Fig. 7.

HPV E6/E7 decreases lnc-CCDST level and elevates DHX9 expression. a DHX9 protein level is increased while MDM2 protein level is decreased in HPV-positive cells. DHX9 and MDM2 in HPV-negative cells (HT-3 and C-33A) and HPV-positive cells (HeLa and SiHa) were examined by Western blotting using the indicated antibodies. b Lnc-CCDST level is upregulated after E6/E7 knockdown. Lnc-CCDST was detected by RT-qPCR in HeLa and SiHa cells transfected with an E6/E7 siRNA pool. GAPDH served as internal control for normalization. Data are mean ± SEM determined from three independent experiments (n = 3), each with four technical replicates. Two-tailed unpaired Student’s t test was used. ***P < 0.001. c DHX9 protein level was decreased after E6/E7 knockdown. Western blotting analysis of DHX9 protein was performed in HeLa and SiHa transfected with an E6/E7 siRNA pool. d DHX9 protein level was increased in C-33 A cells transducing with E6/E7. HPV-negative C-33 A cells were transfected with HPV E6 and E7 plasmids. DHX9, E6, and E7 proteins were examined by Western blotting using the indicated antibodies. e Lnc-CCDST level was decreased in E6/E7 transducing C-33A cells. lnc-CCDST in C-33A cells transfected with E6 and E7 plasmids was detected by RT-qPCR. GAPDH was used as an internal control for normalization. Data are mean ± SEM determined from three independent experiments (n = 3), each with four technical replicates. Two-tailed unpaired Student’s t test was used. *P < 0.05, **P < 0.01, ***P < 0.001. f In normal tissues, the level of lnc-CCDST is significantly lower in HPV16/18-positive group than in HPV16/18-negative group. The lnc-CCDST expression levels were examined in HPV16/18-positive or -negative cervical normal tissues. β-actin was used as an internal control for normalization. Data are mean ± SD, and two-tailed unpaired Student’s t test was used. *P < 0.05. g In cancer tissues, lnc-CCDST is significantly lower in HPV16/18-positive group than in HPV16/18-negative group. The lnc-CCDST expression levels were examined in HPV16/18-positive or -negative CC tissues. β-actin was used as an internal control for normalization. Data are mean ± SD, and two-tailed unpaired Student’s t test was used. *P < 0.05. h A schematic model of this work. Our results (the black arrows) indicate that HPV E6/E7 decreases lnc-CCDST level by an unknown mechanism in cervical cells, which stabilizing DHX9 by reducing the MDM2/DHX9 interaction. We extend the HPV knowledge in the literature (the red arrows) that HPV E6 degrades p53 through E6-AP rather than MDM2, and p53 possesses the ability to transcriptionally activate MDM2, which might explain why E6 also inhibits MDM2 expression in HPV-infected cells. p53 is also inhibited by DHX9 in CC cells but the mechanism remains unclear