Figure 1. Reversal of amino acid-starvation-induced stress increases miR-122 biogenesis in Huh7 cells.

(a,b) Outline of the experimental set-up used (a). Relative level of CAT-1 mRNA in Huh7 cells either fed or starved for 4 h and re-fed with media containing amino acids for another 2 h. CAT-1 mRNA levels were quantified by quantitative reverse transcriptase-PCR with levels in Fed cells taken as 1 (b). (c) Effect of starvation and re-feeding on mature miR-122 levels in Huh7 cells. Total RNA was extracted and 8 μg RNA was used for northern blotting of mature miR-122, let-7a and miR-16. U6 snRNA was used as loading control. (d) Copy number/number of molecules of mature miR-122 and CAT-1 mRNA per Huh7 cell calculated in fed, starved and re-fed Huh7 cells. Estimations were done by real-time-based methods. (e) Increase in mature miR-122 but not that of other non-relevant miRNAs, miR-16, miR-21, miR-24 and miR-125b on relief of starvation. Real-time PCR-based quantification of mature miRNA levels in Huh7 cells starved for amino acids (4 h) and subsequently re-fed (2 h). (f) Increase in mature miR-122 is accompanied by a concomitant decrease in pre-miR-122 on re-feeding the starved cells for 2 h. Cellular small RNA population was isolated by mirVANA kit to minimize possible contamination of pri-miR-122 and real-time-based assays were carried out to quantify pre-miR-122. Pre-miR-122 detected by northern blotting with 15 μg total RNA. Synthetic pre-miR-122 was used as a size marker to determine the position of the pre-miR-122 in the northern blotting. (g) Increase of mature miR-122 on re-feeding of starved cells is reduced in DICER1 knockdown Huh7 cells. miR-16 and miR-21 did not show any significant change. siDICER1-mediated knockdown of DICER1 is confirmed by western blotting. Paired two-tailed Student's t-tests were used for all comparisons. *P<0.05, **P<0.01 and ***P<0.001. Error bars represent s.d. (n⩾3).