Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays

AIM:To identify differentially expressed genes in quiescent and activated hepatic stellate cells(HSCs)and explore their functions.METHODS:HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation.Total RNA and mRNA of quiescent HSCs,and cultureactivated HSCs were extracted,quantified and reversely transcripted into cDNA.The global gene expression profile was analyzed by microarray with Affymetrix rat genechip.Differentially expressed genes were annotated with Gene Ontology(GO)and analyzed with Kyoto encyclopedia of genes and genomes(KEGG)pathway using the Database for Annotation,Visualization and Integrated Discovery.Microarray data were validated by quantitative real-time polymerase chain reaction(qRTPCR).The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi.The expression of Wnt5a in fibrotic liver of a carbon tetrachloride(CCl4)-induced fibrosis rat model was also analyzed with Western blotting.RESULTS:Of the 28 700 genes represented on this chip,2566 genes displayed at least a 2-fold increase or decrease in expression at a P<0.01 level with a false discovery rate.Of these,1396 genes were upregulated,while 1170 genes were downregulated in culture-activated HSCs.These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms.The most enriched GO terms included response to wounding,wound healing,regulation of cell growth,vasculature development and actin cytoskeleton organization.KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs.Wnt5a was significantly increased in cultureactivated HSCs as compared with quiescent HSCs.qRTPCR validated the microarray data.Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation,downregulated expressions of typeⅠcollagen and transforming growth factor-β1.Wnt5a was upregulated in the fibrotic liver of a CCl4-induced fibrosis rat model.CONCLUSION:Wnt5a is involved in the activation of HSCs,and it may serve as a novel therapeutic target in the treatment of liver fibrosis.

Study on differential gene expression profile of serum exosomes in patients with acute cerebral infarction

Objective To analyze the differential gene expression profile of serum exosomes in patients with acute cerebral infarction(ACI)and clarify the changes in gene expression related to cerebral infarction injury and the potential serum markers.Methods Four patients with ACI and five healthy people were enrolled in the PhaseⅠstudy.After serum isolation from peripheral blood,exosomes were extracted with exosomes kits,highthroughput detection of m RNA was performed with gene chips,and differentially expressed m RNAs were screened.Gene Ontology(GO)functional analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed simultaneously.Furthermore,real-time polymerase chain reaction(q RT-PCR)was used to verify the expression levels of the screened differential m RNAs in the serum exosomes collected in PhaseⅡfrom 32 patients each in the ACI case and normal control groups.Results In the PhaseⅠstudy,there were 248 differentially expressed m RNAs(fold change≥2.0,P<0.05)among five patients in the normal control group and four patients in the case group,of which the expression of 242 was upregulated and that of six was downregulated.The results of GO functional enrichment analysis mainly included behavior regulation,cell connection,and antioxidant activity.The results of KEGG pathway enrichment analysis mainly included ribosomes,proteasomes,oxytocin signaling pathways,and oxidative phosphorylation.After researching and screening based on relevant literature,it was found that among the genes with significant differential expression,H3 F3 B m RNA may be associated with and might play an important role in ACI.The q RT-PCR method was used to detect the H3 F3 B mRNA expression in serum exosomes of 32 patients each in the normal control and case groups in PhaseⅡ;the expression was significantly higher in serum exosomes of the case group than in those of the normal control group(P<0.001).H3 F3 B mRNA expression in serum exosomes of the case group positively correlated with age,the National Institutes of Health Stroke Scale(NIHSS)score,and the maximum infarct size(P<0.05).Conclusion ACI can lead to changes in the serum exosomes mRNA expression profile,which may be closely related to the occurrence,development,and prognosis of this condition.These findings will provide direction for research on the molecular mechanism,diagnostic markers,and therapeutic targets of ACI.

Identification of TM9SF2 as a Candidate of the Cell Surface Marker Common to Breast Carcinoma Cells

OBJECTIVE We aimed identification of cell surface molecules, which might serve as diagnostic biomarkers or useful targets for therapies, in breast cancer. METHODS We developed unique DNA microarray coupled with spherical self-organizing map （sSOM） analysis to characterize cells and tissues by the cell surface markers. In the microarray 1,797 probes for human genes coding membrane bound proteins were spotted. With this microarray the gene expression profiles of eight breast carcinoma cell lines were compared to identify the genes that were commonly expressed in breast carcinomas but not in normal cells. RESULTS The gene expression profiles of sSOM from the eight breast carcinoma cell lines were successfully distinguished from that of normal breast tissue derived cells suggesting the presence of genes of interest, sSOMon the data extensively filtered revealed several candidate genes, of which expression was significant in carcinoma cells but low in normal cells. Finally, TM9SF2 was nominated through validations of PCR procedures together with CD24 and ErbB3, which are known breast carcinoma markers. TMgSF2 expression was further confirmed by immunological staining. Interestingly, TMgSF2 was found to be expressed in all the cell lines evaluated while CD24 and ErbB3 were not in all of the carcinoma cells, supporting their relationship in sSOM. Although physiological significance of TMgSF2 is unknown yet, siRNA treatment significantly inhibited the growth of MDA- MB-231 cells. CONCLUSION We propose TM9SF2 as a novel and useful diagnostic marker as well as a potential molecular target specific to breast carcinoma cells covering wide range of breast cancer.

