title: [miR-144 regulates BCG- and rapamycin-induced autophagy by targeting Atg4a in RAW264.7 cells].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  miR-144 can directly inhibit the autophagy-related gene Atg4a expression and participate in the regulation of autophagy process in Mycobacterium tuberculosis infection.
authors: nstitute of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors: Linlin
authors: L
authors: Zhou
authors: Institute of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors: Le
authors: L
authors: Guo
authors: Institute of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors: Jianzhong
authors: J
authors: Tang
authors: Institute of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors: Aijun
authors: A
authors: Zhang
authors: Institute of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors: Xueying
authors: X
authors: Liu
authors: Institute of Laboratory Medicine, Ningxia Medical University, Yinchuan 750004, China.
authors: Guangxian
authors: G
authors: Xu
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects; D001500:BCG Vaccine / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D014376:Tuberculosis / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology / Q000503:physiopathology
chemical_list: D001500:BCG Vaccine; C551064:MIRN144 microRNA, mouse; D035683:MicroRNAs; C000599119:Atg4a protein, mouse; D003546:Cysteine Endopeptidases; D020123:Sirolimus