vIRF1 suppresses miR-218-5p via hypermethylation of the pre-miR-218-1 promoter by inhibiting p53 to increase DNMT1 expression.

(A). Western-blotting of DNMT1 expression in vIRF1-transduced HUVECs. (B). Western-blotting of DNMT1 in KSHV-infected HUVECs. (C). Methylation-specific PCR showing DNA methylation of the pre-miR-218-1 promoter in vIRF1-expressing HUVECs transfected with a mixture of siRNAs targeting DNMT1 (siDNMT1). (D). qPCR showing DNMT1, SLIT2, pre-miR-218-1 and miR-218-5p expression in vIRF1-expressing HUVECs transfected with a mixture of siRNAs targeting DNMT1 (siDNMT1). (E). Western-blotting of DNMT1, HMGB2 and CMPK1 expression in vIRF1-expressing HUVECs transfected with a mixture of siRNAs targeting DNMT1 (siDNMT1). (F). Western-blotting of DNMT1, HMGB2 and CMPK1 expression in vIRF1-expressing HUVECs transfected with pCMV6-Entry-C-Myc-p53 construct. The Western blots were ran with the same samples but in two different gels, with each of them calibrated by independent GAPDH panels. (G). Methylation-specific PCR showing DNA methylation of the pre-miR-218-1 promoter in vIRF1-expressing HUVECs transfected with pCMV6-Entry-C-Myc-p53 construct. (H). qPCR showing pre-miR-218-1 and miR-218-5p expression in vIRF1-expressing HUVECs transfected with pCMV6-Entry-C-Myc-p53 construct. The quantified results represent mean ± SD. * P P P < 0.001, Student's t-test.