Figure 1

miR-375 targets HPV type 16 and 18 transcripts and suppresses E6 and E7 expression. (A) Five putative miR-375 binding sites on HPV16 transcript sequence. Integrated HPV16 viral gene produces polycistronic transcripts consisting of E6, E7, and E1. Two putative sites on E7 region and three putative sites on E1 region of HPV16 transcript were predicted to interact with miR-375. Lines between nucleotides of HPV16 sequence and miR-375 are typical Watson–Crick interactions (A-U and G-C, respectively), and colons are weak, nontypical base-pair interactions. Corresponding specific seed binding site mutants were generated for dual luciferase reporter assays. (B) FL repression effect was analyzed with FL construct that contains both E7 and E1 regions. Five putative miR-375 binding sites were mutated to examine their direct interaction. Cells were cotransfected with miR-375-mimic or non-specific control (NS). Renilla luciferase was used as an internal control to normalize FL activity expression. Results were compared to NS control transfected cells for statistical analyses. (C) Real-time PCR analyses of endogenous E6 and E7 mRNA levels in SiHa (HPV16-positive cervical cancer cell line) transfected with miR-375-mimic. (D) Measurement of endogenous E6 and E7 mRNA level in UPCI:SCC090 (HPV16-positive oropharyngeal cancer cell line) transfected with miR-375-mimic. Final concentration of 25 nM was used for miR-375 inhibitor and NS control, and 50, 100, 200 nM were used for miR-375-mimic transfection. (E) Expression changes in E6 and E7 mRNA in HeLa (HPV18-positive cervical cancer) after miR-375-mimic transfection. Final concentration of 25 nM was used for miR-375 inhibitor, miR-375-mimic, and NS control for transfection. White and black bars indicate E6 and E7 mRNA levels, respectively. Results are expressed as mean ± SD from at least three independent experiments. **p < 0.01 and ****p < 0.0001.