Effect of the PI3K-AKT-mTOR pathway on the drug resistance of osteosarcoma cells and its mechanism
Na Risong1, Guo Shibing2, Sun Liang2, Zhao Zhenqun2, Zhao Wei2, Wang Yuxin2
1Health Center of Cadre, Inner Mongolia People's Hospital, Hohhot 010020, China; 2Department of Orthopedics, the Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, China
Abstract:Objective This study aims to investigate the effect of the phosphatidylinositol 3-hydroxy kinase(PI3K)-protein kinase B(AKT)-mammalian target of rapamycin(mTOR) pathway on the drug resistance of osteosarcoma cells and its mechanism to provide new ideas for the targeted therapy of osteosarcoma.Methods MG-63 cells were cultured in vitro, and MG-63 drug-resistant cell lines were established using the repeated administration of 2 μg/mL cisplatin. The high-expression plasmids of miRNA-22 were transfected into MG-63 drug-resistant cell lines to establish MG-63 drug-resistant cell lines with high miRNA-22 expression. MG-63 cells were the control group, and the MG-63 drug-resistant cells were the drug-resistant group. MG-63 frug-resistant cells treated with 2 μg/mL cisplatin and the drug-resistant MG-63 cells transfected with miRNA-22 were the drug-resistant DDP and the drug-resistant miRNA-22+DDP groups, respectively. The cell proliferation was detected using the thiazoline (MTT) assay. The expression levels of PI3K, AKT, and mTOR mRNA were detected using fluorescence quantitative RT-PCR. The protein expressions of PI3K, AKT, pAKT, mTOR, and p-mTOR were detected using the Western blot.Results (1)The MTT results showed that the absorbance values of the control, drug-resistant, drug-resistant DDP, and drug-resistant miRNA-22+DDP groups were 1.097±0.039, 1.157±0.065, 0.870±0.014, and 0.737±0.029, respectively. The differences were statistically significant (F=67.731, P<0.01). Compared with that of the control group, the absorbance of the drug-resistant DDP and the drug-resistant miRNA-22+DDP groups decreased significantly (all P values<0.01). For the drug-resistant, drug-resistant DDP group, and drug-resistant miRNA-22+DDP group, the absorbance showed a downward trend, and the differences were statistically significant (all P values<0.01). (2)The results of fluorescence quantitative RT-PCR showed that compared with that in the control group, the relative expression of PI3K and AKT in the drug-resistant and the drug-resistant DDP groups increased especially in the drug-resistant group (all P values<0.01). No significant difference was observed between the drug-resistant miRNA-22+DDP and the control groups (all P values>0.01). The relative expression of mTOR significantly increased in the drug-resistant group and significantly decreased in the drug-resistant DDP and the drug-resistant miRNA-22+DDP groups (all P values<0.01). The relative expression levels of PI3K, AKT, and mTOR in the drug-resistant DDP and drug-resistant miRNA-22+DDP groups were lower than those in the drug-resistant group, and all differences were statistically significant (all P values<0.01). The relative expression levels of PI3K and mTOR in the drug-resistant miRNA-22+DDP group were lower than those in the drug-resistant DDP group, and the differences were statistically significant (all P values<0.01). (3) The results of Western blot showed that compared with that in the control group, the relative expression levels of PI3K, AKT, and mTOR proteins in the drug-resistant group increased significantly and that the relative expression levels of PI3K and AKT proteins in the drug-resistant DDP and drug-resistant miRNA-22+DDP groups were significantly reduced. The differences were statistically significant (all P values<0.01). The pairwise comparison among three groups revealed that the relative expression levels of PI3K, AKT, and mTOR proteins in the drug-resistant DDP and drug-resistant miRNA-22+DDP groups were significantly lower than those in the drug-resistant group. The relative expression levels of PI3K and AKT proteins in the drug-resistant miRNA-22+DDP group were significantly lower than those in the drug-resistant DDP group (all P values<0.01).Conclusions The PI3K-AKT-mTOR pathway was involved in the development of osteosarcoma cells' resistance to cisplatin. miRNA-22 reduced the resistance of osteosarcoma cells to cisplatin by downregulating the expression levels of PI3K, AKT, and mTOR in the PI3K-AKT-mTOR pathway.
