OBJECTIVE: To explore the effect and mechanism of miR-30b on cisplatin-resistance of human NK/T cell lymphoma lines SNK-6 and YTS cells. METHODS: Normal NK cells, SNK-6 and YTS cells were cultured, the expression levels of miR-30b and macrophage-derived chemokine (CCL22) were detected by real-time PCR assay, and the CCL22 expression was detected by Western blot. The SNK-6 and YTS cells were transfected with miR-30b mimics and inhibitor respectively, then the effect of cisplatin resistance in SNK-6 and YTS cells was measured by MTT assay, the activity of caspase-3 was detected by caspase-3 assay kit, and the cell apoptosis was analyzed by flow cytometry. Dual-luciferase reporter gene assay was used to determine the targeting relationship between miR-30b and CCL22. Furthermore, the effect of CCL22 on cisplatin-resistance and caspase-3 actirity was also evaluated. RESULTS: Compared with the normal NK cells, the expression levels of miR-30b significantly decreased in both SNK-6 and YTS cells (P＜0.01), but CCL22 mRNA expression increase in both cells (P＜0.01). MiR-30b mimics decreased the cell activity (P＜0.05), down-regulated the cisplatin-resistance (P＜0.05), and increased cell apoptosis and caspase-3 activity (P＜0.05). The effects of miR-30b inhibitor were contrary to the mimics. Up-regulation of miR-30b expression significantly decreased the luciferase activity in CCL22 3'-UTR-transfected NK cells, but not in Mut-CCL22 3'UTR group, suggesting that CCL22 could act as a direct target of miR-30b. The expressions of CCL22 pathway proteins were down-regulated after SNK-6 cells transfected with miR-30b mimics (P＜0.05), while this effect was restored by overexpression of CCL22. Moreover, CCL22 overexpression also increased the cell activity and decreased caspase-3 activity when SNK-6 cells were transfected with miR-30b mimics. CONCLUSION MiR-30b inhibits cisplatin-resistance of human NK/TCL SNK-6 and YTS cells by targeting CCL22.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Chemokine CCL22 ; Cisplatin ; Gene Expression Regulation, Neoplastic ; Humans ; Killer Cells, Natural ; Lymphoma, T-Cell ; genetics ; MicroRNAs ; T-Lymphocytes

Aminoquinolines ; pharmacology ; therapeutic use ; Animals ; Asthma ; drug therapy ; immunology ; pathology ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Count ; Chemokine CCL17 ; Chemokine CCL22 ; Chemokines, CC ; analysis ; genetics ; Cytokines ; biosynthesis ; Flow Cytometry ; Interleukin-4 ; antagonists & inhibitors ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; STAT6 Transcription Factor ; analysis ; antagonists & inhibitors ; genetics ; Signal Transduction ; drug effects

Th-2-biased immune responses are known to play a key role in the pathogenesis of atopic dermatitis. In particular, the macrophage-derived chemokine CCL22 is directly implicated in Th-2-associated skin inflammatory reactions, and its levels are significantly elevated in serum and are correlated with disease severity in atopic dermatitis. In this study, we tested the development of genetic therapeutic options to treat atopic dermatitis using bacteria expressing miRNA. We constructed a recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) for the in vivo knockdown of CCL22. The CCL22 gene was downregulated with CCL22 miRNA in activated lymphocytes. In mice with a cutaneous disease similar to atopic dermatitis, interleukin-4 was inhibited and interferon-gamma was induced after treatments with ST-miRCCL22. Furthermore, CCL22 levels were suppressed in the atopic mice treated with ST-miRCCL22. These results suggest that ST-miRCCL22 may be an effective genetic agent for treating atopic dermatitis.
Animals ; Cell Line ; *Chemokine CCL22/genetics ; Cytokines/blood ; Dermatitis, Atopic/pathology ; Disease Models, Animal ; Female ; Gene Expression Regulation/drug effects ; Gene Silencing ; Immunoglobulin E/blood ; Mice ; *MicroRNAs/genetics/pharmacology ; *Organisms, Genetically Modified/genetics ; *Salmonella typhimurium/genetics/metabolism ; Skin/*drug effects/pathology