Zamani Bahareh, Ramazani Amin, Saberzadeh Jamileh, Rostampour Puria, Takhshid Mohammad Ali
Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Science, Shiraz, Iran.
Mol Biol Res Commun. 2023;12(2):77-85. doi: 10.22099/mbrc.2023.47160.1823.
Epithelial-to-mesenchymal transition (EMT) plays a critical role in colorectal cancer (CRC) metastasis. In the present study, we evaluated the effects of annexin A5 () overexpression on invasiveness as well as the expression of genes involved in EMT of HCT 116 cell line. PCMV6-AC-IRES-GFP plasmid harboring cDNA was constructed. HCT 116 cell line was transfected with recombinant plasmids using Lipofectamine 3000. Fluorescent microscopy was used to determine the efficiency of plasmid transfection. Cell viability was determined using the MTT assay. HCT 116 cell migration was evaluated using wound healing assay and transwell migration assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of genes involved in EMT. The results of RT-qPCR showed overexpression of compared to the control group. overexpression had no significant effects on cell viability but significantly decreased the rate of wound closure in the wound healing assay as well as the number of migrated cells in transwell assay. Furthermore, overexpression decreased the expression of N-cadherin, Snail, Slug, MMP-2, and MMP-9 while the expression of E-cadherin increased following overexpression. However, VEGF expression did not significantly change after overexpression. Results of the present study suggest that overexpression might have inhibitory effects on the metastasis of CRC through modulating the expression of EMT- related genes.
上皮-间质转化（EMT）在结直肠癌（CRC）转移中起关键作用。在本研究中，我们评估了膜联蛋白A5（Annexin A5）过表达对HCT 116细胞系侵袭性以及EMT相关基因表达的影响。构建了携带Annexin A5 cDNA的PCMV6-AC-IRES-GFP质粒。使用Lipofectamine 3000将重组质粒转染至HCT 116细胞系。采用荧光显微镜确定质粒转染效率。使用MTT法测定细胞活力。采用伤口愈合试验和Transwell迁移试验评估HCT 116细胞迁移情况。使用实时定量聚合酶链反应（RT-qPCR）检测EMT相关基因的表达。RT-qPCR结果显示，与对照组相比Annexin A5过表达。Annexin A5过表达对细胞活力无显著影响，但在伤口愈合试验中显著降低伤口闭合率，在Transwell试验中显著减少迁移细胞数量。此外，Annexin A5过表达降低了N-钙黏蛋白、Snail、Slug、基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）的表达，而E-钙黏蛋白的表达在Annexin A5过表达后增加。然而，Annexin A5过表达后血管内皮生长因子（VEGF）表达未显著变化。本研究结果表明，Annexin A5过表达可能通过调节EMT相关基因的表达对结直肠癌转移具有抑制作用。
