Screening For Tumor Related Genes In Hepatocellular Carcinoma

Part One Screening for differentially expressed genes among hepatocellular carcinoma, cirrhotic liver tissue and normal liver by genechip techologyObjective: To compare gene expression profiles between hepatocellular carcinoma(HCC) and normal liver, cirrhotic liver and normal liver, by genechip technology respectively. Differentially expressed genes were gathered and classified. Their biological informations were analyzed, which is help to the diagnosis and treatment of HCC.Methods1 Research subjects:4 specimens of HCC, 1 specimen of cirrhotic liver and 1 specimen of normal liver came from exairesis of the department of liver and gall surgery of the fourth hospital of Hebei Medical university and identified by histology. All patients did not accept any kinds of treatment before operation.2 Research methods: The specimens were put into liquid nitrogen immediately after excision and then were stored in the -80℃ultra cold freezer. Total RNA was isolated from hepatocellular carcinomas, cirrhotic liver tissue and normal liver respectively, and was purified into mRNA by oligotex, and then reverse-transcribed to synthesis two kinds of fluorescences (Cy3-dUTP, Cy5-dUTP) labeled cDNA probes. The targets were mixed together and hybridized with the genes on the genechip contained 4096 genes. The following comparisons were performed: hepatocellular carcinoma versus normal liver; cirrhotic liver versus normal liver. Genes which were differentially expressed between these groups were identified based on signal-to-noise ratios by using GenePix Pro3.0 software. The outcome was gathered and biological informations of these genes were inquested in gene bank. Results1 The expression profile was significantly different not only between HCC and normal liver, but also between cirrhotic liver and normal liver.2 141 genes were found differently expressed between HCCs and normal liver, occupying 3.5% of total sum in genechip, 51 genes were up-regulated and 90 genes were down-regulated in HCC. Up-regulated genes were predominantly associated with cell proliferaton and cell signal transduction, including some genes that were proved to have close relation to tumor lately, such as MCM7, 14-3-3ηand GRB2 et al. Down-regulated genes were mostly associated with substance metabolism. Among the 141 genes, there were 23 genes whose expression tendency consistent with that of cirrhotic liver, compared with normal liver, occupying 0.6% of total sum in genechip; 7 genes were up-regulated, 16 genes were down-regulated, the functions of these genes were related to cell proliferation and fibrogenesis, such as PCNA, SPARC and COL3A1 et al. There were 2 genes significantly differently expressed between HCCs and cirrhotic liver, and they all belonged to metallothionein family. Additionally, there were some function unknown genes waited for deeper research.ConclusionWe could compare the expression profiles among different tissues parallelly in one time by genechip technology, in the hence, this technology can provide great help in the reseach of tumor. Lots of gene clusters were found associated with HCC by this technology in the research, some of these genes were related to the regulation of cell cycle and cell signal transduction, additionally, there were some genes function unkown. Research into these genes systematically will help to clarify the mechanisms of hepatocarcinogenesis, to discover original early diagnosis criteria, to guide treatment proposal and to evaluate prognosis better.Part Two The detection of partly differentially expressed genes by semi-quantitative RT-PCRObjective: Chose some differentially expressed genes among the different liver tissues that proved by the genechip, enlarged the number of specimens, and test the expression of these genes among HCC,adjacent noncancerous cirrhotic liver and normal liver by semi-quantitative reverse transcription-polymerase chain reaction again to verify the outcome of genechip, and to explore the relationship between these genes with hepatocellular carcinoma preliminarily.Methods1 Research subjects: 19 specimens of HCC, 11 specimens of adjacent noncancerous cirrhotic liver, 7 specimens of normal liver identified by histology came from exairesis of the department of liver and gall surgery of the fourth hospital of Hebei Medical university. All patients did not accept any kinds of treatment before operation. The expression of metallothionein 3 mRNA,14-3-3γmRNA,14-3-3ηmRNA,minichromosome maintenance protein 7 mRNA and retinoblastoma like 2 (p130) mRNA were investigate which expressed differently among different liver tissue came from the outcome of gene chip by semi-quantitative reverse transcription-polymerase chain reaction .2 Research methods: The total RNA of HCC,adjacent noncancerous cirrhotic liver and normal liver was isolated respectively, reverse transcripted into cDNA, and corresponding primers were added in and amplified, at the same time, amplifiedβ-ACTIN as inside reference, compared the expressions of these genes among the different liver tissues. Associated the expression of these genes with the clinical performance of HCC patients, the relationship between these genes and HCC was explored preliminarily.Results1 The expression of MT3 mRNA was significantly lower in HCC than in adjacent noncancerous cirrhotic liver and normal liver(p<0.01 or p<0.05, respectively). The expression of MT3 mRNA was lower according to the lower differentiation of tumor (p<0.01 or p<0.05). There was not significantly difference between the adjacent noncancerous cirrhotic liver and normal liver in the expression