Preliminary Screening For Prewarning Biomarkers Of High-risk Population In The Endemic Area Of HCC In Guang-xi

Guang-xi is one of the provinces with a high hepatocellular carcinoma incidence and mortality. Hepatocellular carinoma was often diagnosed at advanced stage with poor prognosis. It is an important prevention and control meature to establish a prediction system, which can predict the risk of HCC of high-risk population in endemic area of HCC in Guang-xi. As the formation of HCC is a multi-factor, multi-mechanism and multi-gene synergistic effect complex process, which multiple gene expression levels change result in abnormal molecular changes within the cells. So far, there is no better molecular marker to be found to become the prewarning biomarkers in screening high-risk population. Since tumor markers are divided into genetic markers and phenotype markers, previous studies are mostly single-factor model,which tend to be concentrated in one or several genes and proteins. It is unable to understand the changes of multiple genes throughout the genome and can not simultaneously understand multiple humoral protein changes, which can not fully reflect the molecular characteristics of hepatocellular carcinoma. In this study, a case-control study which is based on the high-risk groups cohort of the endemic area of HCC in Guang-xi is applied.cDNA microarray and protein chip technology are applied to screen the gene transcription levels and protein levels,which not only to clarify the possible molecular biological mechanisms of development of hepatocellular carcinoma , but also to provide the basis for searching higher specificity and sensitivity prewarning biomarkers of high-risk population.This experimental study consists of four parts.Part onePreliminary screening on differentially expressed genes in the endemic area of HCC in Guang-xiObjective: To detect the expression profiles of differentially expressed genes in peripheral blood between HCC group and the same region normal population group in the endemic area of HCC in Guang-xi, which were classified by bioinformatic anylysis in order to select candidate genes from them.Methods1 research subjects: 9 cases of HCC were selected from the high-risk populations cohort of the endemic area of HCC in Guang-xi. In accordance with the ratio of 1:1, 9 cases were selected from matched normal persons of the same area as control group. All of the subjects were not carried out surgery radiotherapy and chemotherapy and biological therapy when their bloods were collected,which the questionnaive survey and blood collection were informed consent.2 research methods: 5ml EDTA anticoagulated blood were collected.Total RNA were extracted from two group specimens and mRNA were purified,which were reversed transcription to synthesize fluorescent labeled cDNA probes and hybrided with Roche NimbleGen expression profile chip in order to screen out the differentially expressed genes of two groups peripheral blood specimens . Finally, the molecular function, biological processes, cellular components and biological pathways of differentially expressed genes were analyzed by the Gene Ontology, the KEGG and NCBI database. Refered to large number of literature reports, candidate genes were selected.Results1 Gene expression profiles between the HCC peripheral blood and matched normal population peripheral blood were different.2 Compared with the control group, there were 42 differentially expressed genes in peripheral blood,which they were consistent with in 9 HCC patients,accounting for 0.93 ‰ of the total number of genes .Among which included 20 up-regulated genes and 22 down-regulated genes.The most multiples up-regulated genes is GOS2, which raised seven times multiples IGHA1 gene increased 4 times. Two unknown up-regulated genes increased 3-4 times.MXD1, IL1R, SLC22A4 three up-regulated genes were increased three times and the others genes increased two times. 22 genes were down-regulated more than 2 times. Depending on the genes involved in biological processes,These differentially expressed genes were roughly categorized and found that up-regulated genes IL1R2, IGHA1, TOB1, MXD1, PROK2 were involved in cell proliferation and DUSP1, GOS2, IL8, JUNB and PROK2 were involved in cell cycle; JUNB was involved in reproduction; STK17B was involved in apoptosis; SLC22A4 and IRS2 were involved in metabolism and transportion;NFIL3 and ASGR2 were related to signal transduction and transcription; and the function of three genes including LRG1, SLC22A and FLJ36031 were unknown. Downward genes KLRF1, KLRC1 and XCL1 were related to signal transduction; PRF1, GAMA, GAMH and GZMB were involved in immune response; HOP was related to cell differentiation; the functions of 5 down-regulated genes, such as EDG8, KSP37 were unknown.3 By bioinfomatic analysis, 42 differentially