Q61R和V112A突变的HRAS基因在NIH小鼠体内诱发肿瘤的实验研究
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摘要
目的:分析含有HRAS(V112A)基因及其G12C、G13C、Q61R和G12C/G13C/Q61R突变体的质粒DNA在NIH小鼠体内的致癌性。方法:从人结肠癌DiFi细胞中扩增含有V112A突变的HRAS(V112A)基因,采用PCR点突变法在HRAS(V112A)中分别引入G12C[HRAS(V112A/G12C)]、G13C[HRAS(V112A/G13C)]、Q61R[HRAS(V112A/Q61R)]或3位点联合突变[HRAS(V112A/G12C/G13C/Q61R)],将HRAS(V112A)及上述突变基因克隆入真核表达载体pCDNA3.1(+),Western blot确证HRAS(V112A)及各突变体可体外表达后,每种质粒按每只100μg剂量皮内注射6～8周龄雌性NIH小鼠,阴性对照组注射PBS,每组8只小鼠,持续观察小鼠致瘤情况。注射后4个月,取小鼠瘤样组织分别采用PCR和Western blot方法检测病变组织中的HRAS基因及其表达产物。结果:从DiFi细胞中成功扩增出含有V112A突变的HRAS(V112A)基因,并构建4个突变体,体外转染结果表明各HRAS均可在L929细胞中表达。重组质粒注射小鼠后,HRAS(V112A)组有4只小鼠注射后4个月于注射部位出现增生样改变,该病变在1个月后自愈;HRAS(V112A/Q61R)组有1只小鼠于注射后4个月出现明显的瘤样增生。至观察期末,HRAS(V112A/G12C)、HRAS(V112A/G13C)和HRAS(V112A/G12C/G13C/Q61R)组小鼠均未出现可见增生样结节。病变组织的PCR和Western blot检测结果表明HRAS(V112A)组小鼠增生样组织和HRAS(V112A/Q61R)组小鼠肿瘤组织中均有对应的HRAS基因存在和表达。结论:HRAS(V112A)可在NIH小鼠体内导致注射部位皮肤的增生样改变,含有Q61R突变的HRAS(V112A)可在NIH小鼠体内诱导肿瘤形成,含有G12C、G13C及G12C/G13C/Q61R突变的HRAS(V112A)在小鼠体内不会引起明显病变。
OBJECTIVE:To evaluate oncogenicity of HRAS(V112A) and its G12 C,G13C,Q61 R,G12C/G13C/Q61 R mutants in NIH mice.METHODS:HRAS(V112A) containing the V112 A mutation was amplifiedfrom the DiFi human colon cancer cells.G12 C [HRAS(V112A/G12C)],G13 C [HRAS(V112A/G13C)],Q61 R [HRAS(V112A/Q61R)] and 3 sites joint [HRAS(V112A/G12C/G13C/Q61R)] mutants were generated with site directedmutation PCR.After HRAS(V112A) and the above 4 mutants were inserted into pCDNA3.1(+) and the in vitro expression was verified with Western blot,each plasmind was injected into 8 6-8 weeks aged female NIHmice intracutaneously with a dosage of 100 μg per mouse to observe the expression of tumorgenicity.Pathological tissues was assayed with PCR and Western blot was used to detect the presence of the HRAS gene and protein.RESULTS:HRAS(V112A) and the 4 designed mutants were amplified successfully.Aftertransfection of L929,HRAS expression was detected.Four months after the injection,4 mice from the HRAS(V112A) injected group developed hyperplastic lesions and 1 mouse from the HRAS(V112A/Q61R) groupdeveloped tumor.No lesions were observed in the HRAS(V112A/G12C),HRAS(V112A/G13C) and HRAS(V112A/G12C/G13C/Q61R) groups.PCR and Western blot results show the existence and expression of thecorresponding HRAS genes.CONCLUSION:HRAS containing the V112 A mutation could induce hyperplasticlesions in mice,while HRAS containing V112 A and Q61 R mutations could induce tumors in NIH mice.TheHRAS(V112A) containing G12 C,G13C and G12C/G13C/Q61 R did not induce observable lesions.
OBJECTIVE:To evaluate oncogenicity of HRAS(V112A) and its G12 C,G13C,Q61 R,G12C/G13C/Q61 R mutants in NIH mice.METHODS:HRAS(V112A) containing the V112 A mutation was amplifiedfrom the DiFi human colon cancer cells.G12 C [HRAS(V112A/G12C)],G13 C [HRAS(V112A/G13C)],Q61 R [HRAS(V112A/Q61R)] and 3 sites joint [HRAS(V112A/G12C/G13C/Q61R)] mutants were generated with site directedmutation PCR.After HRAS(V112A) and the above 4 mutants were inserted into pCDNA3.1(+) and the in vitro expression was verified with Western blot,each plasmind was injected into 8 6-8 weeks aged female NIHmice intracutaneously with a dosage of 100 μg per mouse to observe the expression of tumorgenicity.Pathological tissues was assayed with PCR and Western blot was used to detect the presence of the HRAS gene and protein.RESULTS:HRAS(V112A) and the 4 designed mutants were amplified successfully.Aftertransfection of L929,HRAS expression was detected.Four months after the injection,4 mice from the HRAS(V112A) injected group developed hyperplastic lesions and 1 mouse from the HRAS(V112A/Q61R) groupdeveloped tumor.No lesions were observed in the HRAS(V112A/G12C),HRAS(V112A/G13C) and HRAS(V112A/G12C/G13C/Q61R) groups.PCR and Western blot results show the existence and expression of thecorresponding HRAS genes.CONCLUSION:HRAS containing the V112 A mutation could induce hyperplasticlesions in mice,while HRAS containing V112 A and Q61 R mutations could induce tumors in NIH mice.TheHRAS(V112A) containing G12 C,G13C and G12C/G13C/Q61 R did not induce observable lesions.
