miR-7 inhibits activation of astrocytes derived from rats via silencing of EGFR / 中国药理学通报

ABSTRACT

Aim To explore the effects of miR-7 on astrocyte activation and the underlying mechanisms. Methods Following isolation and culturing, astro-cytes extracted from rat cortex were treated with culture solution (control group), ciliary neurotrophic factor (CNTF, an agonist of astrocyte activation), miR-7 mimic+CNTF, miR-7 mimic control+CNTF, miR-7 inhibitor+CNTF and miR-7 inhibitor control+CNTF, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA ex-pression of glial fibrillary acidic protein (GFAP) and epidermal growth factor receptor(EGFR). The protein expression of GFAP, EGFR, signal transducers and activators of transcription 3(STAT3) and phosphoryla-ted STAT3 (p-STAT3) was measured using Western blot. Wild type pGL3-EGFR and mutant pGL3-EGFR-m recombinant plasmids were constructed and then co-transfected with miR-7 mimic into HEK293T cells,re-spectively. The luciferase activity of reporter gene was measured. In addition,astrocytes were treated with ei-ther EGFR siRNA or S31-201 (an inhibitor of STAT3),followed by the incubation with miR-7 inhib-itor and CNTF. Both qRT-PCR and Western blot were subsequently used to detect the mRNA and protein lev-els of GFAP. Results The expression levels of GFAP and EGFR as well as p-STAT3/STAT3 ratio in CNTF group were higher than those in control group (P <0.01). When compared with CNTF group,GFAP and EGFR levels and p-STAT3/STAT3 ratio significantly decreased in miR-7 mimic+CNTF group but increased in miR-7 inhibitor+CNTF group(P<0.01). In com-parison with control group, transfection with miR-7 mimic markedly reduced the luciferase activity of wild type EGFR (P <0.01). Moreover, miR-7 inhibitor-induced up-regulation of GFAP expression was almost completely reversed by either EGFR siRNA or S31-201 pretreatment (P<0.01). Conclusion miR-7 antag-onizes the activation of astrocytes from rats by inhibi-ting the EGFR/STAT3 signaling pathway.