目的 探讨抑制FLOT1基因表达对胃癌细胞侵袭、凋亡的影响及机制。方法 将设计合成的FLOT1 siRNA慢病毒载体(si-FLOT1组)转染人胃癌MKN-45细胞,同时转染阴性对照慢病毒载体(阴性组),并设置空白组。Western blotting检测转染48 h后细胞FLOT1、E-cadherin、α-SMA、cleaved caspase 3、Bcl-2和Bax蛋白表达。CCK-8法检测转染24、48、72和96 h的细胞活力。Transwell小室、流式细胞术及DCFH-DA法分别检测转染48 h的细胞侵袭能力、凋亡率及活性氧(ROS)含量。结果 FLOT1 siRNA慢病毒载体可明显抑制MKN-45细胞FLOT1表达,与空白组比较差异具有统计学意义(P<0.05)。si-FLOT1组与空白组比较,细胞活力降低,细胞侵袭能力降低,细胞凋亡率升高,E-cadherin、cleaved caspase 3和Bax蛋白表达升高,α-SMA和Bcl-2蛋白表达降低,ROS含量升高,差异均具有统计学意义(P<0.05)。结论 下调FLOT1基因表达可通过抑制EMT降低胃癌细胞侵袭能力,通过调控凋亡相关蛋白表达及提高细胞ROS含量促进细胞凋亡。
Abstract
OBJECTIVE To investigate inhibitory effect of FLOT1 gene expression on invasion and apoptosis of gastric cancer cells. METHODS The designed FLOT1 siRNA lentiviral vector (si-FLOT1 group) was transfected into human gastric cancer MKN-45 cells, at the same time, the negative control lentivirus vector (negative group) was transfected, and the blank group was set up. Western blotting was used to detect the expression of FLOT1, E-cadherin, α-SMA, cleaved caspase 3, Bcl-2 and Bax proteins. After cells were transfected for 24, 48, 72 and 96 h, cell viability was detected by CCK-8 assay. After cells were transfected for 48 h, transwell chamber, flow cytometry and DCFH-DA assay were used to detect the invasiveness, apoptosis rate and ROS content, respectively. RESULTS FLOT1 siRNA lentiviral vector inhibited significantly the expression of FLOT1 in MKN-45 cells, which was significantly different from the blank group (P<0.05). Compared with si-FLOT1 group and the blank group, the cell vitality decreased, the invasion ability decreased, the apoptosis rate increased, the expression of E-cadherin, cleaved caspase 3 and Bax protein increased, the expression of α-SMA and Bcl-2 protein decreased, and the content of ROS increased, and the difference was statistically significant (P<0.05). CONCLUSION Down regulation of FLOT1 gene expression can reduce the invasiveness of gastric cancer cells by inhibiting EMT, and promote apoptosis by regulating the expression of apoptosis related proteins and increasing the ROS content of cells.
关键词
胃癌 /
FLOT1基因 /
侵袭 /
凋亡 /
上皮细胞间质转化
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Key words
gastric cancer /
FLOT1 gene /
invasion /
apoptosis /
epithelial mesenchyme transformation
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中图分类号：
R965
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参考文献
[1] ZHOU X, WANG W, LI P, et al. Curcumin enhances the effects of 5-fluorouracil and oxaliplatin in inducing gastric cancer cell apoptosis both in vitro and in vivo . Oncol Res, 2016, 23(1):29-34.
[2] KOH M, YONG H Y, KIM E S, et al. A novel role for flotillin-1 in H-ras-regulated breast cancer aggressiveness. Int J Cancer, 2016, 138(5):1232-1245.
[3] LI H, WANG R M, LIU S G, et al. Abnormal expression of FLOT1 correlates with tumor progression and poor survival in patients with non-small cell lung cancer. Tumor Biol, 2014, 35(4):3311-3315.
[4] ZHANG Y, LI J, SONG Y, et al. Flotillin-1 expression in human clear-cell renal cell carcinoma is associated with cancer progression and poor patient survival. Mol Med Reports, 2014, 10(2):860-866.
[5] QIN Y, MIN Z, ZI W, et al. 4.1N is involved in a flotillin-1/β-catenin/Wnt pathway and suppresses cell proliferation and migration in non-small cell lung cancer cell lines. Tumor Biol, 2016, 37(9):12713-12723.
[6] CAO C L, LI T Y, CAO C, et al. Expression and clinical significance of FLOT1 protein in gastric cancer. World Chin J Digestol(世界华人消化杂志), 2015,23(32):5141-5147.
