[miR-144 regulates BCG- and rapamycin-induced autophagy by targeting Atg4a in RAW264.7 cells]

To study the effect of Bacilli Calmette-Guerin (BCG) on miRNAs (miR-21, miR-181a, miR-155 and miR-144) in RAW264.7 cells, and with miR-144 as an example, to verify the relationship of miR-144 and Atg4a, an autophagy-related gene, and study the mechanism underlying the regulatory effect of miR-144 on autophagy in the process of Mycobacterium tuberculosis infection.

RAW264.7 cells were treated respectively with starvation (12 hours), 50 ng/mL rapamycin (2 hours) and 10 nmol/L 3-methyl adenine (3-MA, 12 hours), and then stimulated with BCG for 12, 24 or 48 hours. The macrophages were collected for total RNA extraction. The expression levels of miR-21, miR-181a, miR-155 and miR-144 were detected by real-time quantitative PCR (qRT-PCR). Thereafter, we constructed the recombinant plasmids pMIR-Report-Atg4a and pMIR-Report-Atg4a mut. The targeting effect of miR-144 on Atg4a gene was verified by the dual-luciferase reporter assay system, Western blotting and qRT-PCR.

After BCG stimulated RAW264.7 cells, the expression levels of miR-21, miR-155 and miR-144 were up-regulated, and miR-181a expression was down-regulated. The expressions of miR-21, miR-144, miR-155 and miR-181a increased 64 times, 52 times, 14 times and 1 times in the rapamycin group, respectively; the expressions of miR-21, miR-144, miR-155 and miR-181a decreased 1.22 times, 1.05 times, 1.54 times and 12.5 times in the 3-MA group, respectively. The dual-luciferase reporter assay system and Western blotting demonstrated that miR-144 could suppress Atg4a expression by targeting the specific 3'-untranslated region (3'UTR) sequence of Atg4a gene.

miR-144 can directly inhibit the autophagy-related gene Atg4a expression and participate in the regulation of autophagy process in Mycobacterium tuberculosis infection.