PADI4基因在肿瘤中表达的初步研究

The study of expression of PADI4 in various tumors

目的本研究旨在通过检测PADI4在肿瘤中的表达特征和表达机制,初步探讨PADI4是如何参与肿瘤的发病过程的。方法 1.应用ELISA的方法检测各种恶性肿瘤患者血液中PADI4的表达水平。2.应用ELISA双抗体夹心方法检测各种恶性肿瘤患者血液中瓜氨酸化抗凝血酶的表达水平。3.应用RT-PCR的方法检测PADI4mRNA在卵巢癌组织中的表达。4.应用Taqman实时荧光定量RT-PCR的方法检测PADI4mRNA在卵巢癌组的表达量。5.应用免疫组化的方法检测PADI4蛋白在卵巢癌组织中的表达。结果 1.PADI4在乳腺癌、肝细胞癌、肺癌、食管癌、胃癌、结直肠癌、胰腺癌、卵巢癌、肾细胞癌子宫癌、前列腺癌和膀胱癌各种恶性肿瘤患者血清中与对照组相比有很高的表达,差异有统计学意义(P<0.05)。而在甲状腺癌中PADI4表达比健康血清低。在乳腺癌、肝细胞癌、肺癌、胃癌、结直肠癌、胰腺癌、卵巢癌和肾细胞癌等手术后患者血中PADI4表达明显下降。2.cAT在乳腺癌、肝细胞癌、肺癌、食管癌、胃癌、结直肠癌、胰腺癌、卵巢癌、膀胱癌、甲状腺癌和前列腺癌大多数恶性肿瘤中表达很高(P<0.05)。cAT在恶性肿瘤患者手术后表达下降。3.PADI4和cAT在肿瘤患者血液中表达的相关性通过χ2检验,在肝癌、肺癌、卵巢癌等肿瘤中PADI4表达和cAT的表达密切相关(P<0.01)。4.PADI4mRNA在卵巢癌组织中有表达,良性肿瘤病变组织中未检测到PADI4 mRNA的表达。5.PADI4mRNA在卵巢癌组织中的表达量为1.57×102copies/ml,良性肿瘤病变组织中未检测到PADI4mRNA的表达。6.PADI4蛋白在卵巢癌组织中显著表达,良性肿瘤病变组织中无表达或者微弱表达。结论 1.通过大量临床标本的检测,我们发现在肝癌、肺癌、卵巢癌、子宫癌中PADI4和瓜氨酸化抗凝血酶大量表达,并且二者表达密切相关,表明PAID4可能通过对抗凝血酶瓜氨酸化而参与了某些肿瘤的发病过程。2.通过RT-PCR和Taqman实时荧光定量RT-PCR方法检测了卵巢癌组织和卵巢良性肿瘤组织中PADI4mRNA的表达,发现在卵巢癌组织PADI4mRNA是高表达的,揭示了PADI4和卵巢癌之间的相关性。3.通过免疫组化方法检测了卵巢癌组织和卵巢良性肿瘤组织中PADI4蛋白的表达,发现在卵巢癌癌细胞中PADI4蛋白是显著表达的,表明PADI4在卵巢癌发病过程中发挥了重要作用。4.卵巢癌组织中PADI4在基因转录水平和蛋白翻译水平表达都是升高的,表明PADI4基因转录水平的异常可能导致其蛋白质表达异常,从而启动了了卵巢癌的发病过程。

