How miR-31-5p and miR-33a-5p Regulates SP1/CX43 Expression in Osteoarthritis Disease: Preliminary Insights

Abstract

Osteoarthritis (OA) is a degenerative bone disease that involved micro and macro-environment of joints. To date, there are no radical curative treatments for OA and novel therapies are mandatory. Recent evidence suggests the role of miRNAs in OA progression. In our previous studies, we demonstrated the role of miR-31-5p and miR-33a families in different bone regeneration signaling. Here, we investigated the role of miR-31-5p and miR-33a-5p in OA progression. A different expression of miR-31-5p and miR-33a-5p into osteoblasts and chondrocytes isolated from joint tissues of OA patients classified in based on different Kellgren and Lawrence (KL) grading was highlighted; and through a bioinformatic approach the common miRNAs target Specificity proteins (Sp1) were identified. Sp1 regulates the expression of gap junction protein Connexin43 (Cx43), which in OA drives the modification of i) osteoblasts and chondrocytes genes expression, ii) joint inflammation cytokines releases and iii) cell functions. Concerning this, thanks to gain and loss of function studies, the possible role of Sp1 as a modulator of CX43 expression through miR-31-5p and miR-33a-5p action was also evaluated. Finally, we hypothesize that both miRNAs cooperate to modulate the expression of SP1 in osteoblasts and chondrocytes and interfering, consequently, with CX43 expression, and they might be further investigated as new possible biomarkers for OA.

Keywords: CX43; SP1; chondrocytes; microRNAs; osteoarthritis; osteoblasts.

Figure 1

Investigation of osteoblasts (OB) and chondrocytes (CH) isolated by tissue debris of mild OA and severe OA patients through qRT-PCR expression analysis of (A) miR-31-5p and miR-33a-5p; (B) miR-31-5p OB targets genes: RHO and HIF-1AN; and (C) miR-33a-5p CH targets genes: COL2A1 and HMGA2. Quantitative RT-PCR data are expressed as fold of change (FOI) in miRNAs or gene expression (2^−ΔΔCt) occurred in severe OA-derived cells vs mild OA-derived cells. Data reported were analysed by Student t test: *, p < 0.05, **, p < 0.005, ***, p < 0.0005 between experimental group and represented as mean ± SD (n = 4).

Figure 2

Analysis of miRNAs common target SP1 by the bioinformatics tool miRTargetLink Human. The central node represents Sp1 and other common targets, surrounded by the miRNAs that target Sp1 with strong (green) and weak (blue) evidence.

Figure 3

Analysis of SP1 gene and protein expression on osteoblasts (OB) and chondrocytes (CH) derived by patients with mild OA and severe OA. (A) qRT-PCR analysis of SP1 gene expression. Data are expressed as fold of change (FOI) in gene expression (2^−ΔΔCt) occurred in severe OA vs mild OA isolated cells. Data reported were analysed by Student t test: *, p < 0.05, between experimental group and represented as mean ± SD (n = 4). (B,C) Western blot and densitogram analysis of Sp1 and α-tubulin proteins were performed on total cell extract isolate by primary cells derived by mild- and severe OA groups. (D) Confocal analysis of Sp1 protein expression and localization on OB isolated by mild OA and severe OA patients. In green the Sp1 signals while in blue the nuclei localization.

Figure 4

Study of SP1 gene expression modulation by gain and loss of function assays. Osteoblasts (OB) and chondrocytes (CH) isolated by mild OA patients (mild OA) after 24 h of transfection with: miR-31-5p mimic and miR-31-3p mimic for OB cells, with miR-33a-5p mimic and miR-33a-3p mimic for CH cells and relatives scrambles. Cells were analyzed for the gene expression modulation of SP1 by qRT-PCR analysis. (A) The efficiency of miR-31-5p mimic and miR-31-3p mimic and relatives scrambles transfection were evaluated in transfected mild OA-derived OB versus untransfected ones. (B) The efficiency of miR-33a-5p mimic and miR-33a-3p and relatives scrambles transfection were evaluated in transfected mild OA-derived CH versus untransfected ones. SP1 gene expression evaluated in (C) OB isolated from mild OA patients transfected with mimic-31-5p and negative scramble, in (D) CH isolated from mild OA patients transfected with mimic-33a-5p and negative scramble. Quantitative RT-PCR data are expressed as fold of change (FOI) in miRNAs or gene expression (2^−ΔΔCt) occurred in transfected mild OA isolated cells vs untransfected ones. Data reported were analysed by Student t test: *, p < 0.05, **, p < 0.005, ***, p < 0.0005 between experimental group and represented as mean ± SD (n = 4).

Figure 5

Study of SP1 gene expression modulation by gain and loss of function assays. Osteoblasts (OB) and chondrocytes (CH) isolated by OA patients after 24 h of transfection with miR-31-5p inhibitor for OB cells and with miR-33a-5p inhibitor for CH cells and relatives scrambles were analyzed for the gene expression of SP1 by qRT-PCR analysis. (A) The efficiency of miR-31-5p inhibitor and relative scrambles transfection was evaluated in transfected severe OA-derived OB versus untransfected ones. (B) The efficiency of miR-33a-5p inhibitor and relatives scramble transfection was evaluated in transfected severe OA-derived CH versus untransfected ones. SP1 gene expression evaluated in (C) OB isolated from severe OA patients transfected with miR31-5p inhibitor and negative scramble, and in (D) CH isolated from severe OA patients transfected with miR-33a-5p inhibitor and negative scramble. Quantitative RT-PCR data are expressed as fold of change (FOI) in miRNAs or gene expression (2^−ΔΔCt) occurred in transfected OA derived cells vs untransfected ones. Data reported were analysed by Student t test: *, p < 0.05, **, p < 0.005, between experimental group and represented as mean ± SD (n = 4).

Figure 6

Analysis of CX43 gene and protein expression on osteoblasts (OB) and chondrocytes (CH) derived by patients with severe and mild grade OA. (A) qRT-PCR analysis of CX43 gene expression. (B,C) Western blot and densitogram analysis of Cx43 and α-tubulin proteins were performed on total cell extract isolated by OB primary cells. (D) qRT-PCR analysis of CX43 gene expression in OB isolated by mild OA patients and transfected with miR-31-5p mimic and relative scramble. (E) CX43 gene expression analysis by qRT-PCR in OB isolated by severe OA patients and transfected with miR-31-5p inhibitor and relative scramble. qRT-PCR data are expressed as fold of change (FOI) in gene expression (2^−ΔΔCt) occurred in transfected vs untransfected cells. Data reported were analysed by Student t test: *, p < 0.05, **, p < 0.005, ***, p < 0.0005 between experimental group and represented as mean ± SD (n = 4).

Figure 7

Our hypothesis of the molecular mechanism of miR-31-5p and miR-33a-5p on SP1/CX43 regulations in OA condition.