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A microRNA 221- and 222-mediated feedback loop maintains constitutive activation of NF?B and STAT3 in colorectal cancer cells.

ABSTRACT: BACKGROUND & AIMS:Constitutive activation of the transcription factors nuclear factor ?B (NF-?B) 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-?B and signal transducer and activator of transcription 3 (STAT3) activation in human CRC cell lines. METHODS:CRC cell lines (HCT116 and RKO) were transfected with miR-221 or miR-222 mimics or inhibitors. The activity levels of NF-?B 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. RESULTS:In CRC cell lines, mimics of miR-221 and miR-222 activated NF-?B 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. CONCLUSIONS: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.

SUBMITTER: Liu S

PROVIDER: S-EPMC4839969 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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A microRNA 221- and 222-mediated feedback loop maintains constitutive activation of NFκB and STAT3 in colorectal cancer cells.

Liu Sanhong S Sun Xiaohua X Wang Mingliang M Hou Yingyong Y Zhan Yu Y Jiang Yuhang Y Liu Zhanjie Z Cao Xinwei X Chen Pengfei P Liu Zhi Z Chen Xi X Tao Yu Y Xu Chen C Mao Jie J Cheng Chunyan C Li Cuifeng C Hu Yiming Y Wang Lunshan L Chin Y Eugene YE Shi Yufang Y Siebenlist Ulrich U Zhang Xiaoren X

Gastroenterology 20140612 4

<h4>Background & aims</h4>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.<h4>Methods</h4>CRC cell lines (HCT116 and RKO) were transfected ...[more]

PMID: 24931456

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Project description:BACKGROUND:MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression. They negatively regulate gene expression post-transcriptionally by translational repression and target mRNA degradation. miRNAs have been shown to play crucial roles in muscle development and in regulation of muscle cell proliferation and differentiation. METHODOLOGY/PRINCIPAL FINDINGS:By comparing miRNA expression profiling of proliferating myoblasts versus differentiated myotubes, a number of modulated miRNAs, not previously implicated in regulation of myogenic differentiation, were identified. Among these, miR-221 and miR-222 were strongly down-regulated upon differentiation of both primary and established myogenic cells. Conversely, miR-221 and miR-222 expression was restored in post-mitotic, terminally differentiated myotubes subjected to Src tyrosine kinase activation. By the use of specific inhibitors we provide evidence that expression of miR-221 and miR-222 is under the control of the Ras-MAPK pathway. Both in myoblasts and in myotubes, levels of the cell cycle inhibitor p27 inversely correlated with miR-221 and miR-222 expression, and indeed we show that p27 mRNA is a direct target of these miRNAs in myogenic cells. Ectopic expression of miR-221 and miR-222 in myoblasts undergoing differentiation induced a delay in withdrawal from the cell cycle and in myogenin expression, followed by inhibition of sarcomeric protein accumulation. When miR-221 and miR-222 were expressed in myotubes undergoing maturation, a profound alteration of myofibrillar organization was observed. CONCLUSIONS/SIGNIFICANCE:miR-221 and miR-222 have been found to be modulated during myogenesis and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype. Identification of miRNAs modulating muscle gene expression is crucial for the understanding of the circuits controlling skeletal muscle differentiation and maintenance.

Dataset Information

A microRNA 221- and 222-mediated feedback loop maintains constitutive activation of NF?B and STAT3 in colorectal cancer cells.

Publications

A microRNA 221- and 222-mediated feedback loop maintains constitutive activation of NFκB and STAT3 in colorectal cancer cells.

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