Screening And Functional Studies Of Novel Tumor Markers

Part 1 In Silico Identification of Tumor Markers and the Expression Patterns of the Candidate GenesObjectives:To screen tumor markers through whole genome in silico.Methods:To identify the genes,which are differentially expressed in tumors compared with their corresponding normal tissues,a search was performed using the CGAP cDNA xProfiler based on EST data. E-Northern and RT-PCR was used to analyze the expression profiles of the identified tumor unique genes.Results:According to the cDNA xProfiler results,there were 2801 genes or ESTs unique in tumor tissues,68 of which were known and 2733 of which were unknown.The functions of 40 genes are clear.5 genes among the 40 genes were tumor-associated genes.On chromosome 7 and 10,few tumor markers were identified;while on chromosome 8,21 and X,much more markers were identified.Then,we performed E-Northern to analyze the expression profiles of the 2801 tumor unique genes.9 genes seemed like ideal.We then performed RT-PCR to verify the expression of these 9 genes in tumor and normal cell lines.7 of them could not be detected in 11 cell lines.GABRA3 and HTA were expressed specifically in cancer cells.Conclusions:In silico identification is an excellent and time-saving approache to identify genes that could be used as tumor diagnostic markers,prognostic indicators,and suitable targets for various forms of therapeutic intervention.Tumor associated genes were more "active" on some chromosomes.Part 2 Expression Profile and Function of GABRA3Objectives:To study the expression profile and function of GABRA3 gene and to detect the expression of GABAA receptor subunits in tumors.Methods:RT-PCR and immunohistochemistry were used to analyze the expression profile of GABRA3 gene.RNAi and GABA were used to study the function of GABRA3.Results:GABRA3 was highly expressed in hepatocellular carcinoma line HepG2,and weakly detected in glioma cell line U251 and breast cancer cell lines MCF-7 and MDA-MB-231.In normal tissues, GABRA3 was strongly expressed in brain and placenta,and weakly detected in endometrium and prostate,but undetectable in other tissues. More importantly,the expression of GABRA3 in cancers was rare or less abundant but with the exception of glioma,endometrial cancer,lung caner and liver cancer.By RT-PCR,GABRA3 was detected in 12 out of 18 lung cancer samples(66.67%),but not expressed in corresponding adjacent noncancerous lung tissues.By immunohistochemistry,GABRA3 was detected in 25 out of 60 lung cancer samples(41.67%),but not in 10 corresponding adjacent noncancerous lung tissues.The experimental results demonstrated cytoplasmic and membrane staining.The results further showed the correlation between GABRA3 protein expression and clinical-pathologic variables in lung cancers.GABRA3 protein expression was significantly higher in lower grade of lung cancer (p＜0.05).GABRA3 was also detected in 6 out of 8 liver cancer samples (65%),while not in corresponding adjacent noncancerous liver tissues. We also detected the other subunits of GABAA receptor in lung cancer and liver cancer by RT-PCR.In those samples GABAA-α6,β3,γ2,δ,θ,εandπsubunits were also expressed.GABRA3 may compose functional GABAA receptors with them.In liver cancer cell line HepG2,GAD67 were detected,suggesting that GABA could function in an autocrine/paracrine manner in HCC cells and promote cell growth.GABA promoted the proliferation of HepG2 cells.Knockdown of endogenous GABRA3 expression in HepG2 attenuated HCC cell growth.GABA enhanced the growth of GABRA3 expressing HepG2 cell.On the other hand,the proliferating ability of GABRA3-knockdown HepG2 was not enhanced but lowered by GABA.Conclusions:GABRA3 is overexpressed in lung cancer and hepatoma.GABRA3 protein expression was significantly higher in lower grade of cancer.HepG2 cells could produce GABA.GABA promoted the proliferation of HepG2 cells through GABRA3.In lung cancers and liver cancers,some other GABAA receptor subunits were also expressed. When GABRA3 was knocken down,GABA inhibited the proliferative ability of HepG2 cells through those subunits.Part 3 Expression Profile and Function of HTAObjectives:To study the expression profile and function of HTA.Methods:RT-PCR was used to analyze the expression profile of HTA gene and RNAi was used to study the function of it.Results:HTA was a tumor-specific gene,especially in hepatoma.It was detected in liver cancer cell line HepG2,some liver cancer tissue samples(75%) and some other cancer tissues.But in the normal tissues it was not detected.Knockdown of endogenous HTA gene was performed by small interfering RNA in malignant hepatocyte HepG2.Then we tested the cell proliferative ability of these cells in vitro and in vivo.The results showed that HTA could promote HCC cell growth.HTA is located on chromosome 16q22.3,including 3 exons and 2 introns.The encoded protein is an alkaline hydrophilic protein,whose molecular weight is 10KD,PI is 10.9.This protein may play an important role in signal conduction because it may contain 7 phosphorylation sites and 6 glycosylation sites.The secondary structure of the protein contains 33.7%α-helix and 5.43%β-sheet.HTA encoded protein may contains 4 antigen epitope,which are located at 4-14aa,37-43aa,54-60aa and 65-89aa.Conclusions:HTA was a tumor-specific gene,especially in hepatoma.It plays an important role in HCC cell viability and is a sustained event in the tumorigenic process.