Experimental Study Of Screen And Annotation Of Abnormal Scar Related Genes

ObjectHypertrophic scar and keloid scar are general abnormal scars seen in clinic, which are result of over wound-healing. Though a lot of researches have been performed on abnormal scars, pathogenesis of abnormal scars were still unclear. Above all, it's needed to screen genes related with pathogenesis of abnormal scar. Previous works had focused on single or a few genes limited by traditional biological technologies, thus information about pathogenesis of abnormal scar was fragmentary and unsystematic. Further more, it was difficult to reappear the relationships among abnormal scar related genes. So the key point was to find a technology, which can analysis genes systematically, rapidly, conveniently, accurately, and high through putted. The appearance of microarray made it possible. Microarray technologies provide the means of measuring the expression of thousands of genes or proteins simultaneously. This revolution brings new perspectives for the study of expression networks and their regulation, potentially providing valuable insights into the molecular mechanisms underlying the disease. In this study, we used cDNA microarray to measure genes expression in abnormal scars (hypertrophic scar and keloid scar) vs normal skin, screened different expression genes in each group, part of which were verified by RT-PCR method. We then used a literature mining method to annotate these different expression genes to explain the relationships among these genes and abnormal scars, to explore the pathogenesis of abnormal scars, to find out novel candidate abnormal scars related genes which may take part in the pathogenesis of abnormal scars and as targets of therapy research.Material and methodsIdentificating differentially expressed genes of pathologic scar tissues using microarray-based detection.—-Ml samples were divided into three groups(hypertrophic scar group 3 cases, keloid scar group 3 cases and normal skin group 10 cases). All normal skin tissues were pooled together as one group, which was taken as control of hypertrophic scar group and keloid group. Specimens were obtained at a time of operation and were immediately stored in liquid nitrogen. The cDNA microarray composed of 8,064 clones were purchased from Shenzhen Chipscreen Biosciences, Ltd. Total RNA was extracted by TRIzol Reagent. Equivalent total RNA from corresponding samples were annealed to oligo(dT)i8 and reverse-transcribed in the presence of Cy3-dUTP (hypertrophic scar and keloid) and Cy5-dUTP respectively (normal skinV The mixture of Cv3 and Cy5 labeled probes were hybridized against the chip in a humidified chamber with hybridization solution, at 42°C overnight. The chip was scanned with array scanner. Images were transferred to digital data with Arrayvision 6.0. Ratio was calculated from Cy3 intensity divided by Cy5 intensity of each spot after data normalization. It represented the relative gene expression level between the tested sample and the control. Based on Shenzhen Chipscreen Biosciences, Ltd previous experience with many duplication tests, we chose one signal intensity 5E+08 for valid data selection. Differentially expressed genes were chosen when the density is over 5E+08 and the ratio is over 2 or below 0.5 and differential gene-expression profiling of pathologic scar were constructed based on the consistently differential expressed genes of hypertrophic scar and keloid. We analyzed a part of differentially expressed genes and explored the genesis mechanism of abnormal scar. We also analyzed the inconsistently expressed genes between them.Identification of part of differentially expressed genes by semiquantitative RT-PCR-—To test the results of gene chips, semiquantitative RT-PCR was used to confirm the differential expression of CTGF and TGFBR2 chosen as upregulated genes and ACTG gene was performed as the internal control standard. According to the cDNA sequence of TGFBR2^ CTGF and ACTG, primers were designed and PCR products were identificated by 2% agarose electrophoresis. Original gray data were obtained by density scan of targeted band , and relatively expressed level of two genes was proofread by ACTG expression level of each sample. Data were finally analysed.Annotating different expression genes with literature mining method.-—A mining technique based on the analysis of literature profiles generated by extracting the frequencies of certain terms from thousands of abstracts about these different expression genes stored in the Medline literature database was used to annotate these genes. Terms are then filtered on the basis of both repetitive occurrence and co-occurrence among multiple gene entries and suppositional pathogenesis theory ofabnormal scars to get 23 terms at last. Finally, clustering analysis with Cluster and Treeview program is performed on the retained frequency values, shaping a coherent picture of the functional relationship among these large and heterogeneous lists of genes. Such data treatment also provides information on the nature and pertinence of the associations that were formed.Result and DiscussionResult of microarray hybridization was verified to satisfy demand of repeatability and reliability by synthetical evaluation, which indicated that data of microarray is reliable. Electrophoresis analysis of RT-PCR products indicated that all target genes were amplified specifically. Apparent bars were showed at 455 bp, 446 bp, and 425 bp respectively indicated over expression of TGFBR2, CTGF, and ACTG genes. ACTG gene expressed in each group, and the brightness of bars were approximately same. Over expression of TGFBR2 and CTGF were obvious in hypertrophic scar and keloid scar tissues. Comparing with result of microarray, trend of most RT-PCR results were same with microarray. The results of RT-PCR not only verified the dependences of microarray data but also implied that TGFBR2 and CTGF may play important roles in initiation of abnormal scars.There were 1,027 efficient genes data in hypertrophic scar tissue according to the designed data filtered standards. Among these genes, 171 genes were different expression genes between hypertrophic scar with normal skin, where 99 genes were up-regulated and 72 genes were