Cross-talk Of Immunological Negative Regulator TIPE2and Nuclear Protein PCNP In Leukemia Pathogenesis

IntroductionLeukemia is a disorder from malignant clone of hematopoietic stem cell. Several factors could makethe white cells stop differentiating or maturating, therefore the white cells stay in different developmentalstages but the aberrant hematopoietic stem cells keep cloning limitlessly. Leukemia cells massivelyaccumulate in bone marrow and other hematopoietic organs, inhibit the function of normal hematopoieticcells. The functions of immune system of leukemia patients have been seriously restrained, result inpatients suffered from fever, bleeding, anemia, lately multiple organ failure, and even death. So the immunesystem functions’ recovery is important to improve the survival and life quality of patients with leukemia.Tumor necrosis factor-α-induced protein-8like2, abbreviated as TIPE2, is an important immune negativeregulate factor in recent study, it participate in many cancer’s occurrence and development. TIPE2is highlyexpressed in lymphocyte, suggesting that it maybe play an important role in lymphocyte’s normaldifferentiation and physiological function, whether the gene is related with leukemia’s occurrence anddevelopment has brought our attention.ObjectivesUnderstand the biological function of TIPE2, PCNP and JNK in the process of leukemia, explore therelationship among them.MethodsInsert the TIPE2’s CDS region into the over-expression vector pcDNA3.1(-), transfect it into leukemiaJurkat cells, then screen the stable cell lines by using G418positive culture medium. Detect other TNFAIP8family members’(TNFAIP8, TIPE1, TIPE3) expression by using fluorescence quantitative PCR method.The trypan blue staining and artificial counting method were used to detect cell’s growth condition, and theflow cytometry were used to determine cell cycle and apoptosis. Cell differentiation was tested by usingNBT reagent, and cell migration was checked by using the Transwell-Small-Room. Western blotting wasused to detect the expression of PCNP, JNK1, Caspase3and Caspase8protein after TIPE2-overexpressedResultsThe TIPE2over-expression recombinant plasmid was successfully constructed and stably transfected into Jurkat cells. The TIPE2’s mRNA expression in experiment group is more than30times higher than incontrol group (P <0.05), and TIPE2’s protein expression is also higher. QPCR results showed that TIPE3’sexpression was increased when we over-expressed TIPE2’s mRNA (P <0.05), but TNFAIP8and TIPE1were no effected (P>0.05). Trypan blue staining artificial count results showed that TIPE2’sover-expression can speed up the growth of Jurkat cell (P <0.05), but flow cytometry checking did not findsignificant difference in cell cycle and apoptosis (P>0.05). Western Blotting results also showed that thereis no more activated-Caspase3or activated-Caspase8, these two aspects indicate that TIPE2may be notaffect cell apoptosis. Cell differentiation has no obvious difference, too (P＞0.05). Using theTranswell-Small-Chamber, we found that TIPE2’s over-expression can enhance cell’s migration ability (P <0.05). In the protein level, PCNP’s expression was reduced (P <0.05), but JNK1’s expression wasincreased (P <0.05). Through PCNP and JNK1genes’ silencing or over-expressing, we found that PCNPcan regulate cell’s growth rate by affecting its cell cycle (P <0.05), but not apoptosis, differentiation ormigration (P>0.05). JNK1couldn’t regulate the cell growth, apoptosis, differentiation or migration (P>0.05). Western Blotting results showed that PCNP can influence the expression of TIPE2but not JNK1,meanwhile JNK1can influence the expression of PCNP but not TIPE2.Conclusions1. TIPE2can regulate the growth of Jurkat cell through PCNP and JNK1,but has no effect on cellapoptosis.2. PCNP can regulate the growth of Jurkat cell by regulating the cell cycle, but has no effect onapoptosis.3. The interaction among TIPE2, PCNP and JNK1:①T IPE2and PCNPnegatively regulate each other;②TIPE2positively regulate the expression of JNK1, which did not affect the expression of TIPE2;③JNK1negatively regulate the expression of PCNP, which has no effect on JNK1’s expression.