转录因子SNAI1基因沉默对乳腺癌细胞MCF-7生物学表型的影响
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摘要
目的:以体外培养的人乳腺癌细胞MCF-7为研究对象,研究转录因子snail(SNAI1)基因沉默对MCF-7细胞生物学表型的影响。
方法:针对SNAI1基因序列设计shRNA,并与表达绿色荧光蛋白(Green Fluorescent Protein,GFP)的真核表达载体pGCsilencerTMU6/Neo/GFP连接,转染MCF-7细胞并进行G418筛选,获得质粒相对稳定表达的细胞;通过RT-PCR鉴定SNAI1基因mRNA水平沉默效果;细胞免疫荧光检测SNAI1基因蛋白水平沉默效果及表达定位的改变;倒置显微镜观察细胞形态的改变;细胞计数法检测细胞的增殖能力;细胞划痕法检测细胞的体外迁移能力;Boyden小室法检测细胞的体外侵袭能力;RT-PCR检测上皮标记物claudin-4、claudin-6、claudin-7、E-cadherin和间质标记物MMP-2表达水平的改变。
结果:SNAI1基因沉默组相对于对照组细胞形态发生改变;细胞增殖、迁移能力及侵袭能力减弱;细胞的上皮标志物claudin-4、claudin-7、E-cadherin表达升高,间质标志物MMP-2表达降低。
结论:SNAI1基因沉默可以抑制MCF-7细胞的恶性表型,并通过其相关靶基因的调控逆转EMT过程。
Transcription factor SNAI1 belongs to Zine finger transcription factor super family of snail, more than 50 family members have been described in metazoans, SNAI1 is wide distribution in the human body, including placenta, heart, lung, brain, liver, skeletal muscle and other tissues.There are four C2H2 zinc fingers in the carboxyl terminal of SNAI1, which can bind E-boxes of target genes specially, then inhibit the activity of promoters and induce EMT. In the process of EMT, epithelial cell lose their polarity and cell–cell junctions and acquire mesenchymal characteristics and migratory abilities。The process of EMT plays a key role in various physiological and pathologic processes, such as embryo development, healing of wound, cell differentiation and Neoplasm Metastasis.
SNAI1 plays an important role in the tumorigenesis and tumor invison. In the process of invision of malignant tumor cell from epithelium, such as breast carcinoma, SNAI1 can increase the invasion and metastasis abilities by induce EMT. IN many tissue of tumor, such as breast carcinoma, gastric carcinoma, ancer of pancreas, cancer of the liver, colon carcinoma and melanoma, the expression of SNAI1 is increased besides the modify of cellular localization. At the same time, SNAI1 is associated with the invasion and metastasis abilities of cell lines originate from tumor.
Gene promoter activity assay showed that E-box exist in the upstream of promoter in target genes such as E-cadherin,occludin,claudins and ZO-1, SNAI1 can bind E-boxes specially and then inhibit the activity of promoters to complete the physiological or pathological function. At the same time, SNAI1 can up regulate the epithelium cell makers including desmosomes, MUC-1, CK-18, down regregulate mesenchymal cell makers including Vimenti, Fibronectin, lymphoid enhaneer factor l(LEFI) , zinefinger E-box binding homcobox-l(ZEBI) and MMP-2,MMP-9.