expressed genes were related to reproductive, developmental processes, cell proliferation and differentiation,metabolism, biological rhythms, signal transduction, blood circulation,angiogenesis, apoptosis, immune response, cell cycle, Oxidativestress, cell migration process. Most biological pathways of up-regulated genes were involved in cell proliferation, cytokine pathway and most biological pathways of down-regulated genes were involved in immune response pathway.4 Among 20 up-regulated genes,except 3 genes were unknown,there remained 17 genes, which 12 genes had been reported to occur with malignant tumors.5 Re-screened among 12 up-regulated genes related to malignant tumors and refered to the literature reports, PROK2 and DUSP1 were selected as candidate genes of the next experiments.Conclusions: The rsults showed that it could be a one-time, parallel study method to compare with differential study subjects peripheral blood gene expression profile by using gene chip technology. With this technique, the experiment found that 42 genes were involved in formation of HCC in the endemic arae of HCC in Guang-xi, which many genes related to cell cycle regulation and signal transduction and the functions of some genes were unknown. The study showed that there was difference in the gene expression profiles of peripheral blood of two groups, which indicated the occurrence and development of HCC related to environmental factors and environmental factors could change multi-genes mRNA expression levels. To study systemic these differentially expressed genes would help to clarify the mechanism of the development of HCC in Guangxi and search and find the possible prewarning biomarkers of high-risk groups.Part twoThe validation of differentially expressed gene PROK2 and DUSP1 expression in peripheral blood of the endemic area of HCC in Guang-xiObjective:Acording to the results of gene chip, PROK2 and DUSP1 were picked out.Increased the number of each group sample, the expression of PROK2 and DUSP1 mRNA in two groups were investigated in order to verify the reliability of the results of gene chip and explore the relationship between these genes and HCC in Guang-xi.Methods1 research subjects: 25 cases of liver cancer were selected from thehigh-risk populations cohort of the endemic area of HCC in Guang-xi. In accordance with the ratio of 1:1, 25 cases were selected from matched normal persons of the same area as control group. All of the subjects were not carried out surgery,radiotherapy, chemotherapy and biological therapy when their bloods were collected,which the questionnaive survey and blood collection were informed consent.2 research methods: 5ml EDTA anticoagulated blood were collected.Total RNA were extracted from two group specimens,which were reversed transcription to synthesize cDNA. Fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR) relative quantification method was applied to detect PROK2 and DUSP1 gene mRNA expression in the peripheral blood of two groups subjects.Finally, Combined with epidemiological data, the relationship between these genes and HCC in Guang-xi was explored preliminarily.Results: PROK2 gene mRNA expression level of HCC was 7.21 ± 2.394 and PROK2 gene mRNA expression levels in matched normal groups was 1.24± 0.439. Statistical analysis showed PROK2 gene mRNA expression levels of liver cancer was significantly higher than that of the control group (P<0.01);DUSP1 gene mRNA expression level of HCC was 2.48±0.93 and DUSP1 gene mRNA expression levels in high-risk groups was 0.83 ± 0.327. Statistical analysis showed DUSP1 gene mRNA expression levels of liver cancer was significantly higher than that of the control group( P <0.01).Conclusions1 The upregulation of PROK2 and DUSP1 in HCC peripheral blood indicated that the two genes might be involved in development of HCC in Guang-xi.2 PROK2 and DUSP1 gene played a certain role in liver cancer formation process,which they were related to the MAPK signaling pathway, its mechanism of mutual needed further study.3 PROK2 and DUSP1 gene expression levels were significantly increased in the peripheral blood of HCC,which was expected to enter screening study of prewaming markers in endemic area of HCC in Guang-xi.4 The occurrence and development of HCC was a complex pathological process, which a large number of genes changed and there might be more or less contact between these genes. By appling the case-control study based on the high-risk groups cohort of the endemic area of HCC, the systematic study to these genes would help to elucidate the mechanism of HCC and look for high-risk groups prewarning biomarkers.5 After exlarged the number of sample cases, the results of fluorescent quantitative RT-PCR were consistent with the results of gene chip. It indicated that the gene chip was a viable technology, which its finding had a