Elsevier

Oral Oncology

Volume 51, Issue 12, December 2015, Pages 1103-1112
Oral Oncology

The increase of oncogenic miRNA expression in tongue carcinogenesis of a mouse model

https://doi.org/10.1016/j.oraloncology.2015.10.007Get rights and content

Highlights

  • The dysregulation of oncogenic miRNAs occurs in murine tongue tumorigenesis.

  • Increased miR-21, miR-31 and miR-146a are early markers for the liquid biopsies.

  • The targeting against EGFR or oncogenic miRNAs could be pre-clinically tested.

Summary

Objectives

This study investigated the oncogenic miRNA level in the tissue and biofluids in the Nitroquinoline 1-Oxide (4NQO)-induced mouse tongue carcinogenesis model for potential diagnostic or therapeutic application.

Materials and methods

The histological examination, immunohistochemistry, in situ hybridization, quantitative PCR analysis and bioinformatic algorithms were performed to unravel the signaling activation and miRNA expression in female murine samples.

Results

The increase of miR-21 and miR-31 staining, and EGFR activation paralleled the severity of 4NQO-induced epithelial pathogenesis in tongue epithelium. A progressive increase of miR-21, miR-31 and miR-146a in both saliva and plasma samples was also noted. miR-31 was the earliest emerging miRNA in the saliva. The increase of plasma miR-146a, miR-184 and miR-372 was detectable early in the induction, and it was particularly eminent at the most advanced lesion state. The combined analysis of the multiple oncogenic miRNAs in the plasma signified a potent discriminative capacity between normal and pathological states. As the blockage of EGFR or AKT activation drastically reverted the miR-21, miR-31 and miR-146a expression induced by 4NQO in human oral carcinoma cell lines, the results implicated a mechanistic linkage of the oncogenic miRNAs’ induction through EGFR/AKT activation.

Conclusions

In this study, we show the dysregulation of oncogenic miRNAs in murine tongue tumorigenesis, which simulates human counterparts. Increased multiple miRNAs in the biofluids may be valuable non-invasive markers in detecting oral carcinogenesis at an early stage. This animal model may also be useful for developing liquid biopsies and prevention strategies against oral carcinoma by abrogating EGFR or oncogenic miRNAs.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy in the world. Oral squamous cell carcinoma (OSCC) is the most prevalent subset of HNSCC [1]. The 5-year survival rates for OSCC, essentially unchanged during the past few decades, have remained low for less than 50% of patients after initial therapy [2]. It is likely that this high mortality rate is attributed to the late diagnosis, recurrence and the resistance to therapy. Since the neoplastic process of OSCC undertakes a multistep pathogenesis, an understanding of the molecular mechanism at different steps and the development of an access for early detection or targeting may validate more effective therapy and the reduction of disease relapse.

Longitudinal studies have shown that the genetic abnormalities such as the up-regulation of p53, Ki67 and EGFR, the loss in chromosome 9p, and others are able to predict the malignant transformation of oral premalignant lesions [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Table S1 summarized this panel of markers. To validate an early diagnosis and a targeted therapy against these alterations may abrogate oral carcinogenesis. In addition, various molecules have been identified as potential serum markers referring to diagnosis, metastasis prediction or treatment monitoring [16], [17], [18]. The usage of saliva as a diagnosis tool has emerged as a new approach in developing disease markers since the collection of saliva in humans seems to be practically easier and non-invasive than blood sampling [19], [20]. Apart from this, validating salivary molecules as OSCC markers may confer more diagnosis, prevention and therapy values since tumors may release different types of molecules to its immediately adjacent biofluid. Previous studies have found that many kinds of mRNA, protein or peptide could be salivary markers of OSCC [19], [21], [22]. However, it would be important to employ the combined analysis of multiple markers to reinforce the prediction power [21]. Moreover, a case-control study seems relatively difficult to carry out on human subjects due to the confounding of heterogeneity in race, gender, age and other factors.

