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Cancer Research and Clinic ; (6): 259-263, 2021.
Article in Chinese | WPRIM | ID: wpr-886045


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.

Cancer Research and Clinic ; (6): 253-256, 2019.
Article in Chinese | WPRIM | ID: wpr-746406


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.

Cancer Research and Clinic ; (6): 689-690,694, 2014.
Article in Chinese | WPRIM | ID: wpr-601536


Objective To probe into the content of DNA Topo Ⅱ in osteosarcoma after chemotherapy.Methods 30 patients with osteosarcoma received two courses of chemotherapy treatment before the surgical resection of the tumor tissue.Then immunohistochemistry was used to detect the content of Topo Ⅱ in tissues and detected its relationship in pathology.Results There were 8 out of 30 cases in which Topo Ⅱ was presented positive in osteosarcoma (26.7 %).The protein content of Topo Ⅱ was unrelated to the patient' s age,gender,degree of tumor malignancy,tumor location and translocation or Enneking staging (P > 0.05),but related to patients survival rate (P < 0.05).Conclusion Patients with lower expression of Topo Ⅱ are more likely to have poor prognosis.

Chinese Journal of Biotechnology ; (12): 203-212, 2014.
Article in Chinese | WPRIM | ID: wpr-279529


The aim of this study is to identify the express specificity of bone morphogenetic protein 15 (Bmp15) in porcine. The pBMP15-EGFP reporter vector was constructed from the 2.2 kb fragment of porcine bmp15 promoter to trace the differentiation process of stem cells into oocyte-like cells. We used porcine ovary and Chinese Hamster Ovary cell line (CHO), mouse myoblast cell line (C2C12) and porcine amniotic fluid stem cell (pAFSC) to investigate the expression and regulation of this gene via RT-PCR, immunofluorescence, cell transfection, and microinjection methods. We also used single layer cell differentiation to detect the application potential of bmp15. The results show that bmp15 gene was specifically expressed in the porcine ovary and CHO rather than in C2C12 and pAFSC. In addition, the characteristic of tissue-specific of Bmp15 was detected on CHO instead of other cell lines by transient transfection. We also detected the expression of Bmp15 in oocyte at different development stages by immunofluorescence of fixed paraffin-embedded ovary sections. Furthermore, microinjection results show that bmp15 expressed in oocytes at 18 h of maturation in vitro, and continued up to 4-cell stage embryos. Most importantly, we found that the expression of Bmp15 started at day 12 after inducing pAFSC into oocyte-like cells by transfection; green fluorescent was visible in round cell masses. It indicated that bmp15 has the expression specificity and the pBMP15-EGFP reporter vector can be used to trace Bmp15 action in the differentiation of stem cells into germ cells.

Animals , Bone Morphogenetic Protein 15 , Genetics , CHO Cells , Cell Differentiation , Cricetinae , Cricetulus , Female , Genes, Reporter , Genetic Vectors , Mice , Microinjections , Myoblasts , Cell Biology , Oocytes , Metabolism , Ovary , Metabolism , Stem Cells , Cell Biology , Swine