certain degree of repeatability.Part threePreliminary screening on differentially expressed proteins in the endemic area of HCC in Guang-xiObjective: To pick out from the protein chip results in the serum of liver cancer group and the high-risk population groups, differences in protein IGFBP-2 and P21, increases the number of cases of each group of samples to compare the protein IGFBP-2 and P21 in HCC and expression in the serum of the high-risk population groups, in order to verify the reliability of the results of protein chips, and explore the relationship between these proteins and Guangxi in high incidence area of liver cancer.Methods1 research subjects: 11 cases of HCC were selected from the high-risk populations cohort of the endemic area of HCC in Guang-xi. In accordance with the ratio of 1:1, 11 cases were selected from matched normal persons of the same area as control group. All of the subjects were not carried out surgery,radiotherapy and chemotherapy and biological therapy when their bloods were collected,which the questionnaive survey and blood collection were informed consent.2 research methods: 5ml non-anticoagulant blood were collected. The serums were centrifuged and dliuted from two groups specimens, which were hybrided with protein chips. Scanned the chips and analied the signal and image in order to screen out the different protein in serum of two groups.Results: Serum IGFBP-2 level of HCC was 887.48±225.91pg/ml and Serum IGFBP-2 level in matched normal groups was 558.17±125.97pg/ml.Statistical analysis showed Serum IGFBP-2 level of HCC was higher than that of the control group (P<0.05); Serum p21 relative level of HCC was 1040.97±395.91 and Serum p21 relative level in high-risk groups was 2756.64± 1355.64.Statistical analysis showed Serum p21 level of HCC was lower than that of the control group(P <0.05).Conclusions1 It was differnet of the serum proteins between the HCC peripheral blood and matched normal population.2 Compared with the control group,here were two different proteins in serum, which they were consistent with in HCC patients.They were IGFBP-2 and P21, which regulated cell prpliferation, cell cycle and cell apoptosis.3 The rsults showed that it could be a one-time and high-through screening disease-related proteins by using protein chip technology. With this technique,tthe experiment screened some serum proteins,which might be related to the development of HCC in the endemic arae of HCC in Guang-xi. Volidation studies of these different proteins would help to provide clue and evidence for searching for possible serum prewaming markers of HCC in high incidence of HCC in Guang-xi.Part fourThe validation of different protein IGFBP-2 and P21 in serum of the endemic area of HCC in Guang-xiObjective: Acording to the results of protein chip, IGFBP-2 and P21 were picked out.Increased the number of each group sample, the serum IGFBP-2 and P21 in two groups were compared in order to verify the reliability of the results of protein chip and explore the relationship between these proteins and HCC in Guang-xi.Methods1 research subjects: 44 cases of HCC were selected from the high-risk populations cohort of the endemic area of HCC in Guang-xi. In accordance with the ratio of 1:1, 44 cases were selected from matched normal persons of the same area as control group. All of the subjects were not carried out surgery,radiotherapy and chemotherapy and biological therapy when their bloods were collected,which the questionnaive survey and blood collection were informed consent.2 research methods: 5ml non-anticoagulant blood were collected. The serums were centrifuged and dliuted from two groups specimens, which ELISA was applied to detect IGFBP-2 and P21 level in serum of two groups subjects.Finally, Combined with epidemiological data, the relationship between these proteins and HCC in Guang-xi was explored preliminarily.Results: Serum IGFBP-2 level of HCC was 2846.66±1834.74ng/L and Serum IGFBP-2 level in matched normal groups was 1657.39±577.55 ng/L.Statistical analysis showed Serum IGFBP-2 level of HCC was higher than that of the control group (P<0.05); Serum p21 level of HCC was 2925.81 ±1144.74 ng/ L and Serum p21 level in high-risk groups was 7848.43 ±1834.74ng/ L.Statistical analysis showed Serum p21 level of HCC was lower than that of the control group (P <0.05).Conclusions: In the serum of HCC patients, IGFBP-2 levels increased and P21 levels decreased, which indicated that the two proteins might be involved in the development process of HCC in Guang-xi and might become the prewanring markers of HCC in the endemic area of HCC in Guang-xi. After expanding the number of sample cases, the results of ELISA were consistent with the results of protein chip. It indicated that the protein chip was a viable technology, which its finding had a certain degree of repeatability.