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Figure 3 | Journal of Translational Medicine

Figure 3

From: NF-κB-modulated miR-130a targets TNF-α in cervical cancer cells

Figure 3

miR-130a directly targets and negatively regulates TNF-α expression. (A) Wild type (wt) and mutant complementary TNF-α mRNA 3’UTR sequences are shown compared with the miR-130a sequence. (B and C) EGFP reporter assays were performed to confirm the direct interaction of miR-130a and the TNF-α 3’UTR. HeLa and C33A cells were transfected with an EGFP reporter plasmid and the pcDNA3/pri-miR-130a or ASO-miR-130a plasmids, and the EGFP intensity was measured. (D) In HeLa and C33A cells, TNF-α expression levels were measured by real-time RT-PCR. The endogenous expression levels of β-actin mRNA were used to normalize mRNA expression. (E and F) Alterations in the TNF-α protein levels. miR-130a was overexpressed or its endogenous expression was blocked in both cell lines, and total protein was harvested for Western blot analysis. GAPDH protein expression levels were used to normalize the protein expression data. (G) HeLa and C33A cells were transfected with pcDNA3/pri-miR-130a or control vector and then implanted subcutaneously in the right armpit of nude mice, collected tumor after 14 days and made real-time RT-PCR assay to detect the expression levels of miR-130a in tumors. U6 snRNA was used to normalize gene expression. (H) Immunohistochemistry detected the levels of TNF-α protein in cervical cancer overexpress miR-130a compare with the control group. The histograms show the normalized mean ± SD mRNA levels and protein intensities from three independent experiments. (*P < 0.05, **P < 0.01).

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