Breast carcinemia is the female malignant tumor from the breast ductal epithelium, Whose incidence and mortality raise gradually. Leading cause to death of patients caused by breast cancer is tumor metastasis and recurrence, to date there is no satisfactory treatment. At present, many scholars consider that SNAI1 play a key role in invasion and metastasis of breast carcinoma and other epithelial tumor. But to now, there is no relevant reports about the effect of Gene Silencing of SNAI1 on biological phenotypes in Breast Cell Line MCF-7
In order to determine the effect of SNAI1 on biological phenotypes in Breast Cell Line MCF-7, we designed shRNA fragment targeting on SNAI1 gene and then inserted to eukaryotic expression vector pGCsilencerTMU6/Neo/GFP which express Green Fluorescent Protein, The plasmid was transfected into MCF-7 cells using lipidosome . G418 -resistant cells were expanded in culture as relative stable expression population. Cells were selected to examine the mRNA expression of SNAI1 by RT-PCR ; examine the protein expression of SNAI1 and the change of cell localization by Immunofluorescence; Examine the cell appearance by inverted microscope; detect the cell proliferative ability by cell counting; determine the cell migration ability by wound healing assay; check out the cell incision ability by Boyden assay. Examine the expression of cell epithelium makers claudin-4、claudin-6、claudin-7、E-cadherin and mesenchymal cell maker MMP-2 .The results showe that Partial gene silencing of SNAI1 can change the biological phenotypes of breast cancer cell MCF-7 .The rusults are as follows:
(1) Gene Silence of SNAI1 in MCF-7 resulted in an alteration in phenotype. Cell body was relatively rounded, pseudopodia was significantly shortened, three-dimensional of cells was strengthened.
(2) Gene Silence of SNAI1 in MCF-7 resulted that the proliferative ability of cell was decreased in vitro .Cell count suggested that cells of experimental group growed slowly than other groups, P<0.01, statistically analysis shows significant differerce.
(3) Gene Silence of SNAI1 in MCF-7 resulted that the migration ability of cell was decreased in vitro. Wound healing assay found that the migration ability of SNAI1 silencing cells was extremely lower than other groups, P<0.01, statistically analysis shows significant differerce.
(4) Gene Silence of SNAI1 in MCF-7 resulted that the invasion ability of cell was decreased in vitro. Boyden chamber assay show that control group and the negative control group have a certain number of cell invasion into polycarbonate membrane level, the cells of experimental group didn’t reach to that level, P<0.01, statistically analysis shows significant differerce. At the same time, 3D pictures demonstrate cells of experimental group’s migration distance in Matrigel gel is less than the other two groups.
(5) RT-PCR showed that SNAI1 silence could increase the expression of epithelial markers claudin-4、claudin-7、E-cadherin and decrease the expression of mesenchymal markers MMP-2.
These findings further support the hypothesis that SNAI1 as a transcription repressor factor, decreased expression of which contribute to benign progression of cancer, at least for breast cancer cell MCF-7.
方法:针对SNAI1基因序列设计shRNA,并与表达绿色荧光蛋白(Green Fluorescent Protein,GFP)的真核表达载体pGCsilencerTMU6/Neo/GFP连接,转染MCF-7细胞并进行G418筛选,获得质粒相对稳定表达的细胞;通过RT-PCR鉴定SNAI1基因mRNA水平沉默效果;细胞免疫荧光检测SNAI1基因蛋白水平沉默效果及表达定位的改变;倒置显微镜观察细胞形态的改变;细胞计数法检测细胞的增殖能力;细胞划痕法检测细胞的体外迁移能力;Boyden小室法检测细胞的体外侵袭能力;RT-PCR检测上皮标记物claudin-4、claudin-6、claudin-7、E-cadherin和间质标记物MMP-2表达水平的改变。
结果:SNAI1基因沉默组相对于对照组细胞形态发生改变;细胞增殖、迁移能力及侵袭能力减弱;细胞的上皮标志物claudin-4、claudin-7、E-cadherin表达升高,间质标志物MMP-2表达降低。
结论:SNAI1基因沉默可以抑制MCF-7细胞的恶性表型,并通过其相关靶基因的调控逆转EMT过程。
Transcription factor SNAI1 belongs to Zine finger transcription factor super family of snail, more than 50 family members have been described in metazoans, SNAI1 is wide distribution in the human body, including placenta, heart, lung, brain, liver, skeletal muscle and other tissues.There are four C2H2 zinc fingers in the carboxyl terminal of SNAI1, which can bind E-boxes of target genes specially, then inhibit the activity of promoters and induce EMT. In the process of EMT, epithelial cell lose their polarity and cell–cell junctions and acquire mesenchymal characteristics and migratory abilities。The process of EMT plays a key role in various physiological and pathologic processes, such as embryo development, healing of wound, cell differentiation and Neoplasm Metastasis.
