| ObjectiveGuangxi is high incidence area of primary hepatocellular carcinoma(HCC),the most common malignant tumor with the highest degree of malignancy.Since the 1970 s of the 20 th century mortality of liver cancer has been ranked a variety of malignant tumors.Surgical operation is the first choice for treatment of HCC,but the 5 years recurrence rate is as high as 35.4%-43.5% after surgery.Poor prognosis requires the development of some new therapeutic strategies.Cancer-testis Antigen(CTA)is a kind of antigen which is widely expressed in tumor tissues and only expressed in testis of normal tissues.CTA can be used as a potential therapeutic target for liver cancer because of its specific expression.OY-TES-1 is defined as the 23 th member of CTA family.Studies have confirmed that,OY-TES-1 expression in HCC and other cancer tissues,and OY-TES-1 expression was related to the pathological grade of HCC,and OY-TES-1 antibodies was present in the serum of partial patients with HCC;Cell proliferation,migration and invasion ability is reduced,and cycle arrest and apoptosis increased after downregulation of OY-TES-1 in hepatocellular carcinoma cell.These results show that OY-TES-1 plays an important role in the development of hepatocellular carcinoma,however,no study has reported that(1)whether OY-TES-1 play an important role in the process of normal liver cells Malignantly transformating into hepatoma cells;(2)what is the function domains of OY-TES-1 protein in HCC tissues and cells;(3)Is the OY-TES-1distributing in HCC tissues and cells in the full-length protein or in fragments?How about its subcellular localization?(4)what are the differentially expressed genes affecting the malignant behavior after OY-TES-1 down-regulated in liver cancer cells;(5)the possible mechanism of OY-TES-1 affecting the malignant behavior of hepatoma cells.Therefore,we will use bioinformatics analysis,vector construction,lentivirus vector or expression vector infected hepatoma cells and normal liver cells,flow cytometry experiment technology to explore the relationship of OY-TES-1 and the HCC occurrence,protein structure and possible interaction protein of OY-TES-1,expression and subcellular localization of OY-TES-1 in HCC tissues and cells,differentially expressed genes affecting the malignant behavior of HCC after downregulation of OY-TES-1,and the and OY-TES-1 affect HCC cell malignant biological behavior,and the possible mechanism that OY-TES-1 affecting the malignant biological behavior of HCC,to provide new targets and basis for the treatment of HCC.Methods(1)Construct OY-TES-1 interference and expression lentivirus vector to down-regulate expression of OY-TES-1 in hepatoma cells,and to expression of OY-TES-1 in normal liver cells,and by using CCK-8 colorimetric assay,flow cytometry and Transwell migration assays to detect the influence of OY-TES-1upregulation on cell proliferation,cell cycle,migration and apoptosis of normal liver cell.(2)Analyze the solublity,transmembranous helix,subcellular localization,domain and domain-domain interaction,Gene Ontology annotations and biological interaction networks of OY-TES-1 in HCC,by using a variety of online databases and bioinformatics techniques(3)Using polyclonal antibodies of full-length protein,the N-terminal truncated proteins(amino acid sequence 1-272)and C-terminal truncated proteins(amino acid sequence 273-543)of OY-TES-1 to incubate liver cancer cells,normal liver cells,HCC tissues and the corresponding adjacent tissues respectively,then using the IFC,IFHC,ICC and co-focusing technique to detect the expression and subcellular localization of full-length,C terminal and N-terminal truncated proteins of OY-TES-1.(4)Using RNA interference(RNAi),microarray and DAVID to analyze the expression of genes related to cancer cell proliferation,cell cycle,invasion and apoptosis in HCC after OY-TES-1 downregulation.(5)Analyze the relationship of MT1 F and OY-TES-1 in HCC and its effects on malignant biological behavior of liver cancer,by using Knockdown,Knockin,fluorescence quantitative PCR(q RT-PCR),IFC and flow cytometry.Results(1)Successfully construct the OY-TES-1 overexpression vector of the lentivirus,and achieve OY-TES-1 up-regulated infection in normal liver cells.The morphology,growth,apoptosis,proliferation ability and cell cycle of liver cells were not significantly changed after OY-TES-1 overexpression,while the number of apoptotic cells increased.