LncRNA MIR4435-2HG is downregulated in osteoarthritis and regulates chondrocyte cell proliferation and apoptosis

A total of 78 osteoarthritis (44 cases of knee joint and 34 cases of hip joint; 40 males and 38 females; 60 to 78 years old; mean age 69.0 ± 6.0 years) patients and 58 healthy volunteers (30 males and 28 females; 60 to 77 years; mean age 68.8 ± 5.9 years) were enrolled in Tianjin Hospital from January 2015 to January 2017 to serve as research subjects. All patients were diagnosed by joint fluid analysis, which revealed the existence of inflammation and X-rays, which showed the narrowing of the space between the bones, an indicator of cartilage loss. Patients’ inclusion criteria: (1) new cases of osteoarthritis (stages 3 and 4) (2) patients provided informed consent. Patients’ exclusion criteria: (1) patients complicated with other diseases, such as chronic inflammatory diseases other than osteoarthritis; (2) patients failed to complete treatment; (3) patients who had been treated before admission or currently under medication. The 58 healthy volunteers received systemic physical examinations at the physical health center of Tianjin Hospital, and all parameters were within the normal range. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki, and this study was approved by the Ethics Committee of Tianjin Hospital.

Blood (5 ml) was extracted from both patients and healthy controls 1 day after admission under fasting condition. Synovial fluid was collected from all patients and controls (33 cases of hip and 25 cases of the knee). All patients received treatments including exercise, reducing joint burden, and non-steroidal anti-inflammatory drugs (NSAIDs) such as naproxen. Inflammation was inhibited after 1-month treatment, which was revealed by joint fluid analysis. Blood (5 ml) was also extracted at 1 and 3 months after treatment from each patient under fasting condition. Blood samples were used to prepare plasma samples.

Thirteen cases of synovial membrane from osteoarthritis-affected joints and 8 cases of synovial membrane from non-osteoarthritis-affected joints were obtained from the specimen library of Tianjin Hospital.

Primary chondrocytes (402-05A, Sigma-Aldrich) were used in this study to perform in vitro experiments. Cell culture was performed according to the instructions from Sigma-Aldrich.

To detect the expression of lncRNA MIR4435-2HG, total RNAs were extracted using Trizol reagent (Invitrogen, USA). After reverse transcription (SuperScript IV Reverse Transcriptase kit, Thermo Fisher Scientific., lnc.), SYBR® Green Quantitative RT-qPCR Kit (Sigma-Aldrich) was used to prepare qPCR mixtures. Primers of MIR4435-2HG and endogenous control GAPDH were from Sangon (Shanghai, China). Sequences of primers were: 5′-TGATAAAGGGCTCTGAAAGC-3′ (Forward) and 5′-CACGATGCCTTCACCAGTGT-3′ (reverse) for MIR4435-2HG; 5′-CTGACTTCAACAGCGACAC-3′ (forward) and 5′-TAGCCAAATTCGTTGTCATAC-3′(reverse) for GAPDH. According to 2^−ΔΔCT method, expression of MIR4435-2HG was normalized to GAPDH.

MIR4435-2HG-expression vector and empty vectors (pEGFP-C3) were from Sangon (Shanghai, China). MIR4435-2HG siRNA and negative control (NC) siRNA were from GenePharma (Shanghai, China). Lipofectamine 2000 reagent (Thermo Fisher Scientific., lnc.) was used for all cell transient transfections with 10 nM vector (empty vector as NC group) or 35 nM siRNAs (NC siRNA as NC group). Cells without transfection were control cells. Cells were collected for subsequent experiments at 24 h after transfection.

Cells were collected at 24 h after transfection, and 4 × 10⁴ cells were mixed with 1 ml cell culture medium to make single cell suspensions. Cells were cultivated in a 96-well plate (100 μl each well) at 37 °C, followed by the addition of 10 μl CCK-8 solution (Sigma-Aldrich) at 3 h before the end of cell culture. After that, 10 μl DMSO was added, and the measurement of OD values was performed at 450 nm.

Cells were collected at 24 h after transfection and 4 × 10⁴ cells were mixed with 1 ml serum-free cell culture medium to make single cell suspensions. A 6-well plate was used to cultivate cells (2 ml per well) for 48 h. After that, cells were digested by 0.25% trypsin and stained by Annexin V-FITC (Dojindo, Japan) as well as propidium iodide (PI). Apoptotic cells were separated by flow cytometry.

All experiments were performed in triplicate manner and data were recorded as mean ± standard deviation. Comparisons of expression levels of MIR4435-2HG between two groups were done by unpaired t test. Comparisons of expression levels of MIR4435-2HG in patients between different time points were performed by paired t test. Comparisons of cell apoptosis and proliferation data among the three groups were done by ANOVA (one-way) and Tukey test. The diagnostic analysis was performed through ROC curve analysis with healthy controls as true negative cases and osteoarthritis patients as true positive cases. DP < 0.05 was statistically significant.

RT-qPCR was performed to measure levels of MIR4435-2HG in plasma of osteoarthritis patients and healthy controls. As shown in Fig. 1a, plasma levels of MIR4435-2HG were significantly lower in osteoarthritis patients than in the control group (p < 0.05). Moreover, MIR4435-2HG expression in 13 cases of synovial membrane from osteoarthritis-affected joints was also downregulated comparing to 8 cases of synovial membrane from non-osteoarthritis-affected joints (Fig. 1b). These data suggest that downregulation of MIR4435-2HG is likely involved in osteoarthritis.

MIR4435-2HG in join fluid was also measured by qPCR. It was observed that expression levels of MIR4435-2HG in join fluid were also significantly lower in osteoarthritis patients than in the control group (Fig. 2a, p < 0.05). The diagnostic analysis was performed through ROC curve analysis. As shown in Fig. 2, area under the curve was 0.96, with a standard error of 0.016 and 95% confidence interval of 0.92–0.99 (Fig. 2b, p < 0001). It is suggested that downregulation of MIR4435-2HG may serve as a potential diagnostic biomarker for osteoarthritis.

Plasma levels of MIR4435-2HG were measured before treatment as well as 1 and 3 months after the beginning of treatment. Compared with pretreatment levels, plasma levels of MIR4435-2HG were significantly increased at 1 and 3 months after the beginning of treatment (Fig. 3, p < 0.05). Comparing to plasma levels of MIR4435-2HG at 1 after the beginning of treatment, plasma levels of MIR4435-2HG were significantly increased at 3 months after the beginning of treatment (Fig. 3, p < 0.05).

Overexpression and knockdown of MIR4435-2HG were reached after transfection in chondrocytes (Fig. 4a, p < 0.05). Overexpression of MIR4435-2HG promoted, while MIR4435-2HG knockdown inhibited the proliferation of cells of chondrocytes (Fig. 4b, p < 0.05). In contrast, MIR4435-2HG overexpression inhibited, while MIR4435-2HG knockdown promoted the apoptosis of chondrocytes (Fig. 4c, p < 0.05). Therefore, MIR4435-2HG regulated chondrocyte cell proliferation and apoptosis.