Figure 6. miR619 represses PolH expression by inhibiting the long PolH transcript cleaved at PAS-4 site.

(A) Schematic diagrams illustrating the binding sites of miR619 in the 3’UTR of PolH mRNA. (B) A549 cells were transfected with miRNA control or miR619 for 3 days, followed by qRT-PCR to examine the level of total and long PolH transcript. The level of short PolH transcripts is determined by subtracting the PCR amplicons of long PolH transcripts from the ones of total PolH transcript. The ratio of short vs long PolH transcript is calculated by dividing amplicons of the short PolH transcript by the ones of long PolH transcript. (C) The experiments were performed the same as in (B) except that anti-miR control and anti-miR619 were used. (D) The levels of PolH and actin proteins were examined by western blot analysis using lysates from A549 cells transfected with control miRNA or miR619 for 3 days. (E) A549 cells transfected with control miRNA or miR619 for 3 days, followed by RT-PCR analysis to examine the levels of PolH and actin transcripts. (F) The levels of PolH and actin proteins were examined by western blot analysis using lysates from A549 cells transfected with anti-miRNA control or anti-miR619 for 3 days. (G) A549 cells were transfected with anti-miRNA control or anti-miR619 for 3 days, followed by RT-PCR analysis to measure the levels of PolH and actin transcripts. (H-I) Colony formation assay was performed with isogenic control and PolH-KO A549 cells transfected with control miRNA or miR619, ant then quantified using ColonyArea software plugin (I). (J) The level of miR619 was examined by qRT-PCR in various lung cancer cell lines.