| Objective: TIPE2(tumor necrosis factor-α-induced protein8-like2), is arecently identified gene in experimental autoimmune encephalomyelitis miceby the University of Pennsylvania Professor Youhai H. Chen, which shares ahigh degree of sequence homology with TNFAIP8. TIPE2is a novel negativeregulator of innate and adaptive immunity that maintains immune homeostasis.TIPE2deletion in mice leads to multi-organ inflammation, splenomegaly andpremature death. TIPE2contains a death effector domain (DED) and is able tobinds to caspase-8, to inhibit TNFα-mediated apoptosis that it may play a rolein oncogenesis. Previous studies suggest that TIPE2gene is highly expressedin immune cells and immune tissue, such as lymph nodes, lymphocytes andmonocytes, and so on. Moreover it is also expressed in other non-immunecells, such as glandular epithelial cells, stratified or pseudostratified squamousepithelial cells as well as hepatocytes, neurons and chondrocytes. And someresearch about the characteristics of human TIPE2showed TIPE2may beinvolved in the occurrence and development of many diseases, but the specificmechanism was lagerly unknowen. Recently Luan et al has reported thatnaturally occurring CD4+CD25+Tregs isolated from murine spleens expressedTIPE2, and the TIPE2could enhance the immune suppressive activity ofTregs. The results suggested that TIPE2gene might be related to the immuneregulation mediated by Tregs, but the specific mechanism was unclear. Tregwas a group of immune suppression of T cell subsets, play a critical role inimmunologic self-tolerance, infection as well as antitumor immune responsesand transplantation. It is reported that Treg may secrete immunosuppressivefactors to directly inhibit effector T cell function. In many tumor diseases,extensive infiltration of Treg indicated poor prognosis and survival rate. Tregdeletion or deficency could effectively improve antitumor immunity. Human FOXP3, a member of the forkhead/winged-helix family of transcriptionalregulators, which has been identified as a specific marker of Tregs, couldregulated development, differentiation and maturation of Treg. FOXP3expression was not only in T-cell lines, many tumor cells lines (melanoma,glioma, etc.) also constitutively expressed FOXP3, suggesting that FOXP3contributed to tumor cell immunosuppressive activity. Considering normalexpression of TIPE2gene in the immune system could preventhyper-responsiveness and maintain immune homeostasis, we hypothesizedthat the TIPE2gene and FOXP3might be related to the pathogenesis ofprimary liver cancer (PLC). PLC is one of the most common malignanttumor worldwide, incidence of occult, rapid progress, poor prognosis, and thethird leading cause of cancer death worldwide. In recent years, its incidence isrising. Therefore, to fight cancer has become a worldwide problem.Tumorigenesis is the imbalance between malignant cell proliferation andapoptosis, and immune dysfunction or dysfunction of immune surveillancewas essential to formation and development of tumor. Therefore, the study aimto detect the expression of TIPE2gene and FOXP3in peripheral bloodmononuclear lymphocytes of PLC and liver cancer tissues, on the one hand, toinvestigate whether TIPE2is expressed in the PBMCs and liver cancer tissuesof PLC, on the other hand to explore the relationship between TIPE2andFOXP3in the pathogenesis of PLC, and further explore whether TIPE2regulated development of PLC.Methods1Human subjects and specimensAccording to the criteria of Experts consensus of standard diagnosis andtreatment in PLC in2009and exclude the influence of immunosuppressivedrug on patients, we enrolled60patients (Male/female,43/17) with an averageage of54.83±13.58years for this study, including40cases of PLC patientsand20cases of PLC patients with liver transplantation who were fromDepartment of Traditional and Western Medical Hepatology and HepatobiliarySurgery of the third Hospital of Hebei Medical University, respectively.30 healthy controls (Male/female,13/17) were also recruited with an average of39.23±11.53years.30cases of chronic hepatitis B patients,8cases of chronichepatitis C,5cases of alcoholic liver disease.3ml of anticoagulation bloodfrom all procedures was used to separate PBMCs, and5ml of vein blood wasobtained to separate serum. With its own matching method, The freshspecimens were collected from20cases of PLC patients with livertransplantation, including tumor tissues and adjacent tissues (3-5cm from thetumor tissues),8cases of well differention tumor tissues,7cases ofmoderately differention,5cases of poorly differention. The expression ofTIPE2and FOXP3mRNA in liver cancer tissue were analyzed by RT-PCR.The rest liver tissues were placed at-80℃freezer.2Quantification of TIPE2and FOXP3mRNA expression in PBMCsfrom PLC patients and healthy controls by RT-PCRPeripheral blood was sampled in sodium citrate-containing cellpreparation tubes. PBMCs were separated by Ficoll density gradientcentrifugation. Total RNA was extracted from PBMCs using Trizol. ThemRNA expression levels of TIPE2and FOXP3were analyzed by RT-PCR.The differences of each group were analysis by t test.3Quantification of TIPE2and FOXP3mRNA expression in liver cancertissues from PLC patients by RT-PCR20cases of the fresh specimens were collected from the patients withliver transplantation, including tumor tissues and adjacent tissues. Total RNAwas extracted from liver tissues using Trizol. The mRNA expression levels ofTIPE2and