Screening Of Differentially Expressed Genes In Kaposi's Sarcoma By Suppression Subtractive Hybridization

Kaposi's Sarcoma (KS) is a multiple malignant idiopathic pigmented sarcomas. It grows slowly and often injures patients' skin even internal organs. There are four types KS: Classic KS(CKS)> AIDS Associated KS (AIDS- KS), endemic KS, immunosuppression -associated/ transplantation- associated KS. KS has obvious characteristic of endemicity and ethnology, it occurs mostly in Jews and people of Mediterranean origin and so on. In China, Xinjiang province is one of the highest incidence regions of KS. It is reported that CKS can only be found in Uygur and Kazakh people of Xinjiang in china. Human Herpesivirus-8 (HHV-8) is regarded as an important pathogenicity of KS. Moreover some scientists think that there are some relationship between KS and the disorder of cytokine or hereditary factors, but it is not sure about the exact pathogenesis of KS. On the other hand, some scientists pay more and more attention to the pathogeny of KS because KS is the most susceptible cancer of AIDS and the most common reason of AIDS patients' death. We know that the occurrence and development of sarcoma has closer relationship to the change of gene structure and expression. At present, there are no papers which report about the screening of differentially expressed genes of KS both in China and overseas. Objective To screen the differentially expressed genes between KS' tissues and normal skin tissues, to construct the cDNA gene library about KS, to reveal the pathogenesis of KS from molecular level, are the purposes of this project. Methods we collected KS' tissues and normal skin tissues to be contrast from a same patient. Extracted the total RNA from tissues, synthesized the dscDNA and used Rsa I to cut the genes, then linked them with two different adaptors. These dscDNA fragments from KS and the normal skin tissues were applied to conduct Suppression subtractive hybridization (SSH) as tester and driver to screen the differentially expressed genes, amplified these differentially expressed genes by Polymerase chain reaction (PCR) and then linked them with PGEM-Teasy cloning vector and transformed E. Coli DH5. We obtained the white positive clones by blue and white spot screening. Boiled these positive clones for disrupting them and amplified these genes by PCR. Sequenced these genes at random. Compared the homology with Gene bank by Blast n and Blast x. At the same time, used nested PCR to detect whether the patient infects the HHV-8 or not. Results We obtained multiple differentially expressed genes strips which ranged at 200-500bp by SSH. We have constructed 2 gene libraries that represent the up-regulated and down-regulated genes in KS. 207 clones were sequenced randomly and we get 30 differentially expressed genes. Conclusions We screened the differentially expressed genes of KS and constructed its gene library. We found up-regulated and down-regulated genes that may have some function in KS' pathogenesis. CD164 may conduct the function in KS' pathogenesis through adhesiveness, proliferation of epithelia and vessel endothelial cell, introduction of c-myc signal's transduction, etc; Protooncogene c-myc may be activated by point mutation, over expression and HHV-8 infection; the down-regulated gene, heat stock protein 40 (HSP40) can weaken the activation of the HSP70 and influence it's function and so on; The abnormality of metabolism and alteration of mitochondrial may exist in KS. In addition, we also found some genes that may have particular affection in KS but they are not found in other carcinoma. We need do more further study about those genes' function in the future.