Peripheral Blood Gene Expression Profiling Analysis In Hepatocellular Carcinoma Patients And Establishment Of Gene Diagnostic Model

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. At present, HCC screening mainly depends on detection of serum alpha-fetoprotein (AFP) and ultrasonic diagnosis to monitor high risk group. However, with its low sensitivity and nonspecific elevation in nonmalignant hepatic diseases, we need to explore additional new diagnostic biomarkers that could assist HCC diagnosis.Gene chip is a high sensitivity and high-flux technology for gene expression research. One of the most widely used microarray is expression profiling chip, providing a systematic approach to uncover comprehensive information about the transcription profile of diseases, which has found many applications including discovery of gene functions, tumor related genes, drug evaluation, pathway dissection, and classification of clinical samples.In order to study the whole gene transcription profiling of peripheral blood in HCC patients, Nine HCC patients and five health control individuals matched at age and gender were screened with Affy GeneChip HG-U133 plus 2.0. The results shew that there were 726 significantly differential expression probe sets (P<0.05, FC<2.00),103 probe sets of them were significantly up-regulated and 623 were significantly down-regulated. Among them, the genes such as CXCR4, CX3CR1, TLR4, PTPRC, PTGS2, IFNGR1, LYN, CD74, CALR took part in immunologic process. Many genes participated in transcription regulation process, for instance, PFDN5, JUND, SUB1, ZBTB24, NFKBIA, FOS. The genes, PTPRC, PTGS2, RPS17, RPL39, RPS6, RPS24 and so on, took part in biosynthesis process. To validate the results of the GeneChip, the enlarged the experiment samples, including peripheral blood samples of 40 HCC patients and 21 health control individuals matched with the patients were further studied. Meantime the tumor markers and biochemical parameters of the two groupshave been measured, the results shew that significant difference lay in AFP, ALB, AST, ALT, GGT, ALP (P<0.05). Then 15 genes which were significantly differential expression in the GeneChip experiment:CXCR4, SPAG9, FOS, ANXA1, CALR, IL8, PFN1, HSPA1B, PFDN5, GOS2, GZMA, RPS24, RPL27, GPC3 and HGF were selected to analyze mRNA level in the samples by GeXP technology (a analytical technique to detect multiple genes mRNA in one reaction system), with geometric mean of the two housekeeping genes B2M and P-actin as control, then the fluorescence value of the indexes eliminated the value of the control and we got the relative amount of the target genes. The significant statistical difference were found in the indexes of CXCR4, SPAG9, FOS, ANXA1, CALR, IL8, PFN1, GPC3 and HGF (P<0.05), which was in accordance with results of GeneChip experiment. GZMA was deleted because of many absence data.We set up gene diagnostic models of HCC patients according to the GeneChip experiment results and the validated results. The peripheral blood samples of 40 HCC patients,10 hepatic cirrhosis patients,16 hepatitis patients,32 other hyperplasia Liver disease patients such as hemangiomas of liver, adenocarcinoma of liver, and 21 health control individuals matched with the patients, have been collected for validation studies. First, the tumor markers and biochemical indicators of the five groups have been detected, the results shew that significant difference lay in AFP, ALB, AST, ALT, GGT, ALP (P<0.05). Then mRNA level of the 15 genes were detected by GeXP technology. The results shew that significant statistical difference lay in the indexes of CXCR4, SPAG9, FOS, ANXA1, CALR, IL8, PFN1, GPC3 and HGF (P<0.05), which was in accordance with results of GeneChip experiment. According to the statistical results, we calculated the average accurate rate of the five groups with K neighbor algorithm and 5 fold crossing confirmation, and then set up 5 gene diagnostic models, the model two was made up by nine indexes of PFN1, CALR, SPAG9, IL8, HGF, FOS, GPC3, ANXA1, HSPA1B and was the best diagnostic one with the highest average accurate rate for the five group samples:0.760,0.9800,0.853,0.97,0.936.In summary, we screened the whole gene expression profiling of peripheral blood cells in HCC and validated the results of the GeneChip by GeXP technology. The diagnostic model which can better distinguish the five group samples have been developed. This study could provide some information and technology for studying the mechanism of carcinogenesis and raising early diagnostic rate efficiently in HCC patients.