Gene Expression Profiles in Porcine Tissues of Liver and Kidney

Microarray technologies are widely used all over the world for the gene expression analysis in various tissues, but still this technology has some limitations. The problem of eliminated reproducibility of the results, obtained in different laboratories using different platforms, is very relevant nowadays. For revelation of problems ofmicroarrays, the comparative analysis of hepatic and renal gene expression profiles （GEP） was carried out by using swine protein annotated oligonucleotide microarrays （SPAM）. Revealed differences in GEP between kidney and liver of pigs were correlated with functional and histological distinctions of these organs. It was shown that sources of errors in the comparative analysis of organ-specific GEP could be connected to the cross hybridization of one probe to transcripts （cDNA of mRNA） of different genes and to individual variability in gene expression between animals, related with the changeability of influences of exo- and endogenous regulation factors.

Polyphyllin I inhibits proliferation and metastasis of ovarian cancer cell line HO-8910PM invitro

OBJECTIVE： To study the anticancer mechanism of polyphyllin I （PPI）, a Traditional Chinese Medicine, on the ovarian cancer cell line HO-8910PM in vitro. METHODS： Transwell chamber invasive assays were used to investigate the inhibitory capacity of PPI on HO-8910PM metastasis. Gene expression profiling chips was used to screen differentially ex- pressed genes between experiment group and con- trol group. Reverse transcription PCR and Western blotting were used to determine mRNA and pro- tein levels. RESULTS： With increasing PPI concentration, the metastatic capacity of cells decreased, with signifi- cance differences between the experimental and control groups （P〈0.01） as well as between two concentration groups. Gene expression profiling identified 123 differentially expressed genes, of which 70 were downregulated and 53 were upregu- lated. The genes were involved in multiple signal transduction pathways, including apoptosis, prolif- eration and metastasis. Real-time PCR （RT-PCR） showed that differential genes PIK3C2B, Caspase 9and WntSA were downregulated with increasing PPI, showing an evident dose-effect relationship. The c-Jun was an exception. As the PPI dosage in- creased and the exposure time was extended, c-Jun relative expression showed an upward trend. There were significant differences between the ex- periment and control （P〈0.05）. Western blot analy- ses showed that PPI treatment decreased levels of Caspase 9, WntSA and PIK3C2B and increased acti- vated Caspase 9,c-Jun and p-c-Jun expression levels. CONCLUSION＂ PPI has strong antitumor and anti transfer activity. It can activate c-Jun expression and the JNK signaling pathway, elicit cell apoptosis via the mitochondrial-mediated Caspase activation pathway, and finally inhibit tumor growth and mi- gration in vitro. The downregulation of PIK3C2B and Wnt5A jointly inhibit the proliferation and me- tastasis of HO-8910PM. PPI may be a novel treat- ment for ovarian cancer.

Effect of electro-acupuncture on gene expression in heart of rats with stress-induced pre-hypertension based on gene chip technology

OBJECTIVE:To explore electro-acupuncture's(EA's)effect on gene expression in heart of rats with stress-induced pre-hypertension and try to reveal its biological mechanism based on gene chip technology.METHODS:Twenty-seven Wistar male rats were randomly divided into 3 groups.The stress-induced hypertensive rat model was prepared by electric foot-shocks combined with generated noise.Molding cycle lasted for 14 days and EA intervene was applied on rats in model + EA group during model preparation.Rat Gene 2.0 Sense Target Array technology was used for the determination of gene expression profiles and the screened key genes were verified by real-time quantitative polymerase chain reaction(RT-PCR) method.RESULTS:Compared with blank control group,390 genes were changed in model group;compared with model control group,330 genes were changed in model + EA group.Significance analysis of gene function showed that the differentially expressed genes are those involved in biological process,molecular function and cellular components.RT-PCR result of the screened key genes is consistent with that of gene chip test.CONCLUTION:EA could significantly lower blood pressure of stress-induced pre-hypertension rats and affect its gene expression profile in heart.Genes that related to the contraction of vascular smooth muscle may be involved in EA's anti-hypertensive mechanism.