Characteristic Genomics Of Peripheral Blood Mononuclear Cells Of Hepatocellular Carcinoma Patients With Liver-kidney Yin Deficiency Syndrome

一, BackgroundSyndrome is the summary for pathology with the certain stage of development in disease.Because of the TCM syndromes are derived from a set of interrelated signs and symptomswhich were obtained through the look, smell, cut four clinics. Syndrome lack objectiveindications, clinical grasp a large extent depends on the doctor's diagnosis and treatment. Forthis reason, many scholars expect to rely on modern scientific and technological means todiscover and clarify the syndrome and substance.Since the past20years, there are a lot of biology basic researches about syndrome.Because of many characteristics in gene expression, regulation and the biological effectsproducts have relevant points with the overall view in Chinese medicine physiological andpathological. Therefore, the relationship in TCM with genetic Earned much attention, it mayreveal the basis of the dialectical and the essence of the syndrome from a new angle. Kidneyis the origin of life, constitute the original material maintain the development of embryonic.kidney has a certain identity with the genetic material (DNA). Liver function is completed byliver cells, liver cells by functional gene expression in order to exercise the functions. Theepidemiological survey showed that, syndrome of kidney and liver yin asthenia is one of theoften witness one in in patients with HBV-related hepatocellular carcinoma patients. Clinicalpractice shows that the incidence of liver cancer patients combine with other diseases isrelatively low(the rate in early, medium-term liver cancer patients combined with otherdiseases less than32%). Therefore, the choice of liver cancer patients as research subjects can"dilute" the interference factors of disease "background" interference with the research insyndrome. As the syndrome is the clinical manifestations of the reaction of the body underthe action of pathogenic factors of systemic disease resistance regulation, including thepathogenic and the body's ability to respond two factors. The body's ability to responddepends on the constitution, the constitution is a reflection of the body under normalself-regulating control capability and ability to adapt to the external environment. Therefore,the constitution as a reflection of immune status, may be the essence of the syndrome. Turnexploration from the body's immune system is expected to identify the material basis for thesyndrome. Peripheral blood mononuclear cell as the most common immune cells, may reflectthe syndrome formation in real terms. HCC Patients with or without syndrome of liver-kidney yin deficiency were enrolled into the experimental group and the control group,respectively; their gene expression profiles were evaluated by a whole-genome AffymetrixGeneChip Human Genome U133Plus2.0Array. The differentially expressed mRNAs werethen selected by GO and pathway analysis. Based on the results of GO and pathway analysis,Gene coexpression networks were built according to the normalized signal intensity ofspecific expression genes. Finally, the results from microarray were confirmed by real-timequantitative PCR and Western-blot.二,Objectives1. Deteced the gene expression in peripheral blood mononuclear cell of HCC Patientswith or without syndrome of liver-kidney yin deficiency, Confirmed the existence ofdifferential gene expression between the two groups.2. The differentially expressed mRNAs were then selected by GO and pathway analysis.Based on the results of GO and pathway analysis, Gene coexpression networks were builtaccording to the normalized signal intensity of specific expression genes.3. Analysis syndrome of kidney and liver yin asthenia co-expression network joint withthe results of chip verify, screening significantly differentially expressed genes in syndromeof kidney and liver yin asthenia.4. Finally, the results from microarray were confirmed by real-time quantitative PCRand Western-blot, using verify results to build the diagnosis model of syndrome of kidneyand liver yin asthenia.三,Methods1. HCC Patients with or without syndrome of liver-kidney yin deficiency were enrolledinto the experimental group and the control group, respectively. their gene expression profileswere evaluated by a whole-genome Affymetrix GeneChip Human Genome U133Plus2.0Array. After correction by the random variance model t test (the random variance model(RVM) t-test) to calculate significance level (P value) and false positive rate (False positiverate, FDR) of the chip probe, screening differentially expressed probes between the twogroups.2. The differentially expressed mRNAs were then selected by GO and pathway analysis.Based on the results of GO and pathway analysis, Gene coexpression networks were builtaccording to the normalized signal intensity of specific expression genes. 