A Feedback Loop between MicroRNA 155 (miR-155), Programmed Cell Death 4, and Activation Protein 1 Modulates the Expression of miR-155 and Tumorigenesis in Tongue Cancer

ABSTRACT

MicroRNA 155 (miR-155) is an oncomir, generated as a noncoding RNA from the BIC gene whose promoter activity is mainly controlled via activation protein 1 (AP-1) and NF-κB transcription factors. We found that the expression levels of miR-155 and programmed cell death 4 (Pdcd4) exhibit inverse relationships in tongue cancer cells (SAS and AWL) and tumor tissues compared to their relationships in normal FBM cells and normal tongue tissues, respectively. In silico and in vitro studies with the 3′ untranslated region (UTR) of Pdcd4 via luciferase reporter assays, quantitative PCR (qPCR), and Western blotting showed that miR-155 directly targets Pdcd4 mRNA and blocks its expression. Ectopic expression of Pdcd4 or knockdown of miR-155 in tongue cancer cells predominantly reduces AP-1-dependent transcriptional activity of the BIC promoter and decreases miR-155 expression. In this study, we demonstrate that miR-155 expression is modulated by a feedback loop between Pdcd4, AP-1, and miR-155 which results in enhanced expression of miR-155 with a consequent progression of tongue tumorigenesis. Further, miR-155 knockdown increases apoptosis, arrests the cell cycle, regresses tumor size in xenograft nude mice, and reduces cell viability and colony formation in soft-agar and clonogenic assays. Thus, the restoration of Pdcd4 levels by the use of molecular manipulation such as using a miR-155 sponge has an essential role in the therapeutic intervention of cancers, including tongue cancer.

KEYWORDS:

• AP-1
• BIC
• NF-κB
• Pdcd4
• apoptosis
• miR-155
• miR-155 sponge
• tumor xenografts

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00410-18.

ACKNOWLEDGMENTS

We thank the Department of Biotechnology, Government of India, for the financial support (grant number BT/PR/2672/AGR/36/702/2011) and Indian Institute of Technology Madras (IITM) for all other facilities. We thank the IIT Madras Alumni association for providing funds for travel to perform nude mouse experiments.

We thank Anuj Kaushik for his help with writing the manuscript.

S.Z. performed in vitro and in vivo experiments; V.T. assisted in experiments with nude mice under the supervision of K.S.; V.S. carried out the IHC experiments and scoring under the supervision of R.V.; S.Z. and D.K. analyzed the data; S.Z. and D.K. designed the experiments and analyzed the results; S.Z. and D.K. wrote the manuscript under the overall supervision D.K. All authors approved the submission for publication.