down-regulated.There were 1,096 efficient genes data in keloid scar tissue according to the designed data filtered standards. Among these genes, 277 genes were different expression genes between keloid scar with normal skin, where 163 genes were up-regulated and 114 genes were down-regulated.After combination analysis of these two group data, it's found that there were 206 common different expression genes in hypertrophic scar and keloid scar vs normal skin, where 125 genes were up-regulated and 81genes were down-regulated. There were 26 genes which expressed differently in hypertrophic scar but not in keloid scar, and 60 genes which expressed differently in keloid scar but not in hypertrophic scar.In a general comparison with normal skin, obvious tissue and cell structure change can be found in hypertrophic scar and keloid scar, while several signal transfer and gene regulation pathways were changed at the same time. Among these genes, gene groups with obvious functional character were extracellular matrix genes, cell skeleton protein genes, growth factors and receptors genes, transcription factors, and signal transfer genes, et al. Most of these different expression genes werecommon in hypertrophic scar and keloid scar, which explained the clinical similarity of these two abnormal scars and implied that molecular mechanism underlying these two diseases may be major similar.The 206 common different expressed genes in hypertrophic scar and keloid scar consisted of the different expression genes profile of abnormal scars, reflected the general change of genes expression in abnormal scar at transcriptional level. These 206 genes were belonged approximately to 26 different functional gene families which were related with different biological process such as extracellular matrix, cell factors, signal transferring, cell cycle, and apoptosis.Many extracellular genes such as collagen genes, collagenase genes, and skin pontin protein genes found recently were over expressed in abnormal scars, which were related closely with over aggradation of extracellular matrix in abnormal scars. Over expression of connective tissue growth factors, insulin-like growth factor II, and transforming growth factor-82 were related closely with growth, chemotaxis, synthesis and excretion of ECM composition of fibroblast promoted by growth factors. Connexin protein gene families regulated gap junction intercellular communication were also differently expressed in abnormal scars. Over expression of Cx43 and specific expression of Cx26 and Cx37 in hypertrophic scar and keloid scar implied that Cx gene family are tissue specific genes in abnormal scars and over expression of Cx gene family were related closely with initiation and progression of abnormal scar, which may prevent abnormal scars to become tumor. Over expression of early growth response-1 gene in abnormal scar was related closely with cell growth, differentiation, and apoptosis.197 different expression genes with known names in abnormal scars were further analyzed with literature mining methods. 87,282 records about these genes were extracted from PubMed. After text analysis and data filtering, 23 terms were obtained to be clustered analysis. Results indicated that genes were clustered under terms. For example, genes related with terms "hypoxia" and "proliferation" were clustered into one group, which includes HIF1A, EPAS1, and CITED2. As clustered dendrogram showed, the group genes, especially HIF1A, were related with terms "hypoxia", "proliferation", "tumor", "apoptosis", "fibroblast", and "scar". So we further reviewed all articles related with these three genes, and found that HIF1A played not only a centrum role in physiologic reaction at hypoxic condition, but also an important role in normal development of embryo, and in metabolism, angiogenesis and metastasis of tumor. CITED2 was related with HIF1A. Combing with data mining, literature analysis, and microarray, it can be inferred that HIF1A andCITED2 are two novel genes may related with abnormal scars, and pathway activated by HIF1A under hypoxia, which induced downstream genes, especially genes which inhibit nuclear transcript factor inhibitor protein a (NFKBIA) to activate anti-apoptosis process of NF k B, may play an important role in pathogenesis of abnormal scar, especially keloid scar.From the different expression genes of hypertrophic scar and keloid scar, the different expression of ECM1> S100A2> p63> NFKBIA in keloid scar are of specificity. The S100A2 gene expressed up-regulated in keloid scar but expressed normal in hypertrophic scar, which may explain the mechanisms that keloid scar was more malignancy and more invasive to the vicinity normal constitution. The specific up-regulated of ECM1 in keloid scar, like S100A2 in gene mapping, located in chromosome LP21's epidermis differentiation and compound region. Reveal that these two genes cooperate with each other, their different expressions play important roles in pathogenesis of keloid scar. The p63's down-regulated expression in keloid scar, may weaken Tap63' apoptosis function therefore decrease apoptosis in keloid scar. NFKBIA expressed down-regulated in keloid scar but expressed normal in hypertrophic scar. It' s down-regulated trail off the inhibition to NFkB. Thus NFkB adjust lower apoptosis genes and exert anti- apoptosis function, promote proliferation of keloid scar in sequence. The study reveal that all these genes play important role in keloid scar pathogenesis, may become the specific target gene to keloid scar.Innovation of the study(1) We used cDNA microarray to measure genes expression in abnormal scars (hypertrophic scar and keloid scar) vs normal skin, screened different expression genes in each group, established the differential expressed genes of abnormal scars.(2)Annotating different expression genes with literature mining method, detected some new related genes.(3)Screen and annotation of abnormal scar related genes, discovered valuable information that guide the furthermore study thich may bring light to the mechanisms of abnormal scar.(4) Identificated 4 differentially expressed genes which may become the specific target gene to keloid scar through in difference gene annototion between hypertrophic scar and keloid scar,...