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Objective:To investigate the effect of miRNA-1-3p (miR-1-3p) on expression of myocyte enhancer factor 2A (MEF2A) and the biological function of osteosarcoma cells.Methods:The tumor tissues and adjacent normal tissues of 20 patients with osteosarcoma who were clinically diagnosed in Shanxi Provincial Cancer Hospital from January 2019 to January 2020 were collected, and the expression of miR-1-3p in the samples was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The expression of miR-1-3p in osteosarcoma cell lines U2-OS, SAOS-2, MG63, SW1353 and human normal osteoblast cell line hFOB1.19 was detected by qRT-PCR, then the cell line with the lowest expression of miR-1-3p was selected for follow-up experiments. An overexpression miR-1-3p vector was constructed (miR-1-3p mimcs). The miR-1-3p overexpression group was transfected with miR-1-3p mimcs, and the control group was transfected with empty vector (miR-1-3p nc). CCK-8 method was used to detect the proliferation activity of cells; flow cytometry was used to detect the changes of cell apoptosis and cell cycle. miRwalk database was used to predict the miR-1-3p target gene, and the target gene was verified by dual-luciferase reporter gene assay; Western blot was used to detect the expression of MEF2A protein in cells of each group.Results:Compared with adjacent tissues, the expression of miR-1-3p in osteosarcoma tissues was down-regulated (0.31±0.14 vs. 0.62±0.21), and the difference was statistically significant ( t = 5.31, P<0.01). The expression of miR-1-3p in U2-OS cells was the lowest; compared with the control group, the proliferation activity of U2-OS cells was inhibited in miR-1-3p overexpression group (48 h absorbance value 0.56±0.01 vs. 0.77±0.03, t = 2.77, P<0.01; 72 h absorbance value 0.87±0.02 vs. 1.40±0.03, t = 2.93, P<0.01); G 1/S cell cycle arrest increased [G 1 phase (38.24±0.55)% vs. (32.11±0.80)%, t = 9.27, P = 0.01; S phase (61.24±0.90)% vs. (67.78±0.83)%, t = 7.52, P = 0.02]; early apoptotic rate increased [(11.20±0.12)% vs. (1.50±0.12)%, t = 2.91, P<0.05], miRwalk database predicted that the miR-1-3p target gene was MEF2A. The result of dual-luciferase reporter gene assay showed that miR-1-3p bound to MEF2A 3'UTR, and the luciferase activity of U2-OS cells in miR-1-3p overexpression group was lower than that in the control group (renilla luciferase/firefly luciferase activity ratio 0.53±0.06 vs. 1.00±0.04, t = 4.04, P < 0.05). Western blot showed that the expression of MEF2A protein in U2-OS cells of miR-1-3p overexpression group was lower than that of the control group (protein relative expression 0.41±0.14 vs. 0.77±0.12, t = 3.93, P < 0.05). Conclusions:The low expression of miR-1-3p may be associated with the proliferation, apoptosis and cycle changes of osteosarcoma cells. miR-1-3p can negatively regulate the expression of MEF2A protein and regulate the occurrence and development of osteosarcoma.