(2)Bioinformatics analysis suggested that OY-TES-1 is a soluble protein but not a membrane protein,which may be located in the nucleus or extracellular space;OY-TES-1 can be identified to 6 domains(domain)of Kazal-1,Kazal-2,PBP-sp32,TFIIF-alpha,DDHD and Plasmodium＿Vir,of these domains,only Kazal-1 and-2 were reported to correlate with the occurrence,development and drug sensitivity of variety of cancer.POU2F1 and NANOG have homeobox domain,which can be combined with the Kazal domain,so the POU family protein and NANOG may be one of the interacting proteins of OY-TES-1.(3)For the first time it was found that in hepatocellular carcinoma cells and tissues OY-TES-1 is exist in nucleus with the form of full-length protein.The regulation of OY-TES-1 on the malignant biological behavior of hepatocellular carcinoma may be carried out by its C-terminus.(4)The 1051 differentially expressed genes could be clustered into 10 functional sets(p<0.05)in HCC cells after OY-TES-1 downregulation.217 of1051 genes directly involved in cell proliferation,migration,apoptosis and cell cycle,of 217 genes,the expression of 98 genes was consistent with the malignant biological behavior by down-regulation of OY-TES-1 in liver cancer cells,14 genes have been reported related to cancer pathways;of 1051 genes,834 genes involved in the regulation of metal binding and chelation,ATPase the activity,protein localization,transmembrane transport and phospholipase activity;the 232 genes with exact function specifically divided into 12 functional clustering,including metallothionein,protease,transmembrane protein,ATPase,protein kinase,protein and zinc finger protein;77 differentially expressed genes involved in the regulation of 10 pathways,of which 28 genes regulated biological process of cell proliferation,migration,apoptosis and cell cycle;upregulation of metallothionein(MT)got the highest Enrichment Score and the minimum P value when OY-TES-1 down regulated,which showed that there is the most closely relationship between upregulation of MT and the down-regulation of the malignant biological behavior of liver cancer cells.(5)Successfully constructed and prepared OY-TES-1 interference,MT1 F interference and MT1 F over-expressing lentiviral vector,and infected liver cancer cells to construct some cell models of OY-TES-1 knockdown,OY-TES-1+MT1F double knockdown,MT1 F knockin and OY-TES-1knockdown+MT1F knockin;significantly decreased growth,proliferation and migration capability,S phase arrest,increased apoptosis were observed in 3 cell models of OY-TES-1 knockdown,MT1 F knockin and OY-TES-1knockdown+MT1F knockin;among these 3 groups,they array as OY-TES-1knockdown > OY-TES-1 knockdown+MT1F knockin > MT1 F knockin according their ability of inhibiting the malignant behaviors of liver cancer cell;in OY-TES-1+MT1F double knockdown group,the degree of cell proliferation and migration inhibition,apoptosis promoting and S arrest were lower than those of OY-TES-1 knockdown,MT1 F knockin and OY-TES-1knockdown+MT1F knockin groups.Conclusion(1)The upregulation of OY-TES-1 has no effect on the biological behavior of normal hepatocytes,OY-TES-1 may not be a gene related to the occurrence of HCC.(2)The domain of Kazal in the 6 domains of OY-TES-1 may be the main structural domain of OY-TES-1 affecting the malignant biological behavior of hepatocellular carcinoma.POU family proteins and NANOG may be the interacting proteins of OY-TES-1.(3)OY-TES-1 may exist in the form of the full-length protein in HCC cells and tissues,that was mainly distributed in the nucleus;C-terminus contains only Kazal-1,-2 domains,so the effect of OY-TES-1 on the malignant biological behavior of HCC may be through the Kazal domain of C terminus.(4)The regulation of OY-TES-1 on the malignant biological behavior of HCC cells is not only related to proliferation,migration,cell cycle and apoptosis,but also related to protein degradation,protein activation,epigenetic,structural integrity,transcriptional regulation.The finding showed that downregulation of OY-TES-1 weakening the malignant behavior of HCC cells,it is not only the result of expression changes of genes directly related to cell proliferation,cell cycle,cell death,cell migration and invasion,but also the result of combined effects of multi levels and multi processes,such as protein activity changes induced by metal ion binding or protein kinase,protein expression or degradation changes induced by transcription regulation or epigenetic change,and so on.(5)In liver cancer cell,inhibited cell proliferation,increased apoptosis,decreased migration and S phase arrest caused by downregulation of OY-TES-1may be mediated by upregulation of MT1F(or all MT-1).The regulation of OY-TES-1 on MT1 F in HCC cells is likely to be performed by regulating activity of POU2F1,the transcription factor of MT1F. |
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