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.

No abstract

No abstract

Running title: miR-155/Pdcd4/AP-1 feedback loop in tongue cancer.ABSTRACT miR-155 is an oncomir, generated as a non-coding RNA from BIC gene whose promoter activity is mainly controlled via AP-1 and NF-κB transcription factors. We found that the expression levels of miR-155 and Pdcd4 exhibit inverse relationship in tongue cancer cells (SAS and AWL) and tumor tissues compared to normal FBM cells and normal tongue tissues, respectively. Insilco and In-vitro studies with 3'UTR of Pdcd4 via luciferase reporter assays, qPCR and western blots show 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 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 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 miR-155 sponge have important role in the therapeutic intervention of cancers, including tongue cancer. INTRODUCTIONMicroRNAs (miRs) are small non-coding RNAs of 18-25 nucleotides in length involved in posttranscriptional gene regulation mostly by binding to the 3'-untranslated region (3'UTR) of specific target messenger RNAs (mRNAs), causing mRNA degradation or translational repression (1). A single miR can regulate numerous target mRNAs and conversely, a single mRNA can be targeted by several miRs. miRs have a potential role to play in tumor development and sustenance (oncomirs) by down-regulating tumor suppressor genes (2) but they can also act as tumor suppressors in a highly tissue-specific manner (3). The downregulation of tumor suppressor genes is essential for continuous proliferation and survival of cancer cells. One such important tumor suppressor protein is programmed cell death 4 (Pdcd4), which is downregulated in various cancers of oral (4), breast (5), lung (6), liver (7), brain (8) and colon (9) tissues.Pdcd4 plays an important role in regulating apoptosis, invasion, and metastasis (10-12) and is known to inhibit AP-1-dependent transcription (13,14). miR-155 mediated downregulation of Pdcd4 was suggested earlier (15) and a recent study in lung cancer has claimed that microRNA-155 (miR-155) directly targets Pdcd4 and down-regulates its expression in lung cancer cells (16) but did not provide a convincing evidence for Pdcd4 3'UTR mediated downregulation as a mechanism. MicroRNA-155 (miR-155) was found to be overexpressed in oral (17), breast (18), tongue (19), pancreatic (20), prostatic (21) and thyroid cancers (22). miR-155 over expression has also been implicated in enhanced cell proliferation, invasion and metasta...

Pdcd4 (programmed cell death 4) has been demonstrated to be a tumor suppressor gene and is expressed at higher levels during apoptosis. PDCD4 is down regulated in a subset of oral cancers (tongue and buccal cavity) and an association between its down-regulation by miR-21 and tumorigenesis and invasion in oral carcinoma has been demonstrated. In this study we show that miR-155 directly targets PDCD4 in SAS (tongue squamous carcinoma) cells. We demonstrate a positive feedback loop between miR-155 expression and AP-1 dependent transcription through the down regulation of PDCD4. Analysis of miR-155 promoter (BIC) showed the presence of NF-κB and AP-1 (c-Jun and c-Fos) transcription factor binding sites and PDCD4 is known to inhibit AP-1 dependent transcription. Ectopic expression of PDCD4 in SAS cells drastically reduced the BIC promoter activity via inhibition of AP-1 dependent transcription and down regulated the levels of miR-155. We also generated the Lentiviral based miR-155 sponge stable cells to down regulate miR-155 in SAS cells, and found an increase in PDCD4 protein levels. The activity of BIC promoter was relatively low in miR-155 sponge stable cells compared to control sponge stable cells as analyzed by a dual luciferase reporter assay. We have also seen the reduced rate of proliferation in miR-155 sponge stable cells compared to control sponge stable cells. Thus our data show that miR-155 directly targets PDCD4 and consequently activates AP-1 dependent transcription in BIC promoter and increasing the levels of miR-155. Citation Format: shabir A. zargar, Devarajan Karunagaran. MicroRNA-155 directly targets PDCD4 and activates BIC promoter through AP-1 dependent transcription to replenish miR-155 expression in SAS cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3070. doi:10.1158/1538-7445.AM2015-3070