FOXP3were analyzed by RT-PCR. The differences of each groupwere analysis by t test.4Detection of peripheral blood T lymphocyte subsetsFresh anticoagulation blood were collected from all patients and healthycontrol. The levels of CD3+, CD4+, CD8+T lymphocyte, and CD4/CD8ratiowere analyzed by flow cytometry.5The correlation between TIPE2mRNA expression and clinicalparameters and FOXP3in PLC patients We analyzed the association of TIPE2expression with clinical parameters(AFP, AST/ALT, T lymphocyte subsets and tissues pathological results), andFOXP3by SPSS13.0software.Results1The characteristics of PLC patients and healthy control are summarizedin Table1.2The TIPE2mRNA expression levels in PBMCs and liver cancer tissuesfrom PLC patientsThe results showed that the positive detection rate of TIPE2mRNA (75%)in PBMCs from PLC patients was significantly increased than that in healthycontrol (100%). There was significant difference (P<0.05). The mRNA levelsof TIPE2in PBMCs was dramatically decreased in PLC patients than healthycontrol [0.33(0.39) vs1.16(0.05), P<0.05].The positive detection rate of TIPE2mRNA in tumor tissues and adjacenttissues was75%respectively, there was no significantly difference (P>0.05).While the TIPE2mRNA expression was decreased in tumor tissues thanadjacent tissues (0.25±0.19vs0.26±0.20), but there was no significantdifference (P>0.05).3The FOXP3mRNA expression levels in PBMCs and liver tissues fromPLC patientsThe results showed that the positive detection rate of FOXP3mRNA(92.5%) in PBMCs from PLC patients was significantly increased than that inhealthy control (46.67%). There was significant difference between twogroups (P<0.001). The mRNA levels of FOXP3in PBMCs was dramaticallydecreased in PLC patients than healthy control [5.17(3.13) vs1.10(2.92),P<0.001].The positive detection rate of FOXP3mRNA in tumor tissues andadjacent tissues was85%respectively, there was no significantly difference(P>0.05). While the FOXP3mRNA expression was decreased in tumor tissuesthan adjacent tissues (3.72±2.03vs2.54±1.58). There was significantdifference (P<0.05). 4The correlation between TIPE2mRNA expression and clinicalparametersAccording to the value of AFP and AST/ALT, PLC patients were dividedinto AFP low group (AFP≤400ng/ml) and high group (AFP>400ng/ml). AndAST/ALT low group (AST/ALT≤1), moderate group (1<AST/ALT<2) andhigh group (AST/ALT≥2). Non-parametric Wilcoxon rank sum test was usedto determine the significance of the differences in TIPE2, AFP, AST/ALT. Thelevel of TIPE2mRNA in low groups was much higher than that in high groups.The results were expressed as Median (Interquartile Range):[0.41(0.33) vs0.14(0.13),0.68(0.09) vs0.42(0.17) vs0.08(0.15), P<0.01]. Statistical analysisshowed that there was a negative correlation between the mRNA level ofTIPE2and the value of AFP (r=-0.950, P<0.001). Similar relationship existedfor TIPE2and AST/ALT (r=-0.982, P<0.001).According to the Edmondson-Steiner grade: histopathology resultsshowed:8cases of grade I: well differention;7cases of grade II: moderatelydifferentiated;5cases of grade III: poorly differentiated. The mRNA levels ofTIPE2in each group as follows: grade I (0.42±0.11, P<0.05) vs grade II(0.28±0.13, P<0.05), grade III were not expressed TIPE2.5Detection of T cell subsets in PLC patients and the correlation betweenTIPE2and T cell subsetsThe results revealed that the value of CD3+, CD4+T cell and CD4/CD8ratio were significantly decreased, while CD8+T cell were significantlyincreased in PLC patients.(CD3+,64.63±7.13vs70.97士3.80; CD4+,31.61±4.96vs44.66士3.62; CD8+,31.56±5.27vs25.63士4.03; CD4/CD8,1.05±0.33vs1.78士0.34P<0.001).6The correlation between TIPE2, FOXP3and T cell subsets, respectivelyStatistical analysis showed that there was a positively correlation betweenthe mRNA level of TIPE2and the value of CD3+, CD4+T cell and CD4/CD8ratio in PB (r=0.658, P<0.001; r=0.580, P<0.01; r=0.655, P=0.001), but therewas a negatively correlation between the mRNA level of TIPE2and CD8+Tcells (r=-0.635, P=0.001). Contrary, there was a negatively correlation between the FOXP3level inthe PBMCs and the value of CD3+, CD4+T cell and CD4/CD8ratio (r=-0.640,P=0.001; r=-0.642, P=0.001; r=-0.697, P<0.001), but there was a positivelycorrelation between the FOXP3level and CD8+T cells (r=0.677, P<0.001).7The correlation between TIPE2mRNA and FOXP3mRNA expressionStatistical analysis showed that there was a negatively correlationbetween the mRNA level of TIPE2and the value of FOXP3in PBMCs fromPLC patients (r=-0.940, P <0.01); Similar relationship existed for TIPE2andFOXP3in liver tissues (tumor tissues, r=-0.681, P=0.001; adjacent tissues, r=-0.705, P=0.001). There was a significant difference between each group.Conclusion1TIPE2expression was either lower expression or partly lost in PBMCsand liver cancer tissues from PLC patients.2The mRNA levels of TIPE2in PBMCs was dramatically decreased inPLC patients than healthy control (P<0.05). The TIPE2mRNA expression wasdecreased in tumor tissues than adjacent tissues, but there was no significantdifference (P>0.05), it may relate to the amounts of patients.3The FOXP3mRNA levels in PBMCs was dramatically decreased inPLC patients than healthy control (P<0.001). The FOXP3mRNA expressionwas decreased in tumor tissues than adjacent tissues (P<0.01).4TIPE2may control the pathogenesis of PLC through negativelyregulating immune cell activation and apoptosis. |
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