Fig 1.
Silencing of HPV18 E6/E7 expression by RNA interference.
(A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with si18E6/E7, control siRNA siContr-1, or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53, and p21 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control. (C) Immunoblot analysis of HPV18 E7, total pRb (pRb), phosphorylated pRb (pRb-P), and Cyclin A1 protein levels, 72 h after transfection of HeLa cells with si18E6/E7 or siContr-1. α-Tubulin: loading control.
Fig 2.
Inhibition of endogenous HPV18 E6/E7 expression: Effects on the intracellular miRNA composition of cervical cancer cells.
Small RNA deep sequencing (A—D) and qRT-PCR analyses (E) of cellular miRNAs, 72 h after transfection of HeLa cells with si18E6/E7 or control siRNA siContr-1. (A) Mean read count distribution of mature miRNA sequences in si18E6/E7- and siContr-1-transfected cells (n = 2). Only miRNAs with a mean read count > 1 were considered. (B) The 15 most frequently sequenced cellular miRNAs. Selection based on siContr-1 samples, respective values for the si18E6/E7-treatment are indicated. Data represent mean ± SEM (n = 2). Interrupted x-Axis. (C) Overview on differentially affected (> 1.5-fold) cellular miRNAs, determined by small RNA deep sequencing. RPM values of si18E6/E7-treated samples were calculated relative to the control treatment (siContr-1). Only miRNAs with > 1,000 RPM in each sample were considered (n = 2). (D) Relative quantification of miRNAs in si18E6/E7- versus siContr-1-treated cells as assessed by small RNA deep sequencing (log2 display). Dashed lines: 1.5-fold up- or downregulation (log2(1.5) = 0.585). Only miRNAs with > 1,000 RPM in each sample were considered. Data represent mean ± SEM (n = 2). (E) qRT-PCR analyses of E6/E7-dependent cellular miRNAs identified by small RNA deep sequencing. Cellular miRNA levels were normalized to snRNA RNU6–2 and calculated relative to siContr-1 (log2 display). Dashed lines: 1.5-fold up- or downregulation (log2(1.5) = 0.585). The column color shows regulation in the same (dark grey) or opposite (light grey) direction compared to the small RNA deep sequencing data of the individual miRNAs. Data represent mean ± SEM (n = 2 or 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***)).
Fig 3.
Inhibition of endogenous HPV16 E6/E7 expression: Effects on selected intracellular miRNAs.
(A) Immunoblot analysis of HPV16 E7, HPV16 E6, p53 and p21 protein levels, 72 h after transfection of SiHa cells with si16E6/E7 or control siRNA (siContr-1), or upon mock treatment. α-Tubulin: loading control. (B) qRT-PCR analyses of ten selected cellular miRNAs, 72 h after transfection of SiHa cells with si16E6/E7 or siContr-1. Cellular miRNA levels were normalized to the snRNA RNU6–2 and calculated relative to siContr-1 (log2 display). Dashed lines: 1.5-fold up- or downregulation (log2(1.5) = 0.585). Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*) and p ≤ 0.01 (**)).
Fig 4.
Effects of the p53 status on the E6/E7-dependent modulation of intracellular miRNAs.
(A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7, control siRNA (siContr-1), or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control (mock). Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). (B) Immunoblot analysis of HPV18 E6, p53 and p21 protein levels, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1, or upon mock treatment. α-Tubulin: loading control. (C) qRT-PCR analyses of selected cellular miRNAs, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1. miR-34a-3p, positive control miRNA (p53-inducible). Cellular miRNA levels were normalized to snRNA RNU6–2 and calculated relative to siContr-1 (log2 display). Dashed lines: 1.5-fold up- or downregulation (log2(1.5) = 0.585). Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***)).
Fig 5.
Influence of combined silencing of p21 and HPV18 E6/E7 expression on the senescent phenotype of HPV-positive cancer cells.
(A) qRT-PCR analysis of HPV18 E6/E7 (left panel) and p21 (right panel) mRNA expression, 72 h after transfection of HeLa cells with the indicated siRNAs or in mock-treated cells. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 2 or 3). Asterisks above columns indicate statistically significant differences between the indicated treatments (p ≤ 0.05 (*), p ≤ 0.01 (**)). (B) Immunoblot analysis of HPV18 E7, p53, and p21 protein levels, 72 h after transfection of HeLa cells with the indicated siRNAs or upon mock-treatment. α-Tubulin: loading control. (C + D) Cell cycle distribution analyzed by FACS, 72 h after transfection of HeLa cells with the indicated siRNAs or upon mock treatment. Percentage of cells in the G1, S and G2 cell cycle phases are indicated. Representative samples of one experiment are shown as well as a summary of multiple biological replicates. Data represent mean ± SEM (n = 3). (E) HeLa cells were stained for expression of the senescence marker SA-β-Gal, 168 h after transfection with the indicated siRNAs. Visualization by bright field microscopy.
Fig 6.
Effects of miRNAs of the miR-17~92 cluster on p21 expression in HeLa cells.