SNAI1 plays an important role in the tumorigenesis and tumor invison. In the process of invision of malignant tumor cell from epithelium, such as breast carcinoma, SNAI1 can increase the invasion and metastasis abilities by induce EMT. IN many tissue of tumor, such as breast carcinoma, gastric carcinoma, ancer of pancreas, cancer of the liver, colon carcinoma and melanoma, the expression of SNAI1 is increased besides the modify of cellular localization. At the same time, SNAI1 is associated with the invasion and metastasis abilities of cell lines originate from tumor.
Gene promoter activity assay showed that E-box exist in the upstream of promoter in target genes such as E-cadherin,occludin,claudins and ZO-1, SNAI1 can bind E-boxes specially and then inhibit the activity of promoters to complete the physiological or pathological function. At the same time, SNAI1 can up regulate the epithelium cell makers including desmosomes, MUC-1, CK-18, down regregulate mesenchymal cell makers including Vimenti, Fibronectin, lymphoid enhaneer factor l(LEFI) , zinefinger E-box binding homcobox-l(ZEBI) and MMP-2,MMP-9.
Breast carcinemia is the female malignant tumor from the breast ductal epithelium, Whose incidence and mortality raise gradually. Leading cause to death of patients caused by breast cancer is tumor metastasis and recurrence, to date there is no satisfactory treatment. At present, many scholars consider that SNAI1 play a key role in invasion and metastasis of breast carcinoma and other epithelial tumor. But to now, there is no relevant reports about the effect of Gene Silencing of SNAI1 on biological phenotypes in Breast Cell Line MCF-7
In order to determine the effect of SNAI1 on biological phenotypes in Breast Cell Line MCF-7, we designed shRNA fragment targeting on SNAI1 gene and then inserted to eukaryotic expression vector pGCsilencerTMU6/Neo/GFP which express Green Fluorescent Protein, The plasmid was transfected into MCF-7 cells using lipidosome . G418 -resistant cells were expanded in culture as relative stable expression population. Cells were selected to examine the mRNA expression of SNAI1 by RT-PCR ; examine the protein expression of SNAI1 and the change of cell localization by Immunofluorescence; Examine the cell appearance by inverted microscope; detect the cell proliferative ability by cell counting; determine the cell migration ability by wound healing assay; check out the cell incision ability by Boyden assay. Examine the expression of cell epithelium makers claudin-4、claudin-6、claudin-7、E-cadherin and mesenchymal cell maker MMP-2 .The results showe that Partial gene silencing of SNAI1 can change the biological phenotypes of breast cancer cell MCF-7 .The rusults are as follows:
(1) Gene Silence of SNAI1 in MCF-7 resulted in an alteration in phenotype. Cell body was relatively rounded, pseudopodia was significantly shortened, three-dimensional of cells was strengthened.
(2) Gene Silence of SNAI1 in MCF-7 resulted that the proliferative ability of cell was decreased in vitro .Cell count suggested that cells of experimental group growed slowly than other groups, P<0.01, statistically analysis shows significant differerce.
(3) Gene Silence of SNAI1 in MCF-7 resulted that the migration ability of cell was decreased in vitro. Wound healing assay found that the migration ability of SNAI1 silencing cells was extremely lower than other groups, P<0.01, statistically analysis shows significant differerce.
(4) Gene Silence of SNAI1 in MCF-7 resulted that the invasion ability of cell was decreased in vitro. Boyden chamber assay show that control group and the negative control group have a certain number of cell invasion into polycarbonate membrane level, the cells of experimental group didn’t reach to that level, P<0.01, statistically analysis shows significant differerce. At the same time, 3D pictures demonstrate cells of experimental group’s migration distance in Matrigel gel is less than the other two groups.
(5) RT-PCR showed that SNAI1 silence could increase the expression of epithelial markers claudin-4、claudin-7、E-cadherin and decrease the expression of mesenchymal markers MMP-2.
These findings further support the hypothesis that SNAI1 as a transcription repressor factor, decreased expression of which contribute to benign progression of cancer, at least for breast cancer cell MCF-7.
引文
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[2] KatohM.Pharmacogenomics on gastric cancer [J]. Cancer Biol Ther 2004,3:566-567.
