A microRNA 221– and 222–Mediated Feedback Loop Maintains Constitutive Activation of NFκB and STAT3 in Colorectal Cancer Cells

Abstract

Constitutive activation of the transcription factors nuclear factor κB (NF-κB) and STAT3 is involved in the development and progression of human colorectal cancer (CRC). Little is known about how these factors become activated in cancer cells. We investigated whether microRNA miR-221 and miR-222 regulate NF-κB and signal transducer and activator of transcription 3 (STAT3) activation in human CRC cell lines. CRC cell lines (HCT116 and RKO) were transfected with miR-221 or miR-222 mimics or inhibitors. The activity levels of NF-κB and STAT3 were measured in dual luciferase reporter assays. We used immunoblot and real-time polymerase chain reaction analyses to measure protein and messenger RNA (mRNA) levels. Cells were analyzed by proliferation, viability, and flow cytometry analyses. Mice were given injections of azoxymethane, followed by dextran sodium sulfate, along with control lentivirus or those expressing mRNAs that bind miR-221 and miR-222 (miR-221/miR-222 sponge). The levels of miR-221 and miR-222 as well as RelA, STAT3, and PDLIM2 mRNAs were measured in 57 paired CRC and adjacent nontumor tissues from patients. In CRC cell lines, mimics of miR-221 and miR-222 activated NF-κB and STAT3, further increasing expression of miR-221 and miR-222. miR-221 and miR-222 bound directly to the coding region of RelA mRNA, increasing its stability. miR-221 and miR-222 also reduced the ubiquitination and degradation of the RelA and STAT3 proteins by binding to the 3' untranslated region of PDLIM2 mRNA (PDLIM2 is a nuclear ubiquitin E3 ligase for RelA and STAT3). Incubation of CRC cells with miR-221 and miR-222 inhibitors reduced their proliferation and colony formation compared with control cells. In mice with colitis, injection of lentiviruses expressing miR-221/miR-222 sponges led to formation of fewer tumors than injection of control lentiviruses. Human CRC tissues had higher levels of miR-221 and miR-222 than nontumor colon tissues; increases correlated with increased levels of RelA and STAT3 mRNAs. Levels of PDLIM2 mRNA were lower in CRC than nontumor tissues. In human CRC cells, miR-221 and miR-222 act in a positive feedback loop to increase expression levels of RelA and STAT3. Antagonism of miR-221 and miR-222 reduces growth of colon tumors in mice with colitis.