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Objective:To investigate the related mechanism of miRNA-34a (miR-34a) reverses cisplatin resistance of osteosarcoma through targeted inhibition of high mobility group box 1 (HMGB1).Methods:The MG-63 cisplatin-resistant cell line (MG-63/DDP) was constructed by using dose escalation and intermittent action, and then the successfully constructed MG-63/DDP cells were treated with different concentrations of cisplatin (0, 1, 5, 10, 20, 30 μg/ml), and CCK-8 method was used to detect cell survival rate. The MG-63/DDP cells were transfected respectively and then randomly divided into two groups: the miR-34a overexpression vector group and the miR-34a empty expression vector (miR-34a-NC) group. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the relative expression level of miR-34a. Transfected cells were treated with different concentrations of cisplatin (0, 1, 5, 10, 20, 30 μg/ml), and CCK-8 method was used to detect cell survival rate, flow cytometry was used to detect cell apoptosis. The dual luciferase reporter gene experiment was used to verify whether miR-34a regulated the expression of HMGB1, and Western blotting method was used to detect the HMGB1 protein expression level of the transfected cells. MG-63/DDP cells were transfected respectively and then randomly divided into two groups: HMGB1 gene silencing vector (si-HMGB1) group and its negative control vector (si-NC) group. Western blotting method was used to detect HMGB1 protein expression level and CCK-8 method was used to detect cell survival rate.Results:The MG-63/DDP cell line was successfully constructed. The survival rate of MG-63/DDP cells was higher than that of MG-63 cells when cells were treated with different concentrations of cisplatin (all P < 0.01), and half inhibitory concentration ( IC50) value of MG-63/DDP cells and MG-63 cells on cisplatin was 25.80 μg/ml and 0.47 μg/ml, respectively. qRT-PCR results showed that the relative expression level of miR-34a in MG-63/DDP cells was lower than that in MG-63 cells (0.46±0.04 vs. 1.02±0.05, t = 15.14, P < 0.01); compared with miR-34a-NC group, the relative expression of miR-34a in MG-63/DDP cells was increased in miR-34a overexpression vector group (1.67±0.09 vs. 1.00±0.02, t = -12.58, P < 0.01). Cell survival rate of miR-34a overexpression vector group and miR-34a-NC group was decreased with the increase in the concentration of cisplatin; cell survival rate of miR-34a overexpression vector group was lower than that of miR-34a-NC group when cells were treated with different concentrations of 5- 30 μg/ml cisplatin (all P < 0.01). The apoptotic rate of MG-63/DDP cells in miR-34a-NC group and miR-34a overexpression vector group was (25.1±1.7)% and (42.3±2.3)%, respectively when cells were treated with 20 μg/ml cisplatin; and in miR-34a overexpression vector group, MG-63/DDP cells had a higher rate of apoptosis ( P < 0.01). The dual luciferase reporter gene experiment results showed that compared with miR-34a-NC group, miR-34a overexpression vector group could inhibit the luciferase activity of PGL3- wild-type-HMGB1, and the difference was statistically significant ( t = 6.37, P < 0.01), while miR-34a overexpression vector group had no significant inhibitory effect on the luciferase activity of PGL3- mutant-HMGB1 ( t = 0.35, P = 0.74). The relative expression level of HMGB1 protein in miR-34a overexpression vector group was lower than that in miR-34a-NC group (0.43±0.02 vs. 1.00±0.14, t = 6.98, P < 0.01). Compared with si-NC group, the relative expression level and IC50 value of HMGB1 protein in si-HMGB1 group were reduced (all P < 0.05). Conclusion:Overexpression of miR-34a can enhance the chemosensitivity of osteosarcoma cells MG-63/DDP to cisplatin, and its mechanism may be related to the inhibition of HMGB1 expression.
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Objective To study the correlation between serum miRNA-21 (miR-21) level and chemosensitivity and prognosis of osteosarcoma patients. Methods A total of 68 osteosarcoma patients who were treated in Shanxi Provincial Cancer Hospital from August 2016 to August 2017 were selected. All patients received routine chemotherapy after admission. They were followed up for one year. According to the effectiveness of chemotherapy, they were divided into effective group (52 cases) and ineffective group (16 cases). The general clinicopathological characteristics of the two groups were recorded. Univariate logistic regression model was used to analyze the serum miR-21 level and chemosensitivity in osteosarcoma patients. The prognostic efficacy of serum miR-21 was evaluated by receiver operating characteristic (ROC) curve. Results The serum miR-21 expression in the effective group was higher than that in the ineffective group (6.6 ±0.7 vs. 5.2 ±0.7), and the tumor metastasis rate in the effective group was lower than that in the ineffective group [33.9％ (18/52) vs. 37.5％ (6/16)], and the differences between the two groups were statistically significant (both P< 0.05). There were no significant differences in gender, age, body mass index (BMI), tumor location, size of tumors and TNM stage between the two groups (all P>0.05). Univariate logistic regression model showed that serum miR-21 and metastasis were the factors affecting chemosensitivity (both P<0.05). ROC curve showed that the area under the curve (AUC) of using serum miR-21 to predict the prognosis of osteosarcoma patients was 0.774. Youden index indicated that the best cut-off points of serum miR-21 and chemosensitivity for predicting the prognosis of osteosarcoma patients was 6.25. Conclusion Serum miR-21 level in osteosarcoma patients is significantly correlated with chemosensitivity and prognosis, and it can be used as an effective index to predict the effect of chemotherapy and prognosis of patients with osteosarcoma.