[7] KANG M, REN M P, ZHAO L, et al. miR-485-5p Acts as a negative regulator in gastric cancer progression by targeting flotillin-1. Am J Transl Res, 2015, 7(11):2212-2222.
[8] LI H, ZHANG Y, CHEN S W, et al. Prognostic significance of flotillin1 expression in clinically NO tongue squamous cell cancer. Int J Clin Exp Pathol, 2014, 7(3):996-1003.
[9] ZHANG S H, WANG C J, SHI L, et al. High expression of FLOT1 is associated with progression and poor prognosis in hepatocellular carcinoma. PLoS One, 2013, 8(6):e64709.
[10] CAO S, CUI Y, XIAO H, et al. Upregulation of flotillin-1 promotes invasion and metastasis by activating TGF-β signaling in nasopharyngeal carcinoma. Oncotarget, 2016, 7(4):4252-4264.
[11] LI J. Expression and function of flotillin-1 in small cell lung cancer. Shijiazhuang:Hebei Medical University, 2016.
[12] BREYER J, GIERTH M, SHALEKENOV S, et al. Epithelial-mesenchymal transformation markers E-cadherin and survivin predict progression of stage pTa urothelial bladder carcinoma. World J Urol, 2016, 34(5):709-716.
[13] LIU F, KORC M. Cdk4/6 inhibition induces epithelial-mesenchymal transition and enhances invasiveness in pancreatic cancer cells. Mol Cancer Ther, 2012, 11(10):2138-2148.
[14] SHIN H W, CHO K, KIM D W, et al. Hypoxia-inducible factor 1 mediates nasal polypogenesis by inducing epithelial-to-mesenchymal transition. Am J Respir Critic Care Med, 2012, 185(9):944-954.
[15] SITARZ R, LEGUIT R J, DE LENG W W, et al. Cyclooxygenase-2 mediated regulation of E-cadherin occurs in conventional but not early-onset gastric cancer cell lines. Cell Oncol, 2016, 31(6):475-485.
[16] LIN Y, SHEN L Y, FU H, et al. P21, COX-2, and E-cadherin are potential prognostic factors for esophageal squamous cell carcinoma. Dis Esophagus, 2016, 30(2):1-10.
[17] INOUE H, TSUCHIYA H, MIYAZAKI Y, et al. Podoplanin expressing cancer-associated fibroblasts in oral cancer. Tumor Biol, 2014, 35(11):11345-11352.
[18] LI Z, YANG Y, GAO Y, et al. Elevated expression of flotillin-1 is associated with lymph node metastasis and poor prognosis in early-stage cervical cancer. Am J Cancer Res, 2015, 6(1):38-50.
[19] ZHAO S C, WU Q W, CHENG Y F, et al. Protective effect and mechanism of butylphthalide on SH-SY5Y cell apoptosis induced by MPP+. Chin J Gerontol(中国老年学杂志), 2017, 37(2):316-318.
[20] LI K, XU G, LIU C, et al. Bcl-2/Bax switch was flipped during apoptosis of HCT116 colon cancer cells induced by a traditional Chinese medicine, Banxia Xiexin decoction. Int J Clin Exp Med, 2017, 10(7):10535-10541.
[21] CHECINSKA A, HOOGELAND B S J, RODRIGUEZ J A, et al. Role of XIAP in inhibiting cisplatin-induced caspase activation in non-small cell lung cancer cells. Exp Cell Res, 2016, 313(6):1215-1224.
[22] KOUL M, MEENA S, KUMAR A, et al. Secondary metabolites from endophytic fungus Penicillium pinophilum induce ROS-mediated apoptosis through mitochondrial pathway in pancreatic cancer cells. Planta Med, 2016, 82(4):344-355.
[23] SEO B R, MIN K J, WOO S M, et al. Inhibition of cathepsin S induces mitochondrial ROS that sensitizes TRAIL-mediated apoptosis through p53-mediated downregulation of Bcl-2 and c-FLIP. Antioxid Redox Signal, 2017, 27(4):215-233.
[24] LIANG T, ZHANG X, XUE W, et al. Curcumin induced human gastric cancer BGC-823 cells apoptosis by ROS-Mediated ASK1-MKK4-JNK stress signaling pathway. Int J Mol Sci, 2014, 15(9):15754-15765.
[25] LIU C, SUN H N, LUO Y H, et al. Cryptotanshinone induces ROS-mediated apoptosis in human gastric cancer cells. Oncotarget, 2017, 8(70):115398-115412.
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