人IGFBP-1基因真核表达载体的构建及其对胃癌细胞BGC-823增殖的影响
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摘要
目的构建IGFBP-1真核表达载体,观察IGFBP-1基因真核表达载体转染胃癌细胞株BGC-823后基因的表达及其对BGC-823细胞增殖的影响.方法以GES-1细胞基因组为模板,PCR技术扩增人IGFBP-1的基因片段,克隆人pc DNA3-His-Flag真核表达载体,重组载体pc DNA3-IGFBP-1-His-Flag,并通过PCR、双酶切和测序鉴定.重组质粒经阳离子脂质体法转染BGC-823细胞,RT-PCR和Western blot检测细胞IGFBP-1的表达情况,并通过CCK8法观察IGFBP-1过表达对BGC-823细胞增殖的影响.结果经PCR、双酶切和测序鉴定证明IGFBP-1真核表达载体构建成功,并在胃癌细胞株BGC-823高效表达.CCK8实验显示:IGFBP-1过表达对BGC-823细胞增殖无影响.结论成功构建IGFBP-1真核表达载体,IGFBP-1过表达对BGC-823细胞的增殖无影响,可为进一步研究IGFBP-1生物学功能奠定基础.
Objective To investigate the effect of IGFBP-1 gene on BGC-823 cell proliferation,an eukaryotic expression vector containing IGFBP-1 gene was constructed. Method With GES-1 cell genome as a template,IGFBP-1 gene was amplified by PCR and inserted into the eukaryotic expression plasmid pcDNA3-His-Flag,and then the recombinant plasmid pcDNA3-IGFBP-1-His-Flag was identified by PCR,restriction enzymes digestion and DNA sequencing.Recombinant plasmids pcDNA3-IGFBP-1-His-Flag were transfected into BGC-823 cells by using cationic liposomes. Results RT-PCR and Western blot detected the expression of IGFBP-1 mRNA and protein,and then observed the effect of IGFBP-1 on BGC-823 cells proliferation by CCK8. CCK8 assay showed that IGFBP-1 has no influence on BGC-823 cells proliferation.Conclusion The results indicated that IGFBP-1 eukaryotic expression vector was successfully constructed and IGFBP-1 genes were highly expressed in transfected BGC-823 cells. IGFBP-1 has no influence on BGC-823 cells proliferation. The results provide a basis for the further study of biological functions of IGFBP-1.
Objective To investigate the effect of IGFBP-1 gene on BGC-823 cell proliferation,an eukaryotic expression vector containing IGFBP-1 gene was constructed. Method With GES-1 cell genome as a template,IGFBP-1 gene was amplified by PCR and inserted into the eukaryotic expression plasmid pcDNA3-His-Flag,and then the recombinant plasmid pcDNA3-IGFBP-1-His-Flag was identified by PCR,restriction enzymes digestion and DNA sequencing.Recombinant plasmids pcDNA3-IGFBP-1-His-Flag were transfected into BGC-823 cells by using cationic liposomes. Results RT-PCR and Western blot detected the expression of IGFBP-1 mRNA and protein,and then observed the effect of IGFBP-1 on BGC-823 cells proliferation by CCK8. CCK8 assay showed that IGFBP-1 has no influence on BGC-823 cells proliferation.Conclusion The results indicated that IGFBP-1 eukaryotic expression vector was successfully constructed and IGFBP-1 genes were highly expressed in transfected BGC-823 cells. IGFBP-1 has no influence on BGC-823 cells proliferation. The results provide a basis for the further study of biological functions of IGFBP-1.
引文
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[16] Lim S T,Mikolon D,Stupack D G,et al.FERM control ofFAK function:implications for cancer therapy[J]. CellCycle,2008,7:2306-2314.
[17] Ammoun S,Schmid M C,Zhou L,et al.Insulin-like growthfactor-binding protein-1(IGFBP-1)regulates human sch-wannoma proliferation,adhesion and survival[J]. Onco-gene,2012,31:1710-1722.
[18] Dai B,Ruan B,Wu J,et al. Insulin-like growth factorbinding protein-1 inhibits cancer cell invasion and isassociated with poor prognosis in hepatocellular carc-inoma[J].Int J Clin Exp Pathol,2014,7:5645-5654.
[19] Chesik D,De Keyser J,Bron R,et al.Insulin-like growthfactor binding protein-1 activates integrin-mediated intr-acellular signaling and migration in oligodendrocytes[J].JNeurochem,2010,113:1319-1330.