(A) qRT-PCR analyses of cellular miRNA levels, 72 h after transfection of HeLa cells with the indicated vectors or upon mock treatment. miR-17~92: vector coding for the mir-17~92 cluster; “control”: repective empty expression vector. miRNA levels were normalized to snRNA RNU6–2 and calculated relative to the mock control. miR-17–5p, miR-20a-5p, miR-19b-3p, miR-92a-3p: encoded by the mir-17~92 expression vector; miR-34a-5p: negative control (not encoded by the vector). Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from vector control-treated cells (p ≤ 0.05 (*)). (B) qRT-PCR analysis of p21 mRNA expression, 72 h after transfection of HeLa cells with the indicated vectors or upon mock treatment. mRNA levels were normalized to ACTB and calculated relative to the mock control. Data represent mean ± SEM (n = 4). Asterisks above columns indicate statistically significant differences from vector control-treated cells (p ≤ 0.05 (*)). (C) Immunoblot analysis of p53 and p21 protein levels, 72 h after transfection with the indicated vectors. α-Tubulin: loading control. A representative image is shown with corresponding densitometrically quantified band intensities of p21, normalized to α-Tubulin and calculated relative to mock. (D) miRNA inhibitors against miR-17–5p and miR-20a-5p increase the expression of p21 in HeLa cells. Left panel: Immunoblot analysis of p53 and p21 protein levels, 72 h after transfection of HeLa cells with the indicated miRNA inhibitors, an inhibitor control (‘Inhib. control’), or upon mock treatment. α-Tubulin: loading control. A representative image is shown. Numbers below individual lanes correspond to densitometrically quantified band intensities for p21, normalized to α-Tubulin and calculated relative to the Inhib. control. Right panel: Summary of densitometric quantification of p21 protein signal intensities. Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from Inhib. control-treated cells (p ≤ 0.05 (*), p ≤ 0.01 (**)).
Fig 7.
Characterization of exosomes secreted by HeLa cells used for small RNA deep sequencing.
(A) Immunoblot analysis of total cellular extract (30, 10 and 1 μg) from exosome-producing cells, and of 1 μg protein from exosome preparations. Hsc70, CD63, Annexin-1, CD9 and β-Actin: exosomal markers; EEA1: early endosome marker; GRP78: ER marker. (B) Visualization of exosomes by electron microscopy. Bar corresponds to 100 nm. (C) Characterization of cellular and exosomal RNA. Electropherograms of total RNA isolated from HeLa cells and from RNAse A-treated exosomes. Upper panel: total RNA contents; lower panel: small RNA contents. M = marker. Shown are representative images for siContr-1-treated samples.
Fig 8.
Inhibition of endogenous HPV18 E6/E7 expression: Effects on the miRNA composition of exosomes secreted by cervical cancer cells.
Small RNA deep sequencing (A—D) and qRT-PCR analyses (E) of exosomal miRNAs, 72 h after transfection of HeLa cells with si18E6/E7 or control siRNA (siContr-1), and subsequent exosome purification from the cell culture supernatant. (A) Mean read count distribution of mature miRNA sequences in exosomes released from si18E6/E7- and siContr-1-treated HeLa cells (n = 3). Only miRNAs with a mean read count > 1 were considered. (B) The 15 most frequently sequenced exosomal miRNAs. Selection based on siContr-1 samples, respective values for si18E6/E7-treatment are indicated. Data represent mean ± SEM (n = 3). Interrupted x-Axis. (C) Overview on differentially deregulated (> 1.5-fold) exosomal miRNAs determined by small RNA deep sequencing. RPM values of si18E6/E7-treated samples were calculated relative to the control treatment (siContr-1). Only miRNAs with > 1,000 RPM in each sample were considered (n = 2). (D) Relative quantification of miRNAs in exosomes released from si18E6/E7- versus siContr-1-treated cells, as assessed by small RNA deep sequencing (log2 display). Dashed lines: 1.5-fold up- or downregulation (log2(1.5) = 0.585). Only miRNAs with > 1,000 RPM in each sample were considered. Data represent mean ± SEM (n = 3). (E) qRT-PCR analysis of E6/E7-dependent exosomal miRNAs identified by small RNA deep sequencing. Exosomal miRNA levels were normalized to miR-452–5p and miR-183–5p and calculated relative to siContr-1 (log2 display). Dashed lines: 1.5-fold up- or downregulation (log2(1.5) = 0.585). The column color shows regulation in the same (dark grey) or opposite (light grey) direction compared to the small RNA deep sequencing data of the individual miRNAs. Ct-values > 35 were considered as not detected (n.d.). Data represent mean ± SEM (n = 2 or 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*), p ≤ 0.01 (**)).
Fig 9.
Inhibition of endogenous HPV16 E6/E7 expression: Effects on selected exosomal miRNAs.
qRT-PCR analysis of selected exosomal miRNAs, 72 h after transfection of SiHa cells with si16E6/E7 or control siRNA (siContr-1), and subsequent exosome purification from the cell culture supernatant. Exosomal miRNA levels were normalized to miR-452–5p and miR-183–5p and calculated relative to siContr-1 (log2 display). Dashed lines: 1.5-fold up- or downregulation (log2(1.5) = 0.585). Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***)).
Fig 10.
HPV oncogenes control p21 expression at multiple levels.
E6 can repress p21 transcription at the promoter level by inducing the degradation of the p21 transcriptional activator p53; sustained E6/E7 expression maintains the concentration of miR-17 family members in HPV-positive cancer cells which repress p21 expression by targeting the p21 mRNA; the E7 protein can directly bind to the p21 protein and inhibit its function.