results: In our study, MALAT1 and AKT3 were upregulated while miR-150-5p was downregulated in OA in vitro and vivo. The level of miR-150-5p was negatively correlated with that of MALAT1 or AKT3. More importantly, overexpression of MALAT1 promoted the expression of AKT3 by negatively regulating miR-150-5p. MALAT1 knockdown inhibited cell proliferation, promoted apoptosis, increased MMP-13, ADAMTS-5 expression and decreased collagen II, aggracan expression in IL-1β treated chondrocytes. MALAT1 upregulation or AKT3 overexpression enhanced proliferation, inhibited apoptosis and extracellular matrix (ECM) degradation, which was undermined by overexpression of miR-150-5p. By contrast, miR-150-5p depletion rescued the effect of MALAT1 downregulation or loss of AKT3 on IL-1β-stimulated chondrocytes.

Conclusion: MALAT1 was responsible for cell proliferation, apoptosis, and ECM degradation via miR-150-5p/AKT3 axis.

Keywords: AKT3; MALAT1; MALAT1/miR-150-5p/AKT3; OA; miR-150-5p.

Fig. 1

Different expression levels of MALAT1, miR-150-5p and AKT3 in normal and OA cartilage chondrocytes. a, b The expression levels of MALAT1, miR-150-5p and AKT3 in OA and normal cartilage tissues were detected by qRT-PCR and western blot. Samples were from 42 OA patients and 20 healthy subscribers. c Spearman was used to analyze the relationship between MALAT1, miR-150-5p and AKT3. d, e qRT-PCR and western blot were used to examine the levels of MALAT1, miR-150-5p and AKT3 in chondrocytes with treatment of 10 ng/mL IL-1β for 4, 6, 12, and 24 h. *P < 0.05

Fig. 2

MALAT1 indirectly regulates AKT3 by targeting miR-150-5p. a The surmised binding sites for interaction between miR-150-5p and MALAT1. b Dual luciferase reporter assay was performed to conform the interaction between MALAT1 and miR-150-5p. c Anti-Ago2 RIP assay was carried out to identify the correlation between MALAT1 and miR-150-5p. d The surmised binding sites for correlation between miR-150-5p and AKT3. e Dual luciferase reporter assay was performed to confirm the interaction between miR-150-5p and AKT3. f Anti-Ago2 RIP assay was carried out to identify the correlation between miR-150-5p and AKT3. *P < 0.05

Fig. 3

MALAT1 competitively bound to microRNA-150-5p and indirectly promotes AKT3 expression. a–c Chondrocytes were introduced with miR-150-5p mimic, miR-NC, anti-miR-150-5p, anti-miR-NC, NC, or MALAT1 before IL-1β treatment. qRT-PCR was used to measure the level of miR-150-5p. d–i Western blot analysis of AKT3 in each group. *P < 0.05

Fig. 4

MALAT1 depletion inhibits cell proliferation and enhances cell apoptosis during OA progression. a qRT-PCR of MALAT1 after the transfection of si-MALAT1. 10 ng/mL IL-1β used to induce OA. b MTT assay was applied to measure the proliferation ability of chondrocytes after transfecting with si-MALAT1 and the stimulation of 10 ng/mL IL-1β for 24 h. c, d Apoptosis of chondrocytes was examined by flow cytometry after the same treatment in b. si-NC: treated with si-MALAT1 scramble; control: not treated with IL-1β. e Western blot was used to examine the levels of apoptosis-related protein cleaved-PARP, Bax, and Bcl-2. *P < 0.05

Fig. 5

MALAT1 knockdown promotes the ECM degradation of chondrocytes in OA. The protein expression levels of MMP-13, ADAMTS-5, collagen II and aggrecan detected by western blot after transfecting with si-MALAT1 and stimulating with IL-1β (10 ng/mL) for 24 h. β-actin was used as an internal control. si-NC: treated with si-MALAT1 scramble; Control: not treated with IL-1β. *P < 0.05

Fig. 6

MiR-150-5p reverses the effects of MALAT1 or AKT3 on proliferation and apoptosis in OA chondrocytes. a Western blot was performed to detect the protein level of AKT3 in chondrocytes transfected with si-AKT3, AKT3 overexpression plasmid, or their negative controls before IL-1β treatment. b, c MTT assay and flow cytometry were performed to detect cell proliferation ability and apoptosis, respectively. Chondrocytes were transfection with NC + miR-NC, MALAT1 + miR-NC, NC + miR-150-5p, MALAT1 + miR-150-5p, miR-NC + AKT3, miR-150-5p + AKT3, anti-NC + anti-NC, si-MALAT1 + anti-NC, si-NC + anti-miR-150-5p, si-MALAT1 + anti-miR-150-5p, anti-NC + si-AKT3, and miR-150-5p + si-AKT3 before IL-1β treatment. *P < 0.05

Fig. 7

MiR-150-5p reverses the effects of MALAT1 or AKT3 on ECM degradation in OA. a, b Western blot of MMP-13, ADAMTS-5, collagen II and aggrecan after transfection with NC + miR-NC, MALAT1 + miR-NC, NC + miR-150-5p, MALAT1 + miR-150-5p, miR-NC + AKT3, miR-150-5p + AKT3, anti-NC + anti-NC, si-MALAT1 + anti-NC, si-NC + anti-miR-150-5p, si-MALAT1 + anti-miR-150-5p, anti-NC + si-AKT3, and miR-150-5p + si-AKT3 before IL-1β treatment. *P < 0.05

Fig. 8

The schematic diagram of in MALAT1/MiR-150-5p/AKT3OA axis in OA. Lnc RNA MALAT1 could be act as a ceRNA of miR-150-5p to regulate the expression of AKT3, thus modulating the ECM degradation and cell apoptosis