Expression And Its Significance Of Tsg101 In Lung Cancer Tissue And Cells

IntroductionHuman tumor susceptibility gene 101(TSG101)has been mapped to chromosome 11P115.1-15.2,the whole length of cDNA is 1494bp,including six exons and five introns,which encodes the product with 380 amino and 43KD moleculer weigh.TSG101 protein contains N termination like ubiquitin conjugase,a praline-rich domine(130-205 amino acids),C-termination coiled-coil domain(231-302)and a steady box SB which regulates its steady state.TSG101 protein,both outside of a narrow range can lead to abnormal cell growth.The finding has shown that TSG101that contains C-termination colied-coil domain may act as transcription inhibition factor. Recently,there have been some reportsabout TSG101 in medullocell leukaemia,lymphoma,prostatic carcinoma,cancer of the breast and cervirx,but the findings were different.Our findings show that the expression of TSG101 in lung cancer is much lower than that in normal lung tissues,it changes consistently with the level of cell diferention.But others show that TSG101 were high in some cancer of the chest and cervirx,and may have to do with the abnormality transcripts.Now there have been few reports about the expression of abnormality transcripts in lung cancer.So we investigate the expression TSG101 in lung cancer in order to evaluate the possible roles in the oncogenesis and progression of lung cancer.Materials and Methods1.Samples79 lung cancer tissues samples(48 lung squamous carcinoma and 31 lung adenocarinomas).7 lung cancer cells(SK and QG are squamous cell carcinoma cells,661 and 460 are large cell undifferentiated carcinoma cells,SPC and LTE are adenocarcinoma cells,446 is small cell lung carcinoma cells)2.ReagentsThe anti-human TSG101 mouse monoclonal antibody(sc-7964)was provided by Santa Cruz company(USA).The goat-anti-mouse second antibody(ZB-2305)and DAB agent kit(ZLI-9302)were provided by Beijing zhongshan golden bridge biotechnology company.Trizol was provided by Invitrogen company(Invitrogen).RT-PCR kit were provided by TakaRa company(Dalian).3.Methods(1)ImmunohistochemistryDetect the TSG101 expression by Immunohistochemistry S-P methold.(2)RT-PCRRT-PCR was adopted to detect a common shortened TSG101 transcript because of aberrant alternative splicing and the wild-type transcript in lung cancer lines.(3)Western BlotIncubation with primary antibody(TSG101 1:200),enzyme conjugate incubation(secondry antibody 1:2000).4.Statistical anaysisAll the datas are analyzed with SPSS for Windows 13.0 software.The statistical significance is defined as P＜0.05.Results1.ImmunohistochemistryTSG101 is expressed in lung cancer tissues and localized in cytoplasm and/or nucleus.TSG101 is expressed strongly in normal lung tissues.TSG101 is expressed weekly in cell's cytoplasm in poorly differentiated lung cancer.2.RT-PCRThe wide- type transcripts and shorter transcripts were expressed in all lung cancer cells.But the shorter transcripts was more in small lung carcinoma cells than that in others.And th 3.Western BlotThere are tsg101 in 7 lung cancer cells,and lower in small lung carcinoma cells than that in others.DiscussionTumor susceptibility gene 101(TSG101)was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells.Feng etal reported that TSG101 outside of a narrow range can lead to abnormal growth.TSG101 protein is maintained at an almost constant steady-state leval in cultured murine and human cells.In our study,we find that the expression of TSG101 is significantly lower than in the neighboring noncancerous tissue,The expression rate of TSG101 in well and moderately differentiated cells is significantly higher than that in poorly differentiated cells,the expression teat of TSG101 in patients without lymphatic metastasis is significantly higher than that in those with lymphatic metastasis.However,Maxxwell reported that TSG101 was not mutated in breast cancer,but there were some short splicing transcripts.we find that shorted transcripts which was 900bp(154-1054)delitation.TSG101 did not alter in the process of tumor development,but the splicing of its transcript showed abnormality,the full-length transcripts decrease,so the expression of TSG101 becomes low,and its ability that prevents tumor development decreases and leads to lung cancer.But we did not find shorted protein.We find that the full-length transcripts and TSG101 protein were lower in the small lung carcinoma cells that in others lung cancer cells,and there was more shaoreted transcripts in the small cell lung carcinoma cells.All the dates showed that TSG101 can act differntedly in differnted cancer,and TSG101△154-1054 which acted as cancer development maker,may acted as carcinogenesis.In a word,the formation and development of tumor undergo such pathology process in cluding multiple genes,many procedures,multistage,which requires lots of gene,TSG101 expresses lower in lung cancer.Human pay close attention to the function of TSG101 and its aberrant splicing in tumor tissues and normal tissues.As it is a new candate tumor suppressor gene,we try to find their changing law and provide important clues to detect and cue malignant tumor in time. ConclusionsThe expression of the wide-type and shorted transcripts in the all lung cancer cells,but the shorted transcripts were more in small cell lung carcinoma cells.