Down-regulation of long noncoding RNA HOXA11-AS nullifies the impact of microRNA-506-3p on chondrocytes proliferation and apoptosis in osteoarthritis

Abstract

Objectives: This study was directed towards exploring the impacts of lncRNA HOXA11-AS-mediated microRNA (miR)-506-3p on chondrocytes proliferation and apoptosis in osteoarthritis (OA).

Methods: The articular cartilages were provided by OA patients who received total knee arthroplasty, and Human Chondrocyte (HC)-OA (HCOA) was also attained. The miR-506-3p and HOXA11-AS expressions in articular cartilages from OA patients and HCOA cells were analyzed via qPCR. After gain- and loss-of-function assays in HCOA cells, MTT assay and flow cytometry (FC) were used for assessing cell viability and apoptosis, accordingly. The levels of PIK3CA, AKT, and mTOR as well as AKT and mTOR phosphorylation levels assessed using western blotting (WB). The targeting correlation of HOXA11-AS and miR-506-3p as well as miR-506-3p and PIK3CA was assessed through Dual-Luciferase Reporter gene Assay (DLRA).

Result: The articular cartilages from OA patients and Human Chondrocyte (HC)-OA (HCOA) cells showed increased HOXA11-AS and decreased miR-506-3p. Mechanistically, HOXA11-AS was capable of binding to miR-506-3p to increase PIK3CA, the target gene of miR-506-3p. miR-506-3p suppression facilitated HCOA cell proliferation and reduced their apoptosis, which was nullified by further silencing HOXA11-AS or silencing PIK3CA. The down-regulation of HOXA11-AS disrupted the PI3K/AKT/mTOR pathway, which was counteracted by further miR-506-3p inhibition.

Conclusion: The silencing of HOXA11-AS might block the PI3K/AKT/mTOR pathway through miR-506-3p up-regulation, thereby restricting HCOA cell proliferation and provoking apoptosis.

Keywords: Chondrocytes; Long noncoding rna hoxa11-as; Microrna-506–3p; Osteoarthritis; PI3K/AKT/mTOR pathway; Proliferation.

Fig. 1

HOXA11-AS increase and miR-506–3p decrease are observed in OA articular cartilages. (A) qPCR detection of the HOXA11-AS expression in OA patients’ articular cartilages. (B) The miR-506–3p expression in OA patients’ articular cartilages measured using qPCR. (C) The HOXA11-AS expression in HC—OA and HC cells assessed by qPCR. (D) qPCR to evaluate the miR-506–3p expression in HC—OA and HC cells. (E) Correlation analysis between miR-506–3p and HOXA11-A expression in OA patients’ articular cartilages. *** p < 0.001 vs. normal cartilages or HC cells.

Fig. 2

HOXA11-AS silencing causes repressed HC—OA cell proliferation and enhanced apoptosis. (A) HOXA11-AS expression in HC—OA cells after HOXA11-AS silencing measured using qPCR. (B) HC—OA cell viability after HOXA11-AS silencing detected using MTT assay. (C) HC—OA cell apoptosis rate after HOXA11-AS silencing measured using FC. (D) WB of the expression of apoptosis-relevant proteins in HC—OA cells after HOXA11-AS silencing. *** p < 0.001 vs. the si-NC group.

Fig. 3

HOXA11-AS was capable of binding to miR-506–3p that inversely targets PIK3CA. (A) The predicted binding sites between HOXA11-AS and miR-506–3p as well as miR-506–3p and PIK3CA. (B) The binding correlation evaluated by DLRA and miR-506–3p expression in cells after HOXA11-AS silencing measured by qPCR. (C) DLRA to evaluate the targeting correlation of PIK3CA and miR-506–3p and PIK3CA expression in cells after miR-506–3p mimic transfection. (D) miR-506–3p level following miR-506–3p mimic and/or si-HOXA11-AS transfection assessed using qPCR. (E) PIK3CA expression in cells following si-PIK3CA and/or miR-506–3p inhibitor transfection. a: miR-NC inhibitor; b: si-PIK3CA; c: si-PIK3CA + miR-NC inhibitor; d: si-PIK3CA + miR-506–3p inhibitor. * p < 0.05.

Fig. 4

HOXA11-AS down-regulation inhibits HC—OA cell proliferation and induces apoptosis through orchestrating miR-506–3p/PIK3CA axis. (A) MTT assay to detect HC—OA cell viability after si-HOXA11-AS and miR-506–3p inhibitor transfection. (B) FC of HC—OA cell apoptosis rate after transfection. (C) WB of the expression of apoptosis-relevant proteins in HC—OA cells after transfection. (D) HC—OA cell viability after transfection measured by MTT assay. (E) FC to assess HC—OA cell apoptosis rate after transfection. (F) The expression of apoptosis-relevant proteins in HC—OA cells after transfection evaluated by WB. *p < 0.05.

Fig. 5

HOXA11-AS down-regulation inactivates PI3K/AKT/mTOR pathway through miR-506–3p. (A) PI3K/AKT/mTOR western blot band images. B: Bar graphs of PI3K/AKT/mTOR pathway-related protein expression. 1. The si-NC group; 2. The si-HOXA11-AS group; 3. The si-HOXA11-AS + miR-NC inhibitor group; 4. si-HOXA11-AS + miR-506–3p inhibitor. * p < 0.05.