RNA干扰沉默TGF-β1基因对口腔鳞癌细胞CAL-27生物学行为的影响
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摘要
目的:观察siRNA干扰转化生长因子-β1(TGF-β1)基因对口腔鳞癌CAL-27细胞增殖、迁移、侵袭能力的影响。方法:取体外培养的口腔鳞癌CAL-27细胞,分为观察组和阴性对照组,观察组和阴性对照组分别转染敲除TGF-β1基因的siRNA、阴性对照siRNA。采用实时荧光定量RT-q PCR法检测转染24 h时各组细胞中TGF-β1的表达,通过流式细胞仪分析siRNA对口腔鳞癌细胞周期的影响,用平板克隆和CCK8法观察转染后各组细胞增殖情况及Transwell实验观察转染后各组迁移、侵袭能力,western blot检测转染24 h时各组细胞蛋白。结果:转染24 h时,与阴性对照组相比,观察组细胞TGF-β1相对表达量低,穿膜细胞数少,蛋白相对表达量低。转染72 h、96 h观察组OD值低于阴性对照组。结论:RNA干扰沉默TGF-β1基因能有效抑制口腔鳞癌细胞的增殖、迁移、侵袭,在一定程度上可以逆转口腔鳞癌细胞的恶性表型。
Objective: To investigate the effect of RNA interference( RNAi) targeting transforming growth factor-β1( TGF-β1) gene on the invasion and migration of oral squamous cell carcinoma( OSCC) cellline CAL-27. Methods: The oral squamous cell carcinoma CAL-27 cells cultured in vitro were randomly divided into the observation group,negative control group. CAL-27 cells in the observation group and negative control group were transfected with TGF-β1-siRNA and Negative control siRNA( NC-siRNA) in vitro by using Lipojet. The Real time quantitative polymerase chain reaction( RT-q PCR) was used to detect the expression of TGF-β1 at 24 h after transfection. The distribution of cell cycle phases was determined by flow cytometry. Plate cloning assay and Cell counting kit-8( CCK-8) assay was used to observe the proliferation of the cells after transfection. Transwell assay were used to observe the migration and invasion abilities of the cells after transfection. Western blotting was used to explore the expression of protein at 24 h after transfection. Results: At 24 h after transfection,compared with the negative control group,the expression of TGF-β1 and β-catenin protein was significantly lower,the number of penetrating cells was significantly less. A450 in the observation group was lower than that in the negative control group at 48 and 72 h after transfection. Conclusion: RNA interference targeting TGF-β1 gene could suppress proliferation,invasion and migration of oral squamous cell carcinoma cells effectively,and reverse the malignant phenotypes of oral squamous cell carcinoma cells to some extent.
Objective: To investigate the effect of RNA interference( RNAi) targeting transforming growth factor-β1( TGF-β1) gene on the invasion and migration of oral squamous cell carcinoma( OSCC) cellline CAL-27. Methods: The oral squamous cell carcinoma CAL-27 cells cultured in vitro were randomly divided into the observation group,negative control group. CAL-27 cells in the observation group and negative control group were transfected with TGF-β1-siRNA and Negative control siRNA( NC-siRNA) in vitro by using Lipojet. The Real time quantitative polymerase chain reaction( RT-q PCR) was used to detect the expression of TGF-β1 at 24 h after transfection. The distribution of cell cycle phases was determined by flow cytometry. Plate cloning assay and Cell counting kit-8( CCK-8) assay was used to observe the proliferation of the cells after transfection. Transwell assay were used to observe the migration and invasion abilities of the cells after transfection. Western blotting was used to explore the expression of protein at 24 h after transfection. Results: At 24 h after transfection,compared with the negative control group,the expression of TGF-β1 and β-catenin protein was significantly lower,the number of penetrating cells was significantly less. A450 in the observation group was lower than that in the negative control group at 48 and 72 h after transfection. Conclusion: RNA interference targeting TGF-β1 gene could suppress proliferation,invasion and migration of oral squamous cell carcinoma cells effectively,and reverse the malignant phenotypes of oral squamous cell carcinoma cells to some extent.
引文
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[14]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].Nature,2001,411(6836):494-498.
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[2]Cannon TL,Lai DW,Hirsch D,et al.Squamous cell carcinoma of the oral cavity in nonsmoking women:a nem and unusual complication of chemotherapy for recurrent ovariancancer[J].Oncologist,2012,17(12):1541-1546.
[3]Dibble EH,Alyarez AC,Truong MT,et al.F-FDG metabolic tumor volume and totalglycolytie activity of oral cavity and oropharyngeal squamous call cancer:adding value to clinical staging[J].J Nucl Med,2012,53(5):709-715.
[4]Brenmoehl J,Miller SN,Hofmann C,et al.Transforming growth factorbeta 1 induces intestinal myofibroblast diffrerntiation and modulates ther migration[J].World J Gastroenterol,2009,15(12):1431-1442.
[5]Saji H,Nakamura H,Awut I,et al.Significance of expression of TGFbeta in pulmonar metastasis in non-small cell lung cancer tissues[J].Ann Thorac Cardiovasc Surg,2003,9(5):295-300.
[6]Ji F,Jin X,Jiao CH,et al.FAT10 level in human gastric cancer and its relation with mutant p53 level,lymph node metastasis and TNM staging[J].World J Gastroenterol,2009,15(18):2228-2233.
[7]Lin RL,Zhao LJ.Mechanistic basis and clinical relevance of the role of transforming growth factor-beta in cancer[J].Cancer Biol Med,2015,12(4):385-393.
[8]Eisma RJ,Spire JD,von Biberstein SE,et a1.Decreased expression of transforming growth factor beta receptors on head and neck squamous cell carcinoma tumor cells[J].AM J Surg,1996,172(6):64 l-645.
[9]Subramanian G,Schwarz RE,Higgins L,et al.Targeting endogenous transforming growth factor-βreceptor signaling in SMAD4-deficient human pancreatic carcinoma cells inhibits their invasive phenotype[J].Cancer Res,2004,64(15):5200-5211.
[10]Michl P,Ramjaun AR,Pardo OE,et al.CUTL1 is a target of TGF-βsignaling that enhances cancer cell motility and invasiveness[J].Cancer Cell,2005,7(6):521-532.
[11]Mourskaia AA,Dong Z,Ng S,et al.Transforming growth factor-beta1 is the predominant isoform required for breast cancer cell outgrowth in bone[J].Oncogene,2009,28(7):1005-1015.
[12]Webb CP,Vande Woude GF.Genes that regulate metastasis and angiogenesis[J].J Neurooncol,2000,50(1/2):71-87.
[13]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(391):806-811.
[14]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].Nature,2001,411(6836):494-498.
[15]Chakraborty C.Potentiality of small interfering RNAs(ARNA)as recent therapeutic targets for gene-silencing[J].Current Drug Targets,2007,8(3):469.
[16]Brummelkamp TR,Bernards R,Agami R.A system for stable expression of short interfering RNAs in mammalian cells[J].Science,2002,296(5567):550-553.