Gene Expression Analysis Involved In Pathogenicity Of Multiple Lipoma

BackgroundA lipoma is a benign tumor composed of hyperplastic mature adipose tissue and canoccur at any age according to most of present textbooks, while it's more commonly found inadults from40to50years of age. The great majority of lipomas occur without discomfort,complication or risk of malignant transformation and are simple to diagnose andtreat(resection as the preferred treatment), so the complete research of lipoma hasn't beenpaid high attention and progressed slowly. In clinical work many doctors notice that a singlelipoma can be cured with favourable prognosis, whereas multiple lipoma could impact thephysical and mental health and quality of life of the patients who are always worry about thecontinual development of tumor and suffer from no treatment to real cure the diseasealthough it isn't so serious as to imperil their lives. Besides, most patients with multiplelipoma have inherited tendency. Overall, it's very important and necessary to research thedisease.There is less study on the etiology and pathogenesis of lipoma reported in lots of presentChinese literature as well as English literature mainly focusing on multiple symmetriclipomatosis which has relatively higher incidence in occident than that in China. So there isvery little systemic research of multiple lipoma in China. At present the generallyacknowledged factors that closely related with the occurrence and development of lipoma areas follows:1) Heredity—Some parents with single lipoma have children with multiple lipomawhile some parents with multiple lipoma have children with multiple or single lipoma. Arethey only coincidence or evidence of gene susceptibility?2)Unhealthy lifestyle-such asoverdrink, high fat diet, sit up, et al.3)Overpressure of life or work. In addition, an opinionthat tumorigenic factor of lipoma maybe plays a crucial role was declared in internet. Theysupposed that the factor in the patient's somatocyte was inactive and unable to cause lipomaunder normal circumstance. When the internal environment changed within the body theability of lymphocytes, mononuclear phagocytes and other immune cells to monitor thetumorigenic factor induced decline, coupled with chronic inflammation stimulation, the bodyfat metabolism and other incentive conditions, the factor became more active and bound tocertain gene fragments in cell, forming abnormal gene mutations which result in deposition of adipose tissue and lipoma(http://www.clsbio.com). Though the academic literatures reportedthe supposition haven't been found yet, it's undoubted that lipoma is a consequence ofinteraction of many factors like other tumors. How do these factors cause the occurrence oflipoma finally? Which genes mutate in the process of forming lipoma? Why there is onlyneoplasia but no canceration nearly?With these questions, this project planned to screen out the related differential expressedgene between lipoma and normal adipose tissue by gene chip to elucidate the pathogenesis oflipoma and provide conceivable experience for clinical treatment.ObjectiveThis project planned to screen out the related differential expressed genes between tumortissue and normal adipose tissue around the tumor to discover the pathogenesis of multiplelipoma and the possibility of differential genes' existing and provide conceivable experiencefor clinical treatment.Methods1. Samples collecting: All samples were collected in2lots.1) Three samples werecollected for gene chip in3male outpatients with multiple lipoma who were cured in plasticsurgery department of Changhai hospital(n=3).Each of them had more than5lipomas witheach diameter of less than3cm. Course of disease was control in2years to catch the earlydifferential of gene expression in lipoma. As while normal adipose tissue around the tumorswere collected respectively as contrast sample(n=3). All of6samples were determined byAffymetrix Human GeneChip U133Plus2.0. Paired t-test were performed and differenceswere considered significant when p<0.05. Genomics analysis was used in differential genes.2)The samples of multiple lipoma cases sporadically distributing in crowd(n=3), normaladipose tissue(n=3) were collected to confirm whether those differential expressed genescreened out previously by gene chip did work.2. Analysis of histopathology: By gross observation, pathological section and HE stainingwe analysed the morphologic differential among normal adipose tissue, lipoma andliposarcoma. The samples were collected as follows.3. Screening out of differential expressed genes by gene chip: Cluster analysis wasperformed in the differential expressed genes screened out by6gene chips. The analysisresult was matched and compared with the genes in GenBank Database and screened out6important differential expressed genes associated with cytogenesis, proliferation, signaltransduction of lipoma and other function changes by synthetical function analysis 4. Confirming of related genes in different samples: RT-PCR was performed to confirmthe6important differential expressed genes in6samples of multiple