3). The expression level of TLR-4 (Fig. 3c), but not Bcl-2 (Fig. 3b), was significantly lower in AS T cells than in normal T cells. Although miR-221 was over-expressed in AS T cells and its expression level was correlated significantly with BASRI of lumbar spine in AS patients, the expression of
c-kit was undetectable by Western blotting in Trichostatin A purchase both normal and AS T cells (data not shown). We speculated that miR-221 may play a physiological role in suppressing the protein expression of c-kit in T cells. Thus, we transfected miR-221 inhibitor or scrambled oligonucleotide into AS T cells. The expression level of miR-221 decreased dramatically (fold change: 0·035, P < 0·05) after miR-221 inhibitor transfection (Fig. 4a). However, the expression of c-kit remained undetectable by Western blotting (Fig. 4b). The above results suggest that increased expression of let-7i in AS T cells. We then analysed the expression of let-7i in other systemic autoimmune diseases, including patients with SLE
and RA, for identifying the specificity in AS T cells. We found that the expression of let-7i was not changed in T cells from these patients compared with controls (Fig. 5a). Another interesting finding is that let-7i expression in Jurkat cells was decreased after activation by ionomycin + PMA (Fig. 5b). This may indicate that activated T cells enhance TLR-4 expression. However, further investigation is required to confirm it. For confirming further the roles of let-7i on TLR-4 protein see more expression, we transfected let-7i mimic or scrambled oligonucleotides into Jurkat cells by eletroporation to detect the effects on TLR-4 mRNA and protein expression. The expression levels of let-7i increased dramatically (fold change: 395·78, P < 0·05)
after let-7i mimic transfection (Fig. 6a). Increased let-7i expression did not suppress the mRNA expression of TLR-4 in Jurkat cells (Fig. 6b), whereas the protein expression of TLR-4 in both Jurkat and normal T cells was suppressed significantly by Western blotting, as shown in Fig. 6c,d. Conversely, we Sorafenib manufacturer transfected let-7i inhibitor or scrambled oligonucleotides into Jurkat cells. As expected, the expression level of let-7i was decreased dramatically (fold change: 0·006, P < 0·05) after let-7i inhibitor transfection (Fig. 7a). The decreased let-7i expression did not increase TLR-4 mRNA expression significantly in Jurkat cells (Fig. 7b), but enhanced significantly the protein expression of TLR-4 in Jurkat and AS T cells, as shown in Fig. 7c,d by Western blotting. These results confirmed that let-7i inhibited protein translation rather than mRNA degradation of TLR-4 in Jurkat cells. Bacterial LPS, a TLR-4 agonist, has been proved to play a crucial role in AS pathogenesis , and José et al.