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Coordinated targeting of the EGFR signaling axis by microRNA-27a*.

Wu X, Bhayani MK, Dodge CT, Nicoloso MS, Chen Y, Yan X, Adachi M, Thomas L, Galer CE, Jiffar T, Pickering CR, Kupferman ME, Myers JN, Calin GA, Lai SY - Oncotarget (2013)

Bottom Line: Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a.Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth.Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.

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miR-27a* has putative binding sites in EGFR mRNA and shows decreased expression in HNSCC cell lines and human tumor tissues(A) Identification of specific miR-27a(), −27a*(), −27b(), −27b*(#), −7(), −128() candidate binding sites within EGFR mRNA using in silico screening methods; (B) Hairpin representation of the pre-miR-27a with the sequences of miR-27a* (green) and miR-27a (magenta) highlighted; (C) Decreased expression of mature miR-27a and −27a* by qRT-PCR in 10 HNSCC cell lines and normal oral keratinocytes (OKF-6 and HOK16B). Values normalized to OKF-6, p<0.005; (D) Analysis of miR-27a* RNA in human HNSCC and normal mucosal specimens by qRT-PCR revealed an overall decrease in miR-27a* expression levels in HNSCC, p<0.0001; (E) Comparison of miR-27a* levels in matched normal/HNSCC tissue pairs demonstrated decreased expression in the tumors as compared to matched normal tissue, p<0.01.
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Figure 1: miR-27a* has putative binding sites in EGFR mRNA and shows decreased expression in HNSCC cell lines and human tumor tissues(A) Identification of specific miR-27a(), −27a*(), −27b(), −27b*(#), −7(), −128() candidate binding sites within EGFR mRNA using in silico screening methods; (B) Hairpin representation of the pre-miR-27a with the sequences of miR-27a* (green) and miR-27a (magenta) highlighted; (C) Decreased expression of mature miR-27a and −27a* by qRT-PCR in 10 HNSCC cell lines and normal oral keratinocytes (OKF-6 and HOK16B). Values normalized to OKF-6, p<0.005; (D) Analysis of miR-27a* RNA in human HNSCC and normal mucosal specimens by qRT-PCR revealed an overall decrease in miR-27a* expression levels in HNSCC, p<0.0001; (E) Comparison of miR-27a* levels in matched normal/HNSCC tissue pairs demonstrated decreased expression in the tumors as compared to matched normal tissue, p<0.01.

Mentions: We used software prediction programs to identify candidate binding sites of miRNAs within the EGFR gene and identified miR-7, −27a (miR-27a-3p), −27a*, −27b (miR-27b-3p), −27b* (miR-27b-5p), and −128 (Fig. 1A). Only RNA22 predicted binding sites for the star sequences (miRNA*), which have been generally regarded as non-functional products of miRNA duplex precursor processing (Fig. 1B) [27]. Four potential binding sites were identified for miR-27a* in the 3'-untranslated region (3'UTR) and three in the coding DNA sequence (CDS). MiR-27a, −27b and −7 were predicted to have two target sites in the 3'UTR and one in the CDS. Since miR-27a and 27b had the same seed region sequence, their target sites were the same. Only one target site was identified for miR-27b* or −128 in the CDS.


Coordinated targeting of the EGFR signaling axis by microRNA-27a*.

Wu X, Bhayani MK, Dodge CT, Nicoloso MS, Chen Y, Yan X, Adachi M, Thomas L, Galer CE, Jiffar T, Pickering CR, Kupferman ME, Myers JN, Calin GA, Lai SY - Oncotarget (2013)

miR-27a* has putative binding sites in EGFR mRNA and shows decreased expression in HNSCC cell lines and human tumor tissues(A) Identification of specific miR-27a(), −27a*(), −27b(), −27b*(#), −7(), −128() candidate binding sites within EGFR mRNA using in silico screening methods; (B) Hairpin representation of the pre-miR-27a with the sequences of miR-27a* (green) and miR-27a (magenta) highlighted; (C) Decreased expression of mature miR-27a and −27a* by qRT-PCR in 10 HNSCC cell lines and normal oral keratinocytes (OKF-6 and HOK16B). Values normalized to OKF-6, p<0.005; (D) Analysis of miR-27a* RNA in human HNSCC and normal mucosal specimens by qRT-PCR revealed an overall decrease in miR-27a* expression levels in HNSCC, p<0.0001; (E) Comparison of miR-27a* levels in matched normal/HNSCC tissue pairs demonstrated decreased expression in the tumors as compared to matched normal tissue, p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824521&req=5

Figure 1: miR-27a* has putative binding sites in EGFR mRNA and shows decreased expression in HNSCC cell lines and human tumor tissues(A) Identification of specific miR-27a(), −27a*(), −27b(), −27b*(#), −7(), −128() candidate binding sites within EGFR mRNA using in silico screening methods; (B) Hairpin representation of the pre-miR-27a with the sequences of miR-27a* (green) and miR-27a (magenta) highlighted; (C) Decreased expression of mature miR-27a and −27a* by qRT-PCR in 10 HNSCC cell lines and normal oral keratinocytes (OKF-6 and HOK16B). Values normalized to OKF-6, p<0.005; (D) Analysis of miR-27a* RNA in human HNSCC and normal mucosal specimens by qRT-PCR revealed an overall decrease in miR-27a* expression levels in HNSCC, p<0.0001; (E) Comparison of miR-27a* levels in matched normal/HNSCC tissue pairs demonstrated decreased expression in the tumors as compared to matched normal tissue, p<0.01.
Mentions: We used software prediction programs to identify candidate binding sites of miRNAs within the EGFR gene and identified miR-7, −27a (miR-27a-3p), −27a*, −27b (miR-27b-3p), −27b* (miR-27b-5p), and −128 (Fig. 1A). Only RNA22 predicted binding sites for the star sequences (miRNA*), which have been generally regarded as non-functional products of miRNA duplex precursor processing (Fig. 1B) [27]. Four potential binding sites were identified for miR-27a* in the 3'-untranslated region (3'UTR) and three in the coding DNA sequence (CDS). MiR-27a, −27b and −7 were predicted to have two target sites in the 3'UTR and one in the CDS. Since miR-27a and 27b had the same seed region sequence, their target sites were the same. Only one target site was identified for miR-27b* or −128 in the CDS.

Bottom Line: Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a.Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth.Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.

Show MeSH
Related in: MedlinePlus