lipoma,6samples ofnormal adipose tissue respectively. According to the expressing content and differentia ofthese genes in3kinds of sample, the pathogenic gene for lipoma can be analysed andscreened out finally.ResultsPartⅠ Histopathologic analysis of subcutaneous multiple lipomaAll the samples of multiple lipomas collected in this project had the samehistopathologic features as follows. In the surface, a little of vascular distribution can beobserved in the integrity and thin capsule which enveloped the yellow tumors with certaintenacity. In section plane, there were uniform fat granule and septum made of connectivetissue which separated the tumors into lobules with different sizes. On the other hand, thenormal adipose tissue around the tumors had no capsule and was separated into lobules byfibrous septum whose density was much higher than that of lipomas. Under the microscope,the tumors were consisted of mature adipose cells and separated into lobules with differentsizes by the thin fibrous septum which contained rich vascular supply and enveloping andspreading into the tumors. The adipose cells were round or polygonal with tight arrangementand had the same size and shape in the different lobules. Inside the cells many vacuolesformed after loss of lipid droplets and extruded the nucleolus to be oblate or crescent.Different with the tumors, normal adipose tissue had lots of fibrous septum and loose cellarrangement. But both of them had the near cell size shape and positive red Oil staining of thelipid droplets. Immunohistochemistry showed that there was positive expression of CD31ofthe vessels in fibrous septum of tumors and normal adipose tissue, S100in the cells of normaladipose tissue and Leptin in the tumors.PartⅡ Genomic analysis of genes relatived with pathogenicity in multiple lipomaThe total number of matched gene probes in6samples was54614. Statistical analysisperformed between lipomas and normal adipose tissue showed the number of differentialmatched probes was1776(p<0.05) of which374had a fold change of more than twice.Cluster analysis screened out260differential expressed genes with p<0.05and fold change>2.There were36proliferation-related differential expressed genes including30up-regulatedand6down-regulated genes,2anti-apoptosis genes (ERBB4and NPY5R),10lipid bindinggenes including5up-regulated and5down-regulated genes,1positive lipid metabolic genenamed apolipoprotein B gene (APOB) with7folds down-regulation and1negative lipidmetabolic gene named ABCG1with2.5folds up-regulation. In addition, the expression ofseveral genes involved in proliferation and regulation of tumor cells in lipomas including ESM1, SOX11and HOXD10increased by32.81,31.01and13.99folds respectively, incomparison with normal adipose. By combining the biochemical function and differentialexpression of these genes, ESM1, SOX11, HOXD10, ERBB4, NPY5R and APOB werescreened out for further validation in sporadic cases.PartⅢ Confirming of candidate important pathogenic gene in sporadical casesBy using RT-PCR, the expression of ESM1, SOX11, HOXD10, ERBB4, NPY5R andAPOB in lipomas was validated to accord with the former result of genomic analyses. Thevalidation result showed that the differential expression of these6genes in each sampleaccords with the result of gene chip completely including the differential of signal strength asfollow. ESM1, SOX11and HOXD10were up-regulated by18.51,19.18and20.55foldsrespectively. ERBB4, NPY5R and APOB were down-regulated by16.68,4.99and15.05folds respectively. The result of PCR performed in sporadic cases also accorded with thevariation mode of gene chip as follow. ESM1, SOX11and HOXD10were up-regulated by290.86,6.13and12.2folds respectively. ERBB4, NPY5R and APOB were down-regulatedby5.09,7.19and15.05folds respectively. The results of PCR didn't only validate thedependability and stability of gene chip, but also provide the evidence that the differentialexpression of these6candidate genes exist in sporadic cases.ConclusionThis project screened the differential expressed genes between subcutaneous multiplelipoma and normal adipose tissue firstly and then picked out the genes which had high foldchange including3tumorigenicity-related genes (ESM1, SOX11and HOXD10),2apoptosisgenes (ERBB4and NPY5R) and1lipid metabolic gene (APOB). The further validation ofthese6genes performed in sporadic cases with RT-PCR confirmed preliminarily what causethe occurrence of subcutaneous multiple lipoma as follow. The abnormal high expression oftumorigenicity-related genes improved the proliferation ability of cells and low expression ofapoptosis genes maintained the sustained growth of proliferated cells. In addition, theabnormal storage and metabolism of lipid caused the lipid deposition in cytoplasm. To sumup, it had been confirmed preliminarily that the occurrence of a lipoma isn't the result of thelocal expansible growth of inherent fat cells but the increased number of the proliferated fatcells. This result could be an experiment support for possible revealment of pathogenesis oflipoma and provide conceivable experience for clinical treatment.