3. Expand the sample size, use real-time quantitative PCR to detect important skills ingene expression network vertified microarray validation, Draw ROC curve, calculate the areaunder the curve of differential gene correlation coefficient standardized screeningliver-kidney yin deficiency syndrome were significantly differentially expressed genes basedon differential gene mRNA expression.4. The results from microarray were confirmed by real-time quantitative PCR andWestern-blot, Test results to build the liver-kidney yin deficiency syndrome diagnosis modeland calculate the sensitivity, specificity, positive predictive value, negative predictive value.四,Results1. The results showed that a set of615mRNAs were differentially expressed in the HCCpatients with syndrome of liver-kidney yin deficiency. It also had differentially expressedprobe876between the two groups, which raised387, down489.2. By gene ontology (GO) enrichment analysis, the genes in anti-apoptosis, regulation ofcell cycle, transmembrane transport, etc. were upregulated or downregulated in theexprerimental group. And another functional analysis of mRNAs by KEGG revealed that10signal transduction pathways were upregulated and16were downregulated, such as antigenprocessing and presentation, cell cycle, and protein export, etc. Based on the above results,we constructed coexpression network to determine which genes may play pivotal role inHCC patients with syndrome of liver-kidney yin deficiency.3. Selected60cases of liver cancer patients, through chip results to filter out to18express differences genes which had important ability in gene co-expression network. Theresults showed that the liver and kidney syndrome group lower than the non-liver-kidneysyndrome group in the11cut differences in gene mRNA expression, the same liver andkidney syndrome group8mRNA raised the differential gene expression is higher than thewithout liver and kidney Yin deficiency group. According to the18differentially expressedgenes mRNA expression, draw the ROC curve of differential gene correlation coefficientStandardization Law screened|⊿d egree Normalized|>0.3/under the ROC curve area>0.8,three genes were selected which had significant differences expression in liver cancer liverand kidney syndrome group, they are(SEC62[SEC62homolog (S.cerevisiae)],CCNB1[cyclin B1],BIRC3[baculoviral IAP repeat-containing3]).4. Of another120HCC samples, we found that the mRNA expression of SEC62,CCNB1and BIRC3were significantly lower in HCC patients with syndrome of liver-kidney yin deficiency than those without syndrome of liver-kidney yin deficiency (P<0.001, P<0.001, P=0.001), Also, In the protein expression of SEC62, CCNB1and BIRC3weresignificantly lower in HCC patients (P<0.01). Based on Gene Sec62, CyclinB1, Birc3mRNAexpression in patients to draw ROC curve, area under the curve (0.716,0.682,0.639), Use ofSec62, Cyclin B1, Birc3mRNA joint for the diagnosis liver kidney yin deficiency syndrome,the sensitivity was85%and specificity was78.3%, positive predictive value was79.7%,negative predictive value was83.9%.五,Conclusion1. Gene chip technique allows rapid and high-throughput screening for different geneexpression in HCC patients with or without syndrome of liver-kidney yin deficiency.2. By analyzing the differences in gene function and pathway annotations ofliver-kidney yin deficiency syndrome in peripheral blood mononuclear cell, found "drugmeasurement certificate","different diseases with the same syndrome" and "animal model"approach of the past through the liver and kidney Yin Deficiency of the functions of thedifferentially expressed genes, pathway analysis there are some consistent, were detected inmetabolism, apoptosis-related function or pathway change; by constructing geneco-expression network and found that liver and kidney syndrome group and non-liver-kidneysyndrome group exists between the gene three-dimensional network of relationships. Furtherevidence to explore the body's immune system is expected to identify the material basis forthe syndrome, the material that the syndrome may be based on a set of genes by functionalgroups or protein groups and the expression of specific metabolic component anomaliestogether constitute.3. That liver-kidney yin deficiency syndrome in the peripheral blood mononuclear cellof patients with a small number, but constant difference changes in gene expression, andthese genes in the gene co-expression network in a significant control position, which we callsignificant difference genes.4. The results of this study further confirm our hypothesis on the essence of syndrome,namely, a kind of deviation from the normal state in multi-gene style on the levels of bothmRNA and protein.