[3] Thiery JP. Epithelial -mesenchymal transition in tumor progression [J]. Nat Rev cancer, 2002, 2(6): 442-454.
[4] Peinado H, Ballestar E, Esteller M, et al. Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex[J]. Mol Cell Biol. 2004, 24(1): 306-319.
[5] Pan PH,Yang HZ,Chen Q,et al.The expression of E-cadherin and epidermal growth factor receptor in non-small-cell lung cancer and its clinical significance [J].China Journal of Modern Medicine,2002;12(1):26-32
[6] Van der Wurff AA, Vermeulen SJ, van der Linden EP, et al. Patterns of alpha-and beta-catenin and E-cadherin expression in colorectal adenomas and carcinomas [J]. J Pathol, 1997, 182(3):325-330.
[7] BerxG, Van Roy F. The E-cadherin/catenin complex: an important gatekeeper in breast cancer tumorigenesis and malignant progression [J]. Breast Cancer Res 2001,3 :289-293.
[8] Douglas S Micalizzi, Heide L Ford .Epithelial-mesenchymal transition in development and cancer [J]. Future Oncology . 2009, 5(8):1129-1143.
[9] M. Angela Nieto. The snail superfamily of zinc-finger transcription factors [J]. Nature Reviews Molecular Cell Biology .2002,3, 155-166.
[10] Buzz Baum, Jeffrey Settleman, Margaret P. Quinlan Transitions between epithelial and mesenchymal states in development and disease [C]. Seminars in Cell & Developmental Biology. 2008, 19:294-308.
[11] Jethwa P, Naquvl M, Hardy RG, et al. Over expression of Slug is associated With Malignant Progression of esophageal adenocarcinoma [J]. World Journal of Gastroentrol, 2008, 14(7):1044-1052.
[12] Tadashi Ohkubo, Masayuki Ozawa. The transcription factor Snai downregulates the tight junction components independently of E-cadherin downregulation [J]. Journal of Cell Science.2004, 117 (9): 1675-1685.
[13] Ikenouchi J, Matsuda M, Furuse M, et al. Regulation of tight junctions duringepithelial-mesenchymal transition: Direct repression of the gene expression of claudins/oclaudin by Snail [J]. Cell Sci. 2003, 116:1959-1967.
[14] Jorda M, Olmeda D, Vinyals A, et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor [J]. Cell Sci 2005, 118:3371-3385.
[15] A Miyoshi, Y Kitajima,K Sumi,et al. Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells [J] . British Journal of Cancer ,2004, 90:1265–1273.
[16] Blanco MJ, Moreno-Bueno G, Sarrio D, et al. Correlation of Snail expression with histological grade and lymph node status in breast carcinomas[J]. Oncogene 2002, 21:3241-3246.
[17] Schwartz B, Melnikova VO, Tellez C, et al .Loss of ap-2alpha result in deregulation E-cadherin and mmp-9 and increase in tumorigenicity of colon cancer cells in vivo [J]. Oncogene, 2007,26:404-4058.
[18] Mauro L, Catalano S, Bossi Q Pellegrino M, et al. Evidences that leptin up-regulates e-cadherin expression in breast cancer: Effeets on tumor growth and progression [J].Cancer Res, 2007,67:3412-3421.
[19] Zhou BP, Deng J, Xia W, et al.Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition [J].Nat Cell Biol. 2004, 6(10):931-940.
[20] Manzanares M, Locascio A, Nieto M A .The increasing compleaity of the Snail superfamily in metazoan evolution [J]. Trends Genet, 2001, 17:178-181.
[21] Pisani P, Bray F, Parkin DM. Estimates of the world-wide Prevalence of cancer for 25 Sites in the adult population [J]. Int J Caneer. 2002,97:72-81.
[22] Perz-moren M A, Locascio A, Cano A, et al. A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transition [J]. J Biol Chem, 2001, 276:27424-27431.
[23] Kajita M, McClinic KN, Wade PA. Aberrant expression of the transcription factors snail and slug alters the response to genotoxic stress [J]. Mol Cell Biol 2004, 24:7559-7566.
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