The Effect Of Nuclear Protein PCNP On The Migration And Proliferation Of Murine Dendritic Cells

BackgroundPCNP(PEST-containing nuclear protein) was one kind of nuclear protein that containing a PEST sequence, which was first discovered in 2002. It was studied only as a substrate of the Ubiquitin ligase NIRF(Np95/ICBP90-like RING figer protein) in the two reports. NIRF contained an ubiquitin-like domain, an YDG/SRA domain, a PHD domain and a RING finger domain. The high expression of NIRF was found during normal cell proliferation period, and the low expression of NIRF was found during the G0/G1 period. PEST sequence is rich with the proline(P), glutamic acid(E), serine(S) and threonine(T). The short-lived protein contains the PEST sequence. The functions of these proteins are generally negative controlled by hydrolysis, and mostly through the ubiquitin-mediated degradation. The high expression was also found in tumor cells, for example in HepG2, HT-1080. NIRF with the characteristics of ubiquitin ligase has automatic ubiquitin enzyme activity. The degradation of ubiquitin-dependent protein could regulate a variety of cell cycle processes, including cell cycle progression, DNA damage and mutation and protein transport. The ubiquitin ligase could combine with the specific substrates of the degraded protein PCNP, which affect the function and efficiency of the ubiquitin reaction. PCNP as one kind of nuclear protein can easily be ubiquitinated in 293 T and COS-7 cells. PCNP could be ubiquitinated by NIRF in vivo or in vitro. It is suggested that the NIRF and PCNP signaling axis may play a role in regulating the cell proliferation by the regulation of cell cycle. NIRF gene segment 9p23-24.1 gene were more frequent in many types of tumor cells than that in normal cells, therefore we speculated PCNP and NIRF might have ties with tumor.Dendritic cells(DC) was known as the most powerful professional antigen-presenting cells(APC) in the human body, which was the only antigen-presenting cell to activate naive T cell. The number of DC cells with immature migration capability is tiny. DC cells account for only 1% of mononuclear cells. A trace amount of LPS could stimulate the synthesis and release of cytokines(TNF-α). Our previous study found that the mRNA of PCNP was in a high level of expression in CD8α + DC cells in the spleen of the mouse. It was also found PCNP gene silencing inhibited the proliferation in the DC cells. Currently, the study on PCNP in DC cells is not reported. In this study we try to explore the mechanism of nuclear protein PCNP in DCs. We might provide a better understanding of immune regulation of DCs. Objective 1. To explore the effect of PCNP over-expression and PCNP silencing on the biological function of DC cells. 2. To investigate the effect of treatment factors(LPS and proteasome inhibitor MG – 132) on the biological function of DC cells. Methods1. DC group(DC2.4, the cell line for DC) without treatment: By flow cytometry test, we analyzed the expression of MHC II and costimulatory molecules CD80, CD86 in DC- PCNP-Flag, DC-sh- PCNP and their corresponding Negative Control groups. By Transwell chamber experiments, we studied the cell migration of DC cells transfected with PCNP over-expression and PCNP silencing respectively. By plate cloning assay, we tested the DC cell Colony-forming ability after PCNP silencing and over-expression respectively.2. DC group with treatment:In DC2.4 cells, we added the LPS and proteasome inhibitors(MG- 132) respectively. We observed the morphology of DC cells and studied the colony-forming of DCs cells. We observed the migration using Tran well experiment. Results1. PCNP-Flag and PCNP-EGFP over-expression stable strain was successfully built.2. The stable strain for over-expression of DC-Flag(Negative Control group), DC-PCNP-Flag, DC-EGFP(Negative Control group), DC-PCNP-EGFP, and the stable strain for silencing of DC-RNAi-Negative Control(Negative Control group NC) and DC-sh-PCNP were successfully screened.3. The DC 2.4 morphology had no change when we compared DC-PCNP-Flag stable strain with the Negative Control group; or compared DC-sh-PCNP stable strain with the Negative Control group.4. By flow cytometry test, there was no statistical significance in expression of MHC II surface molecules and costimulatory molecules CD80, CD86 between DC-PCNP-Flag stable strain and the Negative Control group; Compared DC-sh-PCNP stable strain with the Negative Control group,there was no statistical significance in the amount of expression of specific surface costimulatory molecules CD80, CD86 and MHC II.5. By the cell migration assay(cell scratches and Transwell chamber experiments), we found that the migration of DC stable strain with PCNP-Flag decreased, compared with that of Negative Control group(P <0.05), and the migration of DC stable strain with sh-PCNP was enhanced, compared with that of Negative Control group(P <0.01). So, it suggested that PCNP has an effect on the migrate ability of DC cells.6. Plate cloning experiments suggested that PCNP gene silencing inhibit proliferation in the DC cells(P<0.05), whereas PCNP over-expression promoted cell colony-forming ability(P<0.01). So, it suggested that PCNP has an effect on the colony-forming ability of DC cells.7. After adding LPS in DC cells, the migration ability of DC cells were promoted in both PCNP silence group and PCNP over-expression group. There was no difference between the two groups(P>0.05). After adding the MG-132 in DC cells, the migration ability of DC cells were inhibited in both PCNP silence group and PCNP over-expression group P There was no difference between the two groups(P> 0.05). LPS and MG-132 had opposite influence on the migration ability of DC cells. The result indicated that LPS can increase DC migigration, but PCNP is not involved in the migration in LPS treated DC cells.8. After adding LPS in DC cells, the colony-forming ability of DC cells were promoted in both PCNP silence group and PCNP over-expression group. There was no difference between the two groups(P>0.05); After adding the MG-132 in DC cell, the colony-forming ability of DC cells were inhibited in both PCNP silence group and PCNP over-expression group. There was no difference between the two groups(P>0.05). LPS and MG-132 had opposite influence on the colony-forming ability of DC cells(P < 0.01).The result indicated that LPS can increase DC colony-forming ability, but PCNP is not involved in the colony-forming ability in LPS treated DC cells. MG-132 can decrease DC colony-forming ability, but PCNP is not involved in the colony-forming ability in LPS treated DC cells. Conclusions1. Under no LPS stimulation(immature DCs), PCNP has no effect on the morphology and expression of surface molecules of DC cells, but it can suppress migration and promote colony-forming ability of DC cells.2.Under LPS stimulation(mature DCs), PCNP is not involved in the migration, colony-forming ability of DC cells.3. Proteasome inhibitors(MG-132) can inhibit the migration and colony-forming ability of DC cells and LPS is able to promote the migration and colony-forming ability of DC cells.