Apoptosis of osteoblasts, triggered by prolonged or excessive use of glucocorticoids (GCs), has been identified as a dominant contributor to the development of osteoporosis and osteonecrosis. However, the molecular mechanisms underlying GC-induced apoptosis are multifaceted and remain to be fully elucidated. The present study aimed to explore the correlation between dexamethasone (DEX)-induced reactive oxygen species (ROS), autophagy and apoptosis in MC3T3-E1 osteoblast-like cells. Cell viability was assessed using a Cell Counting Kit-8 assay, and flow cytometry was performed to assess cellular apoptosis, cell cycle and ROS production. Immunofluorescence and western blot analysis were respectively used to detect autophagic vacuoles and the expression of proteins, including cyclin D kinase (CDK)2, poly[ADP ribose] polymerase, caspase-3, activating transcription factor (ATF)4, CCAAT/enhancer-binding protein homologous protein (CHOP), Beclin1, microtubule-associated proteins 1A/1B light chain (LC)3B and P62. It was revealed that DEX not only reduced cell viability, but also promoted apoptosis via the activation of endoplasmic reticulum (ER) stress. In addition, DEX induced cell cycle arrest at G0/G1 phase via inhibition of the expression of CDK2, and the production of ROS was activated. Of note, the DEX-mediated changes in viability and apoptosis were attenuated in MC3T3-E1 cells after treatment with 3-methyladenine, which is an autophagy inhibitor. Treatment with the antioxidant N-acetylcysteine abolished the effect of DEX on the proliferation, apoptosis, ER stress and autophagy of MC3T3-E1 cells. In conclusion, the present results indicated that DEX promoted the production of ROS, which enhanced apoptosis through activation of autophagy and ER stress in MC3T3-E1 cells.
Osteosarcoma (OS) is the most common primary malignant tumor of the bone affecting children and adolescents. Chemotherapy is now considered as a standard component of OS treatment, not only for children, but also for adults. However, chemoresistance continues to pose a challenge to therapy. Inhibition of autophagy has been demonstrated to decrease chemoresistance in OS. Moreover, microRNA-22 (miR-22) inhibits autophagy, leading to an improvement in the sensitivity of cisplatin (CDDP) in OS. The aim of the present study was therefore to investigate whether miR-22 could mediate the CDDP resistance of OS cells by inhibiting autophagy via the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. Cell proliferation assay, LC3 flow cytometry assay and monodansylcadaverine staining in MG63 cells and CDDP resistance cells (MG63/CDDP) were performed to explore to role of miR-22 and CDDP in OS chemoresistance. Inoculation of tumor cells in an in vivo model, reverse transcription-quantitative PCR (RT-qPCR) assay, western blot analysis, and immunohistochemistry analysis were performed to investigate the role of miR-22 and CDDP in the PI3K/Akt/mTOR pathway as it is affected by autophagy. The results revealed that miR-22 inhibited the proliferation of MG63 and MG63/CDDP cells, and enhanced the anti-proliferative ability of CDDP in vivo and in vitro. miR-22 mediated the CDDP resistance of OS cells by inhibiting autophagy and decreasing CDDP-induced autophagy via downregulation of the expression of PI3K, Akt, and mTOR at the mRNA level, and the expression of PI3K, phosphorylated (p)-Akt, and p-mTOR at the protein level. It was also convincingly demonstrated that miR-22 mediates the CDDP resistance of OS by inhibiting autophagy via the PI3K/Akt/mTOR pathway. Furthermore, in the MG63 cells that were affected by CDDP, the role of miR-22 was shown to be similar to that of the investigated inhibitor of PI3K (wortmannin) in terms of regulating the PI3K/Akt/mTOR pathway, and wortmannin could also promote the effect of miR-22. Interestingly, CDDP was demonstrated to induce autophagy by inhibiting the PI3K/Akt/mTOR pathway, whereas the pathway was upregulated in the state of chemoresistance. In conclusion, downregulation of the PI3K/Akt/mTOR pathway was shown to assist in the process of preventing chemoresistance.
