Abnormal Expression Of PLAG1 And PLA2G2A In Hepatocellular Carcinoma

Background and ObjectivesHepatocellular carcinoma (HCC) is at the fifth position on the incidence of malignant tumor and the third position on the mortality rate in the world, and more than one half of the cases occur in china. Because of its occult onset, invasion and metastasis fast, high fatality rate, it is so bad to the life and health of human that we should spare no effort to study it. We generally believe that the incidence of HCC is a result of the multi-channel, multi-factor and multi-step, which includes external environment factors (the infection of HBV and HCV and bacteria and parasites, the intake of aflatoxin, drinking, nonalcoholic fatty liver disease, biliary diseases, cirrhosis, etc.) and genetic factors. They may cause hepatic cell injury and regenerative hyperplasias of fibrous tissue profoudly disturb the hepatic parenchymal structure and vascular system, which may directly or indirectly contribute to carcino genesis.Surgical resection is the most effective treatment for HCC compared with the other methods. And combining with radiofrequency ablation therapy, hepatic arterial chemoembolization, radiation therapy and traditional Chinese medicine treatment, it makes the 5-year clinical survival rate increasing gradually. Liver transplantation and biological therapy are also hot spots in clinic. Because the multistage tumorigenesis and progression of HCC is complicated, as they may involve mutation and deletion of tumor suppression genes and oncogenes, abnormal expression of DNA repair genes and immunological associated genes, cellular signal transduction, cell cycle-regulation genes, et al, in the development of pathological processes and many molecular biology researches are seeking another way for the early diagnosis and molecular targeted therapy of HCC. AFP (a-fetoprotein), which is the most originally discovered and the most widely used, is an effective tumor marker, but its false negative rate of HCC can reach more than 30%. At present, there is not any marker can diagnose all of the HCC, only through joint detection to improve the detection rate of HCC. Therefore, it is worth seeking new molecular markers for diagnosis and targeted therapy of HCC, especially in the early stage.We had researched the gene expression profile to analyze the common differentially expression of genes by using oligonucleotide chip.We discovered that Pleomorphic adenoma gene 1 (PLAG1) was consistently up-regulated and that group IIA secretory Phospholipase A2 (PLA2G2A) was consistently down-regulated. PLAG1 was a proto-oncogene that was localized on chromosome 8q12.Its oncogenic activation was a crucial event in various human neoplasias, including pleomorphic adenomas of the salivary glands, lipoblastoma, etc. Its ectopic expression presumably resulted in the deregulation of target genes, leading to uncontrolled cell proliferation. PLA2G2A was identified as a member of the Phospholipase A2 family. It had been proposed to play a role in anti-bacterial defense, inflammation, and Suppressing tumors. Its abnormal expression was discovered in tissues of gastric carcinoma, breast carcinoma, colon carcinoma and so on.In this study, we used the reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry (IHC) methods to detect the differential expression of PLAG1 and PLA2G2A in normal human liver, cirrhosis and HCC, in order to explore the roles of PLAG1 and PLA2G2A in tumorigenesis and progression of HCC and hoped that they could give an ideal candidate gene in the diagnosis of HCC and a molecular targeted therapeutic method.Materials and MethodsThere were nineteen normal liver tissues and fifteen cirrhosis tissues and thirty-nine HCC tissues. All of tissues were got from surgical liver specimens in Henan Provincial People's Hospital from March 2010 to May 2010. HBsAg of cirrhosis tissues and HCC tissues were positive, but HBsAg was negative in normal liver tissues. Diagnose was confirmed by pathology.The total RNA of each sample was extracted according to the manufacture's recommended protocol. And then RT-PCR was carried out according to the protocol. The amplification products were identified by agarose gel electrophoresis, scanned and semi-quantitative analyzed.The proteins which were eluted were subjected to electrophoresis on a Tricine-SDS-PAGE. After transferring to a polyvinylidene difluoride membrane and blocking, the proteins were detected with primary antibodies. Followed by secondary antibodies, final detection was performed by the enhanced chemiluminescence (ECL) Western blot analysis system.All of the specimens were stained with routine HE and classified by pathologist. Then immunohistochemistry were performed in SP method, and the results were judged by three different observers. The pathological section was scanned and photo-taken by Scan Scope Vitural Microscopy System.The data was analyzed statistically with SPSS 17.0 revolved in ANOVA (one-way analysis of variance), LSD-t test,χ2 test, Fisher's exact test and Kruskal-Wallis rank sum test. And the test criteria was 0.05 (α=0.05).Results1. All of the results of the total RNA isolated were fine. The bands of amplification product were clear and the bands of house keeping gene were bright.2. The result indicated that PLAG1 was highly expressed in HCC tissues.The positive rates of PLAG1 mRNA were 87.2%,46.7%, and 26.3% in HCC group, cirrhosis group and control group, respectively. There was a statistically significant difference among the three groups (χ2=22.47, P<0.01).The positive rates of PLAG1 protein in these three groups were 84.6%,40.0% and 15.8%, respectively. There was a statistically significant difference among the three groups (χ2=27.15, P<0.01). The result indicated that PLA2G2A was lowly expressed in HCC tissues.The positive rates of PLA2G2A mRNA were 28.2%, 66.7% and 89.5% in HCC group, cirrhosis group and control group, respectively. There was a statistically significant difference among the three groups (x2=20.83, P<0.01).The positive rates of PLA2G2A protein in these three groups were 25.6%,73.3% and 84.2%, respectively. There was a statistically significant difference among the three groups (x2=21.41, P<0.01). The result of RT-PCR was confirmed by Western blot.3. The immunohistochemistry result indicated that a statistically significant difference in the intensity of PLAG1 protein expression was also noted in different differentiation and pathological grades of HCC (P<0.01).The poor differentiated and the higher nuclear grade of HCC, the higher expression of PLAG1.Conclusions1. On mRNA and protein levels, PLAG1 is highly expressed in HCC tissues, compared with normal liver tissues. It suggests that PLAG1 may play an important role in multistep hepatocarcinogenesis.PLA2G2A is significantly lower expression in HCC tissue, which indicates that the lack expression of PLA2G2A may be related to tumorigenesis and progression of HCC.2. The intensity of PLAG1 expression is associated with clinical pathological characteristics, suggesting that HCC can be distinguished by a significant increase of PLAG1.3.PLAG1 can be used as an ideal candidate gene for the early diagnosis of HCC. PLA2G2A may be a targeted drug for anti-tumor angiogenesis and metastasis. We investigate the molecular mechanism about the abnormal expression of PLAG1 and PLA2G2A, in order to provide a potential targeted therapeutic approach for HCC.