Ahangar Noora Karim, Khalaj-Kondori Mohammad, Alizadeh Nazila, Mokhtarzadeh Ahad, Baghbanzadeh Amir, Shadbad Mahdi Abdoli, Dolatkhah Katayoun, Baradaran Behzad
Department of Animal Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz 5166616471, Iran; Immunology Research Center, Tabriz University of Medical Sciences, Iran.
Department of Animal Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz 5166616471, Iran.
Gene. 2023 Mar 1;855:147086. doi: 10.1016/j.gene.2022.147086. Epub 2022 Dec 16.
Osteosarcoma is common type of bone cancer; however, the prognosis of patients with metastatic osteosarcoma is poor. As a new inhibitory immune checkpoint molecule, HHLA2 is upregulated in osteosarcoma. Herein, we studied the significance of tumor-intrinsic HHLA2 in MG-63 growth. Also, we examined the influence of combined therapy of HHLA2 knockdown with paclitaxel on the apoptosis, cell cycle, migration, and stemness of MG-63 cells.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to study the half-maximal inhibitory concentration (IC50) of paclitaxel and the cytotoxicity of HHLA2-small interfering RNA (siRNA) on MG-63 cells. The apoptosis and cell cycle were analyzed using flow cytometry. The wound-healing and colony formation assays were conducted to investigate the effect of paclitaxel and HHLA2 knockdown on the migration and stemness of MG-63 cells, respectively. QRT-PCR was used to determine the Bax, caspase-3, and Bcl-2 mRNA expression levels.
HHLA2 silencing has enhanced the chemosensitivity of MG-63 cells to paclitaxel. Besides, HHLA2 knockdown has increased the paclitaxel-induced cytotoxic effect on MG-63 cells. In terms of stimulating apoptosis, decreasing clonogenicity, halting the cell cycle at the sub G1 phase, and inhibiting migration, tumor-intrinsic HHLA2 silencing has increased these anti-tumor effects of paclitaxel on MG-63 cells. Besides, HHLA2 knockdown has potentiated paclitaxel-mediated Bcl-2 downregulation and paclitaxel-mediated caspase-3 and Bax upregulation in MG-63 cells.
Tumor-intrinsic HHLA2 knockdown increases the anti-tumoral effect of paclitaxel on MG-63 cells and enhances the chemosensitivity of MG-63 cells to paclitaxel.
骨肉瘤是一种常见的骨癌类型；然而，转移性骨肉瘤患者的预后较差。作为一种新的抑制性免疫检查点分子，HHLA2在骨肉瘤中上调。在此，我们研究了肿瘤内在的HHLA2在MG-63细胞生长中的意义。此外，我们还研究了HHLA2基因敲低联合紫杉醇治疗对MG-63细胞凋亡、细胞周期、迁移和干性的影响。
采用3-(4,5-二甲基噻唑-2)-2,5-二苯基-2H-四唑溴盐(MTT)法研究紫杉醇的半数抑制浓度(IC50)以及HHLA2小干扰RNA(siRNA)对MG-63细胞的细胞毒性。采用流式细胞术分析细胞凋亡和细胞周期。分别进行伤口愈合实验和集落形成实验，以研究紫杉醇和HHLA2基因敲低对MG-63细胞迁移和干性的影响。采用实时定量聚合酶链反应(QRT-PCR)检测Bax、caspase-3和Bcl-2 mRNA表达水平。
HHLA2基因沉默增强了MG-63细胞对紫杉醇的化疗敏感性。此外，HHLA2基因敲低增加了紫杉醇对MG-63细胞的细胞毒性作用。在促进细胞凋亡、降低克隆形成能力、使细胞周期停滞在G1期以下以及抑制迁移方面，肿瘤内在的HHLA2基因沉默增强了紫杉醇对MG-63细胞的这些抗肿瘤作用。此外，HHLA2基因敲低增强了紫杉醇介导的MG-63细胞中Bcl-2下调以及紫杉醇介导的caspase-3和Bax上调。
肿瘤内在的HHLA2基因敲低增加了紫杉醇对MG-63细胞的抗肿瘤作用，并增强了MG-63细胞对紫杉醇的化疗敏感性。