Objective To investigate whether miR‐141 and the sex determination region of Y chromosome box 11 (SOX11) play roles in steroid‐induced avascular necrosis of the femoral head (SANFH), and to explore whether miR‐141 could target SOX11 to influence the proliferation of bone marrow mesenchymal stem cells (BMSC). Methods Bone marrow mesenchymal stem cells (BMSC) were isolated and cultured from 4‐week‐old Sprague Dawley rats. A flow cytometry assay was performed to identify BMSC. BMSC were divided into two groups: a control group and a dexamethasone (DEX) group. BMSC were transfected by miR‐141 mimic, miR‐141 inhibitor, and SOX11. Real‐time polymerase chain reaction (PCR) assay was performed to investigate the mRNA expression of miR‐141 and SOX11. The results were used to determine the effect of transfection and to verify the expression in each group and the association between miR‐141 and SOX11. Luciferase reporter assay revealed the targeted binding site between miR‐141 and the 3′‐untranslated region of SOX11 mRNA. MTT assays were performed to investigate the proliferation of BMSC in the miR‐141 mimic, miR‐141 inhibitor, and SOX11 groups. Result The results of the flow cytometry assay suggested that cells were positive for CD29 and CD90 while negative for CD45. This meant that the isolated and cultured cells were not hematopoietic stem cells. In addition, cell transfection was successful based on the expression of miR‐141 and SOX11. According to the results of real‐time PCR assay, the mRNA expression of miR‐141 in SANFH was upregulated (4.117 ± 0.042 vs 1 ± 0.027, P < 0.001), while SOX11 was downregulated (0.611 ± 0.055 vs 1 ± 0.027, P < 0.001) compared with the control group. Based on the results of the luciferase experiment, MiR‐141 could directly target the expression of SOX11. Inhibition of miR‐141 could upregulate the expression of SOX11 (2.623 ± 0.220 vs 1 ± 0.095, P < 0.001) according to the results of a real‐time PCR assay. MiR‐141 inhibited the proliferation of BMSC (0.618 ± 0.092 vs 1.004 ± 0.082, P < 0.001), while suppression of miR‐141 increased the proliferation of BMSC (0.960 ± 0.095 vs 0.742 ± 0.091, P < 0.001). Furthermore, according to the results of the MTT assay, SOX11 promoted the proliferation of BMSC (1.064 ± 0.093 vs 0.747 ± 0.090, P < 0.001). Conclusion MiR‐141 inhibited the proliferation of BMSC in SANFH by targeting SOX11. Inhibition of miR‐141 upregulated the expression of SOX11 and promoted the proliferation of BMSC. MiR‐141 and SOX11 could be new targets for investigating the mechanism of SANFH.
osteosarcoma (oS) is a primary malignant tumor of bone tissue. effective chemotherapy may improve the survival of patients with OS. MicroRNAs (miRs) serve significant roles in the regulatory function of tumorigenesis and chemosensitivity of different types of cancer. mir-22 has been revealed to inhibit the proliferation and migration of oS cells, as well as increasing their sensitivity to cisplatin (cddP). The mechanisms of action behind the functions of mir-22 in oS drug resistance require investigation. Therefore, in the present study, the human oS cell lines (MG-63, u2oS, Saos2 and oS9901) and a drug-resistant cell line (MG-63/cddP) were cultured. cell proliferation, apoptosis and autophagy assays were performed to investigate the proliferation, apoptosis and autophagy of cell lines transfected with mir-22 mimic. reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to investigate the expression levels of associated genes. The results revealed that mir-22 inhibited the proliferation of MG-63 cells and MG-63/cddP cells, and enhanced the anti-proliferative ability of cddP. mir-22 induced apoptosis and inhibited autophagy of MG-63 cells and MG-63/cddP cells. apoptosis-related genes, including caspase-3 and Bcl-2-associated X protein were upregulated, while B-cell lymphoma-2 was downregulated in both cell lines transfected with the mir-22 mimic. autophagy protein 5, beclin1 and microtubules-associated protein 1 light chain 3 were downregulated in both cell lines transfected with mir-22 mimic. Furthermore, the in vitro and in vivo expression levels of metadherin (MTdH) in the oS/oS-cddP-resistant models were downregulated following transfection with the mir-22 mimic. Therefore, the results of the present study suggested that mir-22 promoted cddP sensitivity by inhibiting autophagy and inducing apoptosis in oS cells, while MTdH may serve a positive role in inducing cddP resistance of oS cells.
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