Esmailzadeh Sahar, Mansoori Behzad, Mohammadi Ali, Shanehbandi Dariush, Baradaran Behzad
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
J Gastrointest Cancer. 2017 Jun;48(2):156-163. doi: 10.1007/s12029-016-9871-z.
Overexpression of HMGA2, known as small non-histone chromosomal protein, is associated with progression of various tumors, including colorectal cancers. The aim of this study was to investigate the effect of a specific HMGA2 siRNA on apoptosis and cell cycle of HCT-116 (colorectal carcinoma) cells.
The cells were transfected with siRNAs using a transfection reagent. The cytotoxic effects of HMGA2 siRNA on colorectal carcinoma cells were determined using MTT assay. Relative HMGA2 mRNA and protein levels were measured by QRT-PCR and western blotting, respectively. Apoptosis was measured by a TUNEL test based on labeling of DNA strand breaks. We also evaluated caspase-3, caspase-8, and caspase-9 expression by QRTPCR to determine which pathway is involved in apoptosis. Cell cycle was assessed by FACS and cell cycle analysis using PI DNA staining.
HMGA2 siRNA significantly reduced both mRNA and protein expression levels 48 h after transfection and dose-dependent manner in colorectal carcinoma cells. We also showed that the silencing of HMGA2 led to the induction of apoptosis through intrinsic pathway and cell cycle arrest in G2/M phases of interphase in HCT-116 cells in vitro.
These results propose that HMGA2 might play an important role in the progression of colorectal carcinoma and might be a potential therapeutic target for trigger apoptosis and cell cycle arrest in colorectal carcinoma.
HMGA2作为一种小的非组蛋白染色体蛋白，其过表达与包括结直肠癌在内的多种肿瘤的进展相关。本研究的目的是探讨特异性HMGA2小干扰RNA（siRNA）对HCT-116（结直肠癌细胞）细胞凋亡和细胞周期的影响。
使用转染试剂将siRNA转染至细胞。采用MTT法测定HMGA2 siRNA对结直肠癌细胞的细胞毒性作用。分别通过实时定量聚合酶链反应（QRT-PCR）和蛋白质印迹法检测相对HMGA2 mRNA和蛋白水平。基于DNA链断裂标记，通过TUNEL试验检测细胞凋亡。我们还通过QRT-PCR评估半胱天冬酶-3、半胱天冬酶-8和半胱天冬酶-9的表达，以确定凋亡涉及哪条途径。通过流式细胞术（FACS）和使用碘化丙啶（PI）DNA染色的细胞周期分析评估细胞周期。
转染后48小时，HMGA2 siRNA在结直肠癌细胞中以剂量依赖方式显著降低mRNA和蛋白表达水平。我们还表明，在体外，HMGA2的沉默通过内在途径诱导HCT-116细胞凋亡，并导致细胞周期在间期的G2/M期停滞。
这些结果表明，HMGA2可能在结直肠癌进展中起重要作用，可能是触发结直肠癌凋亡和细胞周期停滞的潜在治疗靶点。
