LncRNA RMRP promotes chondrocyte injury by regulating the FOXC1/RBP4 axis

Abstract

Introduction: The main pathological feature of osteoarthritis (OA) is chondrocyte injury. LncRNA mitochondrial RNA processing endoribonuclease (RMRP) has been shown to be a chondrogenic differentiation factor. This study aimed to explore the role of RMRP in chondrocyte injury.

Material and methods: Cell counting kit-8 (CCK-8) and TUNEL assays were used to determine lipopolysaccharide (LPS)-induced chondrocyte viability and apoptosis, respectively. The interaction between RMRP and FOXC1 was analyzed by RIP and RNA pull-down. Dual luciferase reporter and ChIP were employed to analyze the interaction between FOXC1 and RBP4. The levels of RMRP, FOXC1, RBP4, apoptosis-related and extracellular matrix (ECM)-related genes were detected by RT-qPCR and western blot. ELISA assay was used for detection of inflammatory cytokines in LPS-induced chondrocytes.

Results: The levels of RMRP, FOXC1 and RBP4 were significantly upregulated in OA cartilage tissues and LPS-induced chondrocytes. Knockdown of RMRP inhibited chondrocyte apoptosis and inflammation under LPS. RMRP interacted with FOXC1 and promoted RBP4 expression. FOXC1 could upregulate RBP4 and promote LPS-induced chondrocyte apoptosis and inflammation. Similarly, RMRP combined with FOXC1 and aggravated apoptosis and inflammation in LPS-treated chondrocytes.

Conclusions: RMRP promoted upregulation of RBP4 and activation of the JNK signaling pathway by binding to FOXC1, thereby accelerating LPS-induced apoptosis and inflammation in chondrocytes.

Keywords: FOXC1; RBP4; chondrocyte injury; lncRNA RMRP; osteoarthritis.

Fig. 1

RMRP, FOXC1 and RBP4 were upregulated in osteoarthritis (OA) and LPS-induced chondrocytes. Knee cartilage tissues (human) were collected from patients with OA (n = 10) and amputees without a history of OA (n = 10). A) RT-qPCR was used to detect the level of RMRP in the cartilage tissues (n = 10). *p < 0.05, **p < 0.01 and ***p < 0.001 B) Expression of FOCX1 and RBP4 in cartilage tissues was detected by western blot (n = 10). The OA cell models were constructed by treating chondrocytes derived from knee cartilage tissue of amputees without a history of OA with 5 μg/ml of LPS for 12 hours. C) CCK-8 assay was used to analyze cell viability (n = 3). D) RT-qPCR was employed to detect the expression of RMRP (n = 3). E) FOCX1 and RBP4 protein levels were checked by western blot (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 2

RMRP knockdown inhibited LPS-induced chondrocyte apoptosis and inflammation. sh-RMRP or sh-NC was transfected into chondrocytes isolated from cartilage tissues of amputees with no history of osteoarthritis (OA) and treated with 5 μg/ml LPS. A) RMRP levels were detected by RT-qPCR (n = 3). B, C) Cell viability and apoptosis were assessed by CCK-8 and TUNEL assays (scale bar = 100 μm) (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001. D) Levels of TNF-α, IL-1β, IL-6 and IL-8 were examined by ELISA kit (n = 3). E) Western blot was used to analyze the expression of Bax, Bcl-2, caspase 3, MMP13, collagen II, aggrecan, JNK and p-JNK (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 3

RMRP bound to FOXC1 and promoted RBP4 expression. A, B) RIP and RNA pull-down assays were employed to verify the interaction between RMRP and FOXC1 (n = 3). C) JASPAR analyzed the potential binding site between FOXC1 and the RBP4 promoter region. D, E) The binding relationship between FOXC1 and RBP4 was examined by dual luciferase reporter and ChIP (n = 3). **p < 0.01 and ***p < 0.001. F, G) RT-qPCR and western blot were used to detect the RBP4 level (n = 3). **p < 0.01 and ***p < 0.001

Fig. 4

FOXC1 upregulated RBP4 to promote apoptosis and inflammation in LPS-induced chondrocytes. LPS-treated chondrocytes were transfected with sh-RBP4 and/or oe-FOXC1. A) Expression of RBP4 and FOXC1 was detected by RT-qPCR (n = 3). B) Cell viability was assessed using CCK-8 assay (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001. C) TUNEL assay was used to assess apoptosis (scale bar = 100 μm) (n = 3). D) Levels of TNF-α, IL-1β, IL-6, and IL-8 were detected using an ELISA kit (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001. E) Expression of FOXC1, RBP4, Bax, Bcl-2, caspase 3, MMP13, collagen II, aggrecan, JNK, and p-JNK was detected by western blot (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001. E) Expression of FOXC1, RBP4, Bax, Bcl-2, caspase 3, MMP13, collagen II, aggrecan, JNK, and p-JNK was detected by western blot (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 5

RMRP combined with FOXC1 to promote apoptosis and inflammation of osteoarthritis (OA) chondrocytes. LPS-treated chondrocytes were transfected with sh-FOXC1 and oe-RMRP. A) RT-qPCR was used to determine the expression of RMRP and FOXC1 (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001. B) CCK-8 was used to detect cell viability (n = 3). C) Cell apoptosis was checked by TUNEL assay (scale bar = 100 μm) (n = 3). D) Levels of TNF-α, IL-1β, IL-6 and IL-8 were detected by ELISA kit (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 E) Western blot was used to detect the expression of FOXC1, RBP4, Bax, Bcl-2, caspase 3, MMP13, collagen II, aggrecan, JNK and p-JNK (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 E) Western blot was used to detect the expression of FOXC1, RBP4, Bax, Bcl-2, caspase 3, MMP13, collagen II, aggrecan, JNK and p-JNK (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001