HPV16 E6蛋白阳性和阴性乳癌组织中p53基因检测

Detection of p53 gene in HPV16 E6 protein-positive and-negative breast carcinoma tissue

目的:研究人乳头瘤病毒16型(HPV16)E6蛋白阳性(E6+)和阴性(E6-)乳癌组织中p53基因的状态和表达。方法:以经PCR筛选证实HPV16 DNA阳性的56例乳癌组织(Western blotting证实38例HPV16 E6+,18例E6-)为研究对象,PCR法扩增p53基因保守核心区DNA序列,测序后与Genbank公布的野生型p53(wtp53)基因序列比对;采用RT-PCR和Western blotting方法分别检测p53 mRNA和P53蛋白。结果:HPV16 E6+和HPV16 E6-乳癌组织中,p53基因突变率分别为15.8%(6/38)和72.2%(13/18),2者比较差异有统计学意义(χ2=17.352,P<0.001);p53基因突变主要发生在第5、6、7和8外显子;第248和273位密码子突变频率较高;除1例无义突变和1例同义突变外,其余均为错义突变。98.2%(55/56)可检测出p53 mRNA。HPV16E6+/wtp53乳癌组织中P53蛋白阳性率9.4%(3/32),低于HPV16 E6+/mtp53和E6-/wtp53乳癌组织中的5/6和4/5(P<0.01)。结论:HPV16 E6+乳癌组织中基因突变不是p53灭活的主要机制,而E6-乳癌组织中p53灭活的主要方式是基因突变;HPV16 E6可通过降解wtP53蛋白而在乳癌发生发展中发挥作用。

Aim：To investigate the status and expression of p53 gene in human papillomavirus 16 type（HPV16） E6 protein-positive（E6＋） and-negative（E6-） breast carcinoma tissue.Methods：56 HPV16 DNA-positive breast carcinoma,including 38 cases with HPV16 E6＋ and 18 cases with HPV16 E6-certified by Western blotting assay,were subjected to detect p53 DNA in the conserved core domain by PCR and sequencing,p53 mRNA by RT-PCR,and P53 protein by Western blotting assay.Results：The mutation rates of p53 gene in HPV16 E6＋ and HPV1E6- breast carcinoma tissue were 15.8% （6/38） and 72.2% （13/18） ,respectively, there was significant difference （χ^2 = 17. 352, P 〈 0. 001 ）. Almost all mutations were present in exon 5, exon 6, exon 7, and exon 8. The mutations of 248 and 273 eodons revealed higher frequeney. One mutation was a nonsense mutation,one was a samesense mutation, whereas all other were missense mutations. 55 eases expressed p53 mRNA. The positive rate of P53 protein in HPV16 E6 ＋/wtp53 breast eareinoma tissue was 9.4% （3/32） ,lower than 5/6 in HPV16 E6^＋/ mtp53 breast carcinoma tissue and 4/5 in E6-/wtp53 breast carcinoma tissue（ P 〈 0. 01 ）. Conclusion ： The gene mutation may be the main mechanism for inactivation of p53 in HPV16 E6 - breast carcinoma,but not for HPV16 E6- breast carcinoma. HPV16 E6 may play an important role in the carcinogenesis and pro- gression of breast carcinoma by degrading wtP53 protein.