of MT3 mRNA. 2 The expression of 14-3-3γmRNA was significantly lower in HCC than in adjacent noncancerous cirrhotic liver and normal liver(p<0.01), the expression of 14-3-3ηmRNA was significantly higher in HCC than in adjacent noncancerous cirrhotic liver and normal liver(p<0.05).3 The expression of MCM7 mRNA was significantly higher in HCC than in adjacent noncancerous cirrhotic liver and normal liver(p<0.05). The expression of RBL2/P130 mRNA was significantly lower in HCC than in adjacent noncancerous cirrhotic liver and normal liver (p<0.01). The lower the differentiation grade was, the higher the expression of MCM7 mRNA was; the lower the differentiation was, the lower the expression of RBL2/P130 mRNA was. The expression of MCM7 mRNA was negatively correlated to the expression of RBL2/P130 mRNA (r=-0.649,p=0.003).Conclusions1 MT3 gene was closely related to the development of HCC, whose expression level could reflect the extent of malignancy in HCC.2 The disregulation of 14-3-3 gene family play a role in the development of HCC, whose mechanism need further research.3 MCM7 gene may be used as a new proliferation marker in HCC. The inactivation of RBL2/P130 gene enhanced the development of HCC.4 The genesis and development of hepatocellular carcinoma is a complex pathologic process, lots of genes changed during the period, and there may exist lots of communication among these genes. It is helpful to the diagnosis and prevention of hepatocellular carcinoma to research into these genes systematically.5 Under the condition of enlarged number of specimens, the conclusions of the research by semi-quantitative reverse transcription-polymerase chain reaction were coincide with the outcomes of genechip essentially. And so, genechip is a feasible research technology, and its outcome possesses repeatability.Part Three The expression and clinical significance of MCM7 protein,RBL2/P130 protein and Cyclin D1 protein in hepatocellular carcinoma Objective: To study the expression of MCM7 protein,RBL2(P130) protein and Cyclin D1 protein in hepatocellular carcinoma and explore their relationship and clinical significance.Methods1 Research subjects: 44 specimens of HCC, 26 specimens of adjacent noncancerous cirrhotic liver, 18 specimens of normal liver identified by histology came from exairesis of the department of liver and gall surgery of the fourth hospital of Hebei Medical university. All patients did not accept any kinds of treatment before operation.2 Research methods: The specimes were fixed by 10% formaldehyde and embeded in paraffin after excision. The expressions of MCM7 protein, RBL2/P130 protein and Cyclin D1 protein among the different liver tisuues were assessed by the technology of immunohistochemistry staining. The clinical data of HCC patients was gathered, and the interrelationship among these proteins was explored, and the relationship between the expression of MCM7 protein and the clinical performance of HCC patients was investigated.Results1 The expression of MCM7 protein among the groupsMCM7 protein did not express in adjacent noncancerous cirrhotic liver and normal liver; the positive rate of MCM7 in HCC was 72.7% (32/44), nucelus stained yellow. The expression of MCM7 protein was not related to age, sex, portal vein tumor thrombosis, the number of tumor foci and HbsAg. The positive rate of MCM7 protein was significantly higher in the group of tumor size≥5cm(χ2=6.57,P<0.05)than in the group of tumor size<5cm(χ2=6.57,P<0.05); The positive rate of MCM7 protein in the tumor without envelope was 100%. The positive rate of MCM7 protein in the tumor with unintact envelope was 88.2%, which was significantly higher than in the tumor with intact envelope(χ2=5.59, P<0.05); The positive rate of MCM7 protein was positive related to the TNM stage of tumor and negative related to the grade of pathological differentiation grade(P<0.05). The positive rate of MCM7 protein was significantly higher in the group of AFP≥400ug/L than in the group of AFP<400ug/L(χ2=6.38,P<0.05).2 The expression of RBL2/P130 protein among the groups The positive rate of RBL2/P130 protein was 34.1% in HCC which was significantly lower than in adjacent noncancerous cirrhotic liver (69.2%) and in normal liver (72.2%), nucleus and (or) cytoplasm stained yellow(χ2=8.09 orχ2=7.5, P<0.01).3 The expression of Cylin D1 protein among the groups The positive rate of Cylin D1 protein was 68.2% in HCC which was significantly higher than in adjacent noncancerous cirrhotic liver (34.6%) and in normal liver (27.8%), nucleus and (or) cytoplasm stained yellow (χ2=7.46 orχ2=8.48, P<0.01).4 The correlation among the expression of MCM7 protein, RBL2/P130 protein and Cyclin D1 protein in HCCThe expression level of MCM7 protein was positively correlated to the expression level of Cyclin D1 protein in HCC (r=0.568, P<0.01), and was negatively correlated to the expression level of RBL2/P130 protein in HCC (r=-0.636, P < 0.01); The expression level of Cyclin D1 protein was negatively correlated to the expression level of RBL2/P130 protein in HCC (r=-0.538, P<0.01).Conclusions1 The level of the expression of MCM7 protein could reflect the reproductive activity and the grade of malignancy of HCC. Monitoring its expression in HCC would bring more help to the early diagnosis of tumor, to forecast the recurrence and evaluate the prognosis in HCC.2 The activation of Cyclin D1 and the suppression of RBL2/P130 may promote the development of HCC.3 The activation of Cyclin D1/CDK4-RBL2/P130-MCM7 regulation pathway maybe play an important role in the development of HCC. Specifically suppress its activation maybe helpful to the treatment of HCC.