探討 YT 細胞株中調控 T-bet 之 microRNA

Abstract

Cytokines and transcription factors specific to the T helper (Th) cell lineages are crucial for driving the development of Th cell subsets via specific signaling pathways. The concept of type I vs. type II differentiation was extensively studied in Th cells. Two transcription factors, T-box expressed in T cells (T-bet) and GATA binding protein 3 (GATA-3), function as master regulators for the development of Th1 and Th2 cells, respectively. These subtype- specific transcription factors of Th cells involved in Th1- and Th2- specific cytokines expression by activating cytokine gene promoters. Because the differentiation of NK cells has not been well defined, we extended the concept of T cell differentiation to NK cells. For this reason, we used YT and NK92 lymphoma cell lines of NK cell origin to examine the role of T-bet and GATA3 in development process of NK cells. To investigate the role of transcription factors, T-bet and GATA3, in differentiation process of NK cells, we analyzed gene expression profiles of NK-92 and YT cell lines by microarray and Western blotting. We found that T-bet was obviously weak in the YT cell line despite strong mRNA expression. The expression pattern suggested that T-bet might be controlled at the level of translation in the YT cell line. In addition, we also performed Mass Spectrometry/MALDI-TOF and antibody mapping, and the results showed that T-bet could have isoforms. The preliminary results hinted that we should focus our research on posttranscriptional control of T-bet. The translational control of mRNA is usually regulated by elements in their 3’ or 5’-untranslated region (3’UTR/5’UTR). At first, we utilized different constructs of T-bet mutants to verify the translational block directed by the putative RNA pseudoknot of 5’UTR in YT cells; on the other hand, we found that T-bet protein was obviously up-regulated in the construct of 3’UTR deletion stable clone (Δ3’UTR-YT). The result suggested that the 3’UTR of T-bet plays an important role in the translational control. MicroRNAs (miRNAs) regulate gene expression by partially or completely base-pairing with the 3’UTR of their target mRNAs and repressing gene translation or inducing mRNA degradation. To examine the possibility of T-bet regulation by miRNAs, we searched the miRNA database for potential miRNA candidates targeting the 3’UTR of T-bet, and 29 Homo sapiens miRNAs were predicted as putative regulators of T-bet. We performed qRT-PCR to identify significant miRNAs whose expression was the most significant in YT, Δ3’UTR-YT and NK92 cell lines. We found 11 Homo sapiens miRNAs with significant expression diversity between YT and NK92 cells. miR-593* was up-regulated most and miR-133a was down-regulated most in YT cells. Moreover, we also analyzed the expression patterns of 39 EBV miRNAs because all the cell lines are EBV-positive. Interestingly, we found that each of the 12 significant EBV-miRNAs was much higher in YT cells than in NK92 cells. Recent studies have shown that viral-encoded miRNA may invole in the immunomodulatory mechanism. The further work will be done to elucidate the roles of these significant miRNAs in the process of posttranscriptional regulation of T-bet. The study of the structure-mediated and miRNA-directed T-bet regulation will be helpful for us to understand the highly complex pattern of gene regulation the development of NK cells.