miR-1303 regulates autophagy related gene Atg2B expression.

<p><b>(A)</b> PBMacs were transfected with miR-1303 mimics (40 nM) or negative control mimics (40 nM) for 24 hrs, followed by BCG (MOI = 1) treatment for 24 hrs. Cell lysates were examined by Western blot using anti-Atg2B or anti-actin antibodies, as indicated. The results shown are representative of independent experiments using PBMac from four individuals. The ratio of Atg2B/actin in each sample was compared with that in cells treated with Mock and negative control mimics. <b>(B)</b> PBMacs were transfected with miR-1303 mimics (40 nM) or negative control mimics (40 nM) for 24 hrs, followed by BCG (MOI = 1) treatment for 1 hr. Total RNA was extracted and reverse-transcribed into cDNA. The Atg2B mRNA levels normalized to that of GAPDH were analyzed by quantitative PCR using the cDNA. The Atg2B mRNA levels in cells were expressed as fold change to those in Mock-negative control mimics-treated cells. The data are expressed as the mean ±SEM of independent experiments using PBMacs from eight individuals. n.s., not significant. <b>(C)</b> Schematic representations of the miR-1303 putative target sites (sensor) and mutated target sites (mutant) of the 3’UTR of Atg2B which were cloned into the pGL-3 luciferase control construct. <b>(D)</b> HEK293T/17 cells were transfected with empty pGL3 luciferase control vector (control) or pGL3 luciferase control vector with miR-1303 putative target sites on Atg2B (sensor) for 4 hrs and then transfected with negative control mimics or miR-1303 mimics (40 nM) for 14 hrs. pRL-TK renilla luciferase reporter vector were transfected as transfection control. Luciferase activities of cell lysates were assayed using Dual-Luciferase Reporter assay System. The luciferase activity ratio of pGL3/pRL-TK in each sample was compared with that in cells transfected with control and negative control mimics. The data are expressed as the mean±SEM of six independent experiments. n.s., not significant. *, p<0.05. **, p<0.01. <b>(E)</b> HEK293T/17 cells were transfected with empty pGL3 luciferase control vector (control) or pGL3 luciferase control vector with miR-1303 mutated target sites on Atg2B (mutant) for 4 hrs and then transfected with negative control mimics or miR-1303 mimics (40 nM) for 14 hrs. pRL-TK renilla luciferase reporter vector were transfected as transfection control. Luciferase activities of cell lysates were assayed using Dual-Luciferase Reporter assay System. The luciferase activity ratio of pGL3/pRL-TK in each sample was compared with that in cells transfected with control and negative control mimics. The data are expressed as the mean±SEM of four independent experiments. n.s., not significant.