Objective：Tumorgenesis,development and metastasis of tumor is a multistep and complex process involved by multiple genes and factors.To find the gene changed in malignant tumor,study their mechanism and illustrate their effect in the development of tumor may provide new markers for tumor staging,for assessment of the relative risk of tumor formation and open new opportunities for therapeutic intervention.The peptidylarginine deiminase type 4（PAD4/PADI4） gene encodes an enzyme that catalyzes protein citrullination,the posttranslational modification of peptidylarginine to citrulline in the presence of calcium ions.To date,five human isoforms of peptidylarginine deiminase（PADI1,2,3,4 and 6） have been identified and they are encoded by a cluster of genes at chromosome 1p36.13.This gene was originally isolated from cell line HL-60,a myeloid precursor cell line.The 2236bp of PADI4 cDNA encode 663 amino acids that have 50%-55% sequence identity with the other three known PADIs.Immunohistochemical studies have detected PADI4 inneutrophils and eosinophils of human peripheral blood.Many studies have indicated that the human PADI4 was associated with rheumatiod arthiritis（RA）.PADI4 played an important role in the process of autoimmune and tissue damage of the diease.AT,a 52kDa plasma-derived glycoprotein,is a principal plasma inhibitor of coagulation proteases,especially thrombin.Recently,our in vitro incubation demonstrated that recombinant human PAD4 protein can inactivate thrombin-inhibitory activity of human antithrombin through citrullination.We also detected a higher level of citrullinated antithrombin in RA plasma by sandwich ELISA.Therefore,we suggested that extensive expression of PADI4 in RA synovial tissue citrullinate antithrombin,and citrullinated antithrombin lost the thrombin-inhibiting activity.The elevated thrombin activity was confirmed to stimultlate angiogenesis by up-regulated of expression of vascular endothelial growth factor receptor,which might lead to the excessive angiogenesis,fibirin deposition and inflammation of the RA synovial tissue.Many studies report that tumor tissue and RA synovium have many features in common,including extreme angiogenesis,excessive fibrin deposition,disordered cell proliferation,and high coagulation activity.In 2005,we firstly reported that PADI4 had significant expression in various tumor tissues,especially in various adenocarcinoma.Western blotting with anti PADI4 antibody and immunostaining with anti citrulline antibody confirmed the expression of the enzyme in these tumors.Thus,we postulate that antithrombin mighte be citrullinated by a high abundance of PADI4 in the tumor tissue,and were being released to the blood of patients with tumor.The purpose of this experiment is to investigate if and how PADI4 is expressed in various tumors.Methods：1.We detected the expression of PADI4 in the blood of patients with various malignant tumors by ELISA.2.We detected the expression of cAT in the blood of patients with various malignant tumors by Sandwich ELISA.3.We analyzed the expression of PADI4mRNA in the ovary carcinoma tissue using RT-PCR.4.We analyzed the quantitative mRNA of PADI4 in the ovary carcinoma tissue using Taqman real time RT-PCR.5.We finally measured the expressional level of PADI4 protein in the ovary carcinoma tissue by immunohistochemistry.Results：1.The expression of PADI4 in sera of malignances was compared with that in the health controls.The PADI4 level was significantly increased in the blood of patients with breast carcinoma,hepatocellular carcinoma,lung cancer,esophageal carcinoma,gastric cancer,colon cancer,rectal cancer,pancreatic cancer,ovary carcinoma,renal cell carcinoma,cervical carcinoma,prostate carcinoma and bladder carcinoma（P0.05）,except for thyroid carcinoma.The level of PADI4 was considerably declined after tumor excision surgery in samples of breast carcinoma,hepatocellular carcinoma,lung cancer,esophageal carcinoma,gastric cancer,colon cancer,rectal cancer,pancreatic cancer,ovary carcinoma（P0.05）.2.Compared with the samples of the health,the level of cAT was significantly elevated in sera of most malignancy tumors,including breast carcinoma,hepatocellular carcinoma,lung cancer,esophageal carcinoma,gastric cancer,colon cancer,rectal cancer,pancreatic cancer,ovary carcinoma,bladder carcinoma,and prostate carcinoma（P0.05）.The level of cAT was considerably declined after tumor excision surgery in blood of most malignancies we tested.3.Using χ2 test,the correlation of the PADI4 and cAT levels in blood of the malignant tumors was analyzed.The expressional level of PADI4 was largely associated with that of cAT in hepatocellular carcinoma、lung cancer、ovarian cancer（P0.01）.4.The mRNA expression of PADI4 was detected in ovary carcinoma tissues but not in the benign samples.5.The quantity of PADI4 transcripts in the ovary carcinoma tissues was 1.57×102 copies/ml and was negatively detected in the control ones.6.Immunohistochemistry results showed that PADI4 protein strongly expressed in the ovary carcinoma tissues.No significant signal was detected in the benign samples.Conclusions：1.Statistics analysis revealed that PADI4 and cAT levels were significantly increased in blood of the most malignant tumors compared with the health controls.Also the expressional level of PADI4 was largely associated with that of cAT in the same samples.This suggested that PADI4 could be involved in carcinogenesis by post-translational modification of antithrombin.2.The mRNA expression of PADI4 was measured by RT-PCR and Taqman real time RT-PCR.The results showed that the relationship between PADI4 gene and ovary carcinoma.3.We detected the significant expression of PADI4 protein in tumor cells of the ovary carcinoma tissues but not in the controls,which implied that PADI4 played an important role in the pathogenesis of ovary carcinoma.4.The increased transcriptional and translational levels of PADI4 in the ovarian cancers suggested that the abnormally transcriptional level of PADI4 would lead to the elevated protein level of it,which invoke the onset of this desease.