MicroRNAs (miRNAs) are non-coding RNAs that play regulatory roles in carcinogenesis. A wide variety of miRNAs, which could be oncogenes or suppressors, were disrupted in OSCC or HNSCC [1], [19], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. The increased expression of oncogenic miRNAs including miR-21, miR-31, miR-146a, miR-184, miR-372 and others in OSCC tissues and in patients’ biofluids such as plasma or saliva has been observed [1], [19], [27], [28], [29], [30], [31], [32], [34], [35], [36], [37], [38]. Table S2 summarizes miRNAs potential for OSCC diagnosis using plasma or saliva samples. As the plasma or salivary miR-31 was elevated in early stage OSCC and the miR-31 up-regulation has become evident in premalignant lesions precedent to OSCC, miR-31 in biofluids could be a potential early marker of OSCC [19], [29], [39]. However, whether the up-regulation of other miRNAs is also an early event of oral carcinogenesis, and whether their levels in circulation or saliva can be used for early diagnosis, remains to be elucidated. Furthermore, accessing the level of multiple miRNAs expression in different biofluids concomitantly on the basis of the same type of assay may facilitate a combined analysis to yield more powerful diagnostic validation.

4-Nitroquinoline 1-Oxide (4-NQO) is a water-soluble reagent that causes bulky DNA adduct, mutagenesis and tumor induction. 4NQO-induced murine carcinomas in tongue and esophagus reiterate many of the pathological and genotypic features in human tumor counterparts [4], [6], [10], [40], [41]. This model also allows the assessments of biofluids to investigate the biochemical or metabolic mechanisms underlying neoplastic development [42], [43]. We have identified that the transgenic mice expressing oncogenic miRNAs in squamous epithelium enhanced the 4NQO induced tumorigenesis [44], [45]. The findings substantiated the contribution of oncogenic miRNAs to murine SCC formation induced by chemicals. The expression of oncogenic miRNAs in tissues, saliva and plasma at each step of oral malignant transformation induced by 4NQO was analyzed to address whether salivary or circulatory oncogenic miRNAs could be utilized as markers in this multistep tongue carcinogenesis model.

Section snippets

4NQO-induced mouse tumorigenesis and sampling

To induce tongue tumorigenesis of C57BL/6 mice, 100 mg/ml of 4NQO (Sigma–Aldrich, St Louise, MO) was added to the drinking water of 6-week-old young adult female mice for 14 weeks [44], [45]. The mice were left for tumor development for 14 additional weeks after termination of treatment. Mice taking water without 4NQO addition were controls. Since mice become offensive during tumor induction, female mice are used to avoid the unexpected loss of mouse in experiments. The treatment modality is

Basic biochemistry and cachexia related phenomenon

4NQO-treated mouse groups were found to have a decreased dietary intake since week 14 (Fig. 1B), while fluid consumption significantly decreased in comparison with the control group during the entire courses of experiments (Fig. 1C). The body weight of test mice drastically decreased at 16 weeks and reached about 60% of the body weight of control mice at 28 weeks (Fig. 1D). The decrease of bone mineral content seems to parallel the loss of body weight in test mice (Fig. 1E). Modules measuring soft

Discussion

Our previous studies specified the elevation of miR-31 in tissue, saliva and plasma of early stage OSCC [19], [29], [46]. Furthermore, miR-31 staining was found in preneoplastic oral epithelial cells [39]. Our cross analysis also identified the more elevated salivary miR-31 than plasma miR-31 in the same individual [29]. The results of miR-31 analysis in this animal study are in agreement with those human studies. We identified the initiation of miR-31 staining across the full thickness of

Competing interests

The authors declare that they have no competing interests.

Authors’ contribution

Kao Y.Y. carried out the animal study and molecular analysis. Tu H.F. carried out the statistical analysis and bioinformatics algorithm. Kao S.Y. participated in the design of the study and the statistical analysis. Chang K.W. and Lin S.C. designed and coordinated the study, and drafted the manuscript. All authors read and approved this manuscript.

Acknowledgements

We acknowledge Professor Chia-Hua Kuo for providing technical assistance and Ms. Diane Hwu for manuscript editing. This study was supported by grant MOST-102-2628-B-010-011-MY3 from Ministry of Science and Technology, Aim for the Top University Plan from Department of Education, and Health and Welfare Surcharge of tobacco products and grant MOHW104-TDU-B-211-124-001 from Ministry of Health and Welfare for Excellence for Cancer Research, Taiwan.

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