[20] Chakraborty C,Gleeson L M,Mc Kinnon T,et al.Regulationof human trophoblast migration and invasiveness[J].Can JPhysiol Pharmacol,2002,80:116-124.
[21] Perks C M,Newcomb P V,Norman M R,et al.Effect ofinsulin-like growth factor binding protein-1 on integrinsignalling and the induction of apoptosis in human breastcancer cells[J].J Mol Endocrinol,1999,22:141-150.
[2] Jones J I,Doerr M E,Clemmons D R. Cell migration:interactions among integrins,IGFs and IGFBPs[J]. ProgGrowth Factor Res,1995,6:319-327.
[3] Lee P D,Giudice L C,Conover C A,et al.Insulin-like growthfactor binding protein-1:recent findings and new directions[J].Proc Soc Exp Biol Med,1997,216:319-357.
[4] Bereket A,Lang C H,Wilson T A. Alterations in thegrowth hormone-insulin-like growth factor axis in insulindependent diabetes mellitus[J]. Horm Metab Res,1999,31:172-181.
[6] Xue M,Fang Y,Sun G,et al. IGFBP3,a transcriptionaltarget of homeobox D10,is correlated with the prognosis ofgastric cancer[J].PLoS One,2013,8:e81423.
[7]张雪英,吉兆宁.胃癌中IGFBP-2、IGFI、IGFIR的表达及意义[J].皖南医学院学报,2008,27(2):98-101.
[8] Jones J I,Clemmons D R.Insulin-like growth factors andtheir binding proteins:biological actions[J].Endocr Rev,1995,16:3-34.
[9] Ferry R J Jr,Katz L E,Grimberg A,et al.Cellular actionsof insulin-like growth factor binding proteins[J]. HormMetab Res,1999,31:192-202.
[10] Baxter R C.Insulin-like growth factor(IGF)-binding pro-teins:interactions with IGFs and intrinsic bioactivities[J].Am J Physiol Endocrinol Metab,2000,278:E967-976.
[11] Clemmons D R.Insulin-like growth factor binding proteinsand their role in controlling IGF actions[J]. CytokineGrowth Factor Rev,1997,8:45-62.
[12] Elgin R G,Busby W H Jr,Clemmons D R.An insulin-like growth factor(IGF)binding protein enhances thebiologic response to IGF-I[J]. Proc Natl Acad Sci U SA,1987,84:3254-3258.
[13] Zhao L L,Galiano R D,Cox G N,et al.Effects of insulin-like growth factor-I and insulin-like growth factorbinding protein-1 on wound healing in a dermal ulcermodel[J].Wound Repair Regen,1995,3:316-321.
[14] Brandt K,Grunler J,Brismar K,et al.Effects of IGFBP-1and IGFBP-2 and their fragments on migration and IGF-induced proliferation of human dermal fibroblasts[J].Growth Horm IGF Res,2015,25:34-40.
[15] Wang J,Razuvaev A,Folkersen L,et al.The expressionof IGFs and IGF binding proteins in human carotidatherosclerosis,and the possible role of IGF bindingprotein-1 in the regulation of smooth muscle cell prolif-eration[J].Atherosclerosis,2012,220:102-109.
[16] Lim S T,Mikolon D,Stupack D G,et al.FERM control ofFAK function:implications for cancer therapy[J]. CellCycle,2008,7:2306-2314.
[17] Ammoun S,Schmid M C,Zhou L,et al.Insulin-like growthfactor-binding protein-1(IGFBP-1)regulates human sch-wannoma proliferation,adhesion and survival[J]. Onco-gene,2012,31:1710-1722.
[18] Dai B,Ruan B,Wu J,et al. Insulin-like growth factorbinding protein-1 inhibits cancer cell invasion and isassociated with poor prognosis in hepatocellular carc-inoma[J].Int J Clin Exp Pathol,2014,7:5645-5654.
[19] Chesik D,De Keyser J,Bron R,et al.Insulin-like growthfactor binding protein-1 activates integrin-mediated intr-acellular signaling and migration in oligodendrocytes[J].JNeurochem,2010,113:1319-1330.
[20] Chakraborty C,Gleeson L M,Mc Kinnon T,et al.Regulationof human trophoblast migration and invasiveness[J].Can JPhysiol Pharmacol,2002,80:116-124.
[21] Perks C M,Newcomb P V,Norman M R,et al.Effect ofinsulin-like growth factor binding protein-1 on integrinsignalling and the induction of apoptosis in human breastcancer cells[J].J Mol Endocrinol,1999,22:141-150.