| [Background]It has been reported that some viral proteins in DNA tumor virus such as EBV/HBV can combine with the cell proteins, in which the proteins inhibiting cell proliferation and promoting cell differentiation in the host cell should be inactivated and the host cell can be resulted in malignant transformed. We previously identified a short palate, lung and nasal epithelium clone gene (SPLUNC1), which was a tissue-specific gene of nasopharyngeal epithelia. We also found that SPLUNC1was down-regulated in nasopharyngeal carcinoma (NPC) and proposed that SPLUNC1was a tumor suppression gene candidate. Our previous studies showed that SPLUNC1could accelerate the lysis and apoptosis of EBV infected cells and initiated antibody-dependent cell-mediated cytotoxicity, which participated in clearance process of EBV and also decreased the expression of EBV encoded Latent membrane protein1(LMP1) and consequently inhibited the tumorgencity of EBV. From the differential analysis of the microRNA (miRNA) expression profiles of over-expressed SPLUNC1NPC cells, miR-141expression could be regulated by SPLUNC1in NPC cells. Bioinformatics analysis revealed that probably phosphatase and tensin homolog deleted on chromosome ten (PTEN) was one of the target genes of miR-141. And then whether PTEN and its downstream molecules could be regulated by miR-141and whether EBV have effects on the expression of SPLUNC1is yet unknown.In this study, we have indentified SPLUNC1as a negative regulatory molecule of LMP1, and the expression of SPLUNC1also can be down-regulated by EBV infection in NPC cells. We have also showed that SPLUNC1can regulate PTEN and Akt signaling pathway through targeting miR-141and that LMP1could regulate the Akt signaling pathway by down-regulating SPLUNC1.[The carcinogenesis of EBV can been inhibited by SPLUNCl,in turn, EBV also suppresed the expression of SPLUNC1in NPC.]We employed co-culture system between B cells and NPC cells to obtain EBV-infected NPC cells and detected the expression level of key genes of EBV in NPC cells by RT-PCR. The results revealed that SPLUNC1could inhibit the expression of EBV related-genes, such as EBER, BZLF and LMP1. We also found that SPLUNC1increased responsively one day after that SPLUNC1-overexpressing HNE2cells contacted with EBV. However, the mRNA level of SPLUNC1decreased gradually after2days. The abovementioned results indicate that SPLUNC1can inhibit the expression of EBV key genes, and also the level of SPLUNC can be regulated by EBV infection.[SPLUNC1regulates NPC cell proliferation or apoptosis through miR-141-PTEN-Akt pathway]Previous analysis of miRNA expression profiles showed that miR-141was down-regulated by SPLUNC1. To confirm the result, we detected the expression of miR-141after SPLUNC1was over-expressed or knocked down. The results showed that SPLUNC1actually could decrease the expression of miR-141. We also used qRT-PCR to measure miR-141and SPLUNC1mRNA levels in NPC tissue samples. miR-141showed increased expression levels in NPC tissues compared with nasopharyngeal epithelial tissues, whereas SPLUNC1showed an inverse expression pattern. And the expression of miR-141negatively correlated with SPLUNC1.Bioinformatics software predicted that miR-141could bind to the3’ untranslated region (UTR) of PTEN. We also demonstrated that the expression of PTEN could be down-regulated by miR-141. Meanwhile, expression levels of PTEN were also found to be down-regulated in NPC tissues in contrast to nasopharyngeal epithelial tissues.Given abovementioned results, we explored the expression of PTEN in SPLUNC1-over-expressed NPC cells and NPC cells treated with ATAR. We demonstrated that SPLUNC1could increase the expression of PTEN, but not have effects on localization of PTEN. These results were furtherly confirmed by the knockdown of SPLUNC1. To determine whether SPLUNC1could regulate the expression of PTEN through targeting miR-141, we assessed the expression of PTEN in SPLUNC1-overexpressed NPC cells, which were transfected with the miR-141mimics. The result showed that the up-regulation of PTEN by SPLUNC1was partially involved with miR-141.Considering that PTEN be participated in the signaling pathway of proliferation and apoptosis mainly through regulating Akt pathway, we assessed the activation of Akt in SPLUNC1-overexpressed NPC cells and revealed that Akt phosphorylation were inhibited by SPLUNC1. The downstream molecule, phosphorylated MDM2, was also inhibited by SPLUNC1, but GSK3β and total MDM2. Then we used the PTEN inhibitor BVP and found that the activity of BVP was partly inhibited by SPLUNC1.Next we detected that SPLUNC1can result in tumor cell growth arrest at the G1to S transition of the cell cycle, increase critical cell cycle regulatory proteins p27expression significantly and decrease the expression of cyclin D1, D2, D3, E2, and cyclin-dependent kinase2(CDK2). We revealed that SPLUNC1could efficiently inhibit the BVP-induced proliferation. We also observed that SPLUNC1could inhibit tumorgenesis through PTEN-Akt pathway in vivo.[SPLUNC1is involvement in the miR-141-PTEN-Akt pathway and its expression interference by EBV/LMP1]To clarify the mechanism that SPLUNC1was down-regulated by EBV, We determined the effects of EBV major tumorgenetic gene LMP1on the expression of SPLUNC1. The results showed that LMP1could inhibit the expression of SPLUNC1, up-regulate the level of miR141. And then PTEN expression was decreased and the Akt pathway was activated. However, when SPLUNC1was re-expressed in the LMP1-overexpressed cells, LMP1expression was inhibited and PTEN was increased again.In conclusion, the carcinogenesis of EBV could be inhibited by SPLUNC1, and in turn, EBV and its major tumorgenetic gene LMP1also could suppress the expression of SPLUNC1in NPC cells. SPLUNC1could regulate NPC cell proliferation or apoptosis through PTEN-Akt pathway by targeting miR-141. Thus EBV/LMP1regulated cell progression and apoptosis probably through SPLUNC1and its downstream molecules. |
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