娜日松, 郭世炳, 孙亮, 赵振群, 赵伟, 王玉鑫. PI3K-AKT-mTOR通路对骨肉瘤细胞顺铂耐药性的影响及其机制[J]. 中华解剖与临床杂志, 2021, 26(1): 88-93.
Na Risong, Guo Shibing, Sun Liang, Zhao Zhenqun, Zhao Wei, Wang Yuxin. Effect of the PI3K-AKT-mTOR pathway on the drug resistance of osteosarcoma cells and its mechanism. Chinese Journal of Anatomy and Clinics, 2021, 26(1): 88-93.
Nugent M. MicroRNA function and dysregulation in bone tumors: the evidence to date[J]. Cancer Manag Res, 2014, 6: 15-25. DOI:10.2147/CMAR.S53928.
[2]
孙亮, 郭世炳, 娜日松, 等. 微小RNA-22通过靶向调控异黏蛋白表达影响骨肉瘤细胞化疗敏感性[J]. 中华实验外科杂志, 2019, 36(11): 1959-1962. DOI:10.3760/cma.j.issn.1001-9030.2019.11.011.Sun L, Guo SB, Na RS, et al. MicroRNA-22 influenced on chemosensitivity of osteosarcoma cells by targeting metadherin[J]. Chin J Exp Surg, 2019, 36(11): 1959-1962. DOI:10.3760/cma.j.issn.1001-9030.2019.11.011.
[3]
赵家义. PI3K-AKT-mTOR信号通路抑制剂与肿瘤免疫治疗[J]. 中国肿瘤生物治疗杂志, 2017, 24(12): 1424-1430. DOI:10.3872/j.issn.1007-385x.2017.12.015.Zhao JY. Inhibitors of PI3K-AKT-mTOR signaling pathway and cancer immunotherapy[J]. Chin J Cancer Biother, 2017, 24(12): 1424-1430. DOI:10.3872/j.issn.1007-385x.2017.12.015.
[4]
Ma JF, Kalle MV , Plautz Q , et al. Relaxin promotes in vitro tumour growth, invasion and angiogenesis of human Saos-2 osteosarcoma cells by AKT/VEGF pathway[J]. European review for medical and pharmacological sciences, 2013, 17(10): 1345-1350.
Bousquet M, Noirot C, Accadbled F, et al. Whole-exome sequencing in osteosarcoma reveals important heterogeneity of genetic alterations[J]. Ann Oncol, 2016, 27(4): 738-744. DOI:10.1093/annonc/mdw009.
[7]
Yang JZ, Ma SR, Rong XL, et al. Characterization of multidrug-resistant osteosarcoma sublines and the molecular mechanisms of resistance[J]. Mol Med Rep, 2016, 14(4): 3269-3276. DOI:10.3892/mmr.2016.5590.
[8]
Chen R, Wang G, Zheng Y, et al. Drug resistance-related microRNAs in osteosarcoma: translating basic evidence into therapeutic strategies[J]. J Cell Mol Med, 2019, 23(4): 2280-2292. DOI:10.1111/jcmm.14064.
[9]
杨豪, 曹臣, 王力军. 长链非编码RNA LINC-PINT通过靶向miR-524-5p调控骨肉瘤细胞增殖凋亡的分子机制[J]. 中华肿瘤杂志, 2020, 42(4): 325-330. DOI:10.3760/cma.j.cn112152-20190726-00471.Yang H, Cao C, Wang LJ. LncRNA LINC-PINT regulating prolifetion and apoptosis of osteosarcoma cells by targeting miR-524-5p[J]. Chin J Oncol, 2020, 42(4): 325-330.DOI:10.3760/cma.j.cn112152-20190726-00471.
