Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells
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 (**)).