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. 2015 Nov 5;6(11):e1960. doi: 10.1038/cddis.2015.267

Figure 3.

Figure 3

The expression of NLC1-C is inhibited by miR-320a and miR-383. (a and b) Knockdown of Dicer and Ago2 increased NLC1-C expression. NT2 cells were transfected with control siRNA (si-NC) or Dicer or Ago2 siRNAs separately. The expression of NLC1-C was detected by quantitative real-time PCR after transfection for 48 h. β-Actin was used as an internal reference. (c) Immunoblotting analysis of the interaction of NLC1-C with Ago2. Proteins from NT2 cells extracts were pulled down with the biotinylated RNAs, subjected to SDS-PAGE, and analysed by immunoblotting with Ago2 antibody. (d) NLC1-C interacts with Ago2 in vivo. Ago2 was immunoprecipitated from untreated NT2 cells and co-precipitated RNAs were detected by qRT-PCR using primers for NLC1-C and β-actin (as negative control). IP enrichment was determined as the amount of RNA associated in the Ago2 IP relative to IgG control. (e) The sequences of miR-320a and miR-383 complement with NLC1-C. (f and g) The expression of NLC1-C was repressed by miR-320a and miR-383. Quantitative real-time PCR analysis of NLC1-C expression after NT2 and NCCIT cells were transfected with indicated miRNAs for 48 h. Data are represented as mean±S.D. from three independent experiments. *P<0.05 or **P<0.01 compared with control RNA. (h). The expression of NLC1-C was repressed by miR-320a and miR-383. Total RNAs were collected from NT2 and NCCIT cells transfected with the indicated miRNAs individually and then resolved on a denatured agarose gel. Transcripts of different types of expressed RNAs were probed with Dig-labelled antisense NLC1-C and β-actin. β-Actin was used as an internal reference Equal amounts of total RNAs were loaded onto an agarose gel and rRNAs were used as the loading control. (i) miR-320a affected the interaction of Ago2 with NLC1-C in NT2 cells. Ago2 was immunoprecipitated from NT2 cells transfected with the indicated miRNAs and co-precipitated RNAs were detected by qRT-PCR using primers for NLC1-C and β-actin (as negative control). IP enrichment was determined as the amount of RNA associated in the Ago2 IP relative to IgG control. Data are represented as mean±S.D. from three independent experiments. *P<0.05 compared with control RNA. (j) The structure of pMIR-REPORT miRNA Expression Reporter Vector. (k) miR-320a and miR-383 repressed NLC1-C luciferase activity and the mutation rescued the luciferase activity