[10]
Wang L, Jiang J, Sun G, et al. Effects of lncRNA TUSC7 on the malignant biological behavior of osteosarcoma cells via regulation of miR-375[J]. Oncol Lett, 2020, 20(5): 133. DOI:10.3892/ol.2020.11994.
[11]
宋瑞鹏, 赵亮, 谭洪宇, 等. 磷酸肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路下p53基因对骨肉瘤细胞增殖及转移的抑制作用[J]. 中华实验外科杂志, 2018, 35(1): 26-28. DOI:10.3760/ema.j.issn.1001-9030.2018.01.010.Song RP, Zhao L, Tan HY, et al. Inhibitory effect of p53 on proliferation and metastasis of osteosarcoma via the phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin pathway[J]. Chin J Exp Surg, 2018, 35(1): 26-28.DOI:10.3760/ema.j.issn.1001-9030.2018.01.010.
[12]
Duo J, Ma Y, Wang G, et al. Metformin synergistically enhances antitumor activity of histone deacetylase inhibitor trichostatin a against osteosarcoma cell line[J]. DNA Cell Biol, 2013, 32(4): 156-164. DOI:10.1089/dna.2012.1926.
[13]
刘春杰, 刘宇, 邓学坤, 等. miR-32通过调控PI3K/Akt信号通路影响骨肉瘤细胞的增殖[J]. 河北医科大学学报, 2018, 39(6): 662-667. DOI:10.3969/j.issn.1007-3205.2018.06.011.Liu CJ, Liu Y, Deng XK, et al. miR-32 affects proliferation of osteosarcoma cells by regulating PI3K/Akt signaling pathway[J]. Journal of Heibei Medical University, 2018, 39(6): 662-667. DOI:10.3969/j.issn.1007-3205.2018.06.011.
[14]
罗鸿斌, 刘蔚楠, 林建华, 等. mTOR抑制剂FIM-A对人骨肉瘤细胞株MG-63的抑制作用及其机制[J]. 中国肿瘤生物治疗杂志, 2013, 20(4): 419-424. DOI:10.3872/j.issn.1007-385X.2013.04.007.Luo HB, Liu WN, Lin JH, et al. Inhibitory effect of FIM-A, a mTOR inhibitor, on the proliferation and apoptosis of human MG-63 osteosarcoma cell line and its mechanism[J]. Chin J Cancer Biother, 2013, 20(4): 419-424. DOI:10.3872/j.issn.1007-385X.2013.04.007.
[15]
Zhang H, Jiang H, Zhang H, et al. Anti-tumor efficacy of phellamurin in osteosarcoma cells: Involvement of the PI3K/AKT/mTOR pathway[J]. Eur J Pharmacol, 2019, 858: 172477. DOI:10.1016/j.ejphar.2019.172477.
[16]
Ma H, Su R, Feng H, et al. Long noncoding RNA UCA1 promotes osteosarcoma metastasis through CREB1-mediated epithelial-mesenchymal transition and activating PI3K/AKT/mTOR pathway[J]. J Bone Oncol, 2019, 16: 100228. DOI: 10.1016/j.jbo.2019.100228.
[17]
Xu R, Liu S, Chen H, et al. MicroRNA-30a downregulation contributes to chemoresistance of osteosarcoma cells through activating Beclin-1-mediated autophagy[J]. Oncol Rep, 2016, 35(3): 1757-1763. DOI:10.3892/or.2015.4497.
[18]
Zhou X, Natino D, Zhai X, et al. MicroRNA-22 inhibits the proliferation and migration, and increases the cisplatin sensitivity, of osteosarcoma cells[J]. Mol Med Rep, 2018, 17(5): 7209-7217. DOI:10.3892/mmr.2018.8790.