引文
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[2]ZHANG Z T,PAK J,HUANG H Y,et al.Role of haras activation in superficial papillary pathway of urothelial tumor formation[J].Oncogene,2001,20(16):1973-1980.
[3]LAND H,PARADA L F,WEINBERG R A.Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes[J].Nature,1983,304(5927):596-602.
[4]SHENG L,CAI F,ZHU Y,et al.Oncogenicity of DNAin vivo:Tumor induction with expression plasmids for activated h-ras and c-myc[J].Biologicals,2008,36(3):184-197.
[5]SHENG-FOWLER L,CAI F,FU H,et al.Tumors induced in mice by direct inoculation of plasmid DNAexpressing both activated h-ras and c-myc[J].Int J Biol Sci,2010,6(2):151-162.
[6]SHENG-FOWLER L,TU W,FU H,et al.A mouse strain defective in both t cells and nk cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated h-ras and c-myc[J].PLoS One,2014,9(10):e108926.
[7]ZHENG L,BAUMANN U,REYMOND J L.An efficient one-step site-directed and site-saturation mutagenesis protocol[J].Nucleic Acids Res.2004,32(14):e115.
[8]ASHLEY K,RAMI T K,KASRA K,et al.Expression of leptin and its long form receptor at the porcine maternal-fetal interface:contrasting healthy and arresting conceptus attachment sites during early and mid-pregnancy[J].Reprod Biol Endocrinol,2014,12:91.
[9]SEPP R,SZABóI,UDA H,et al.Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR[J].J Clin Pathol,1994,47(4):318-323.
[10]LENCH N,STANIER P,WILLIAMSON R.Simple noninvasive method to obtain DNA for gene analysis[J].Lancet,1988,1(8599):1356-1358.
[11]FUJITA J,KRAUS M H,ONOUE H,et al.Activated H-ras oncogenes in human kidney tumors[J].Cancer Res,1988,48(18):5251-5255.
[12]SRIVASTAVA S K,YUASA Y,REYNOLDS S H,et al.Effects of two major activating lesions on the structure and conformation of human ras oncogene products[J].Proc Natl Acad Sci U S A,1985,82(1):38-42.
[13]VISVADER J E,LINDEMAN G J.Cancer stem cells in solid tumours:Accumulating evidence and unresolved questions[J].Nat Rev Cancer,2008,8(10):755-768.
[14]MARTA T G,BENJAMIN O,FRANCOIS L,et al.Tumour Heterogeneity:The Key Advantages of SingleCell Analysis[J].Int J Mol Sci,2016,17(12):E2142.doi:10.3390/ijms17122142.
[15]KNOWLES M A,WILLIAMSON M.Mutation of h-ras is infrequent in bladder cancer:Confirmation by singlestrand conformation polymorphism analysis,designed restriction fragment length polymorphisms,and direct sequencing[J].Cancer Res,1993,53(1):133-139.
[16]MURUGAN A K,HONG N T,CUC TT,et al.Detection of two novel mutations and relatively high incidence of h-ras mutations in vietnamese oral cancer[J].Oral Oncol,2009,45(10):161-166.
[17]CROWDER C,KOPANTZEV E,WILLIAMS K,et al.An unusual h-ras mutant isolated from a human multiple myeloma line leads to transformation and factorindependent cell growth[J].Oncogene,2003,22(5):649-659.
[18]B E H,DANIEL C,DEXTER P,et al.Modeling of RAS complexes supports roles in cancer for less studied partners[J].BMC Biophys.2017,10(Sup 1):5.doi:10.1186/s13628-017-0037-6.
[19]AHMADIAN M R,WIESMULLER L,LAUTWEIN A,et al.Structural differences in the minimal catalytic domains of the gtpase-activating proteins p120 gap and neurofibromin[J].J Biol Chem,1996,271(27):16409-16415.
[20]MAEGLEY K A,ADMIRAAL S J,HERSCHLAG D.Ras-catalyzed hydrolysis of gtp:A new perspective from model studies[J].Proc Natl Acad Sci U S A,1996,93(16):8160-8166.
[21]GREMER L,GILSBACH B,AHMADIAN M R,et al.Fluoride complexes of oncogenic ras mutants to study the ras-rasgap interaction[J].Biol Chem,2008,389(9):1163-1171.
[22]FRANKEN S M,SCHEIDIG A J,KRENGEL U,et al.Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21h-ras[J].Biochemistry,1993,32(33):8411-8420.
[23]SEEBURG P H,COLBY W W,CAPON D J,et al.Biological properties of human c-ha-ras1 genes mutated at codon 12[J].Nature,1984,312(5989):71-75.
[24]BUHRMAN G,KUMAR V S,CIRIT M,et al.Allosteric modulation of ras-gtp is linked to signal transduction through raf kinase[J].J Biol Chem,2011,286(5):3323-3331.
[25]VOGEL U S,DIXON R A,SCHABER M D,et al.Cloning of bovine gap and its interaction with oncogenic ras p21[J].Nature,1988,335(6185):90-93.
[26]GIBBS J B,SCHABER M D,ALLARD W J,et al.Purification of ras gtpase activating protein from bovine brain[J].Proc Natl Acad Sci U S A,1988,85(14):5026-5030.
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