|Year : 2015 | Volume
| Issue : 6 | Page : 216-221
MicoRNA-451 is a novel tumor suppressor via targeting c-myc in head and neck squamous cell carcinomas
Huimin Wang, Guozheng Zhang, Zhiyan Wu, Baocai Lu, Dongjie Yuan, Xiao Li, Zhenmin Lu
Department of Otolaryngology, Head and Neck Surgery, The First Affiliated Hospital of Xinxiang Medical College, Xinxiang 453100, Henan Province, China
|Date of Web Publication||26-Oct-2015|
Department of Otolaryngology, Head and Neck Surgery, The First Affiliated Hospital of Xinxiang Medical College, Xinxiang 453100, Henan Province
Source of Support: None, Conflict of Interest: None
Objective: Head and neck squamous cell carcinoma (HNSCC) represents as a common malignancy with increasing incidence in the worldwide. The fact of its poor survival rate urgently requires developing efficient predictive biomarkers for clinical use. MicroRNAs (miRNAs) recently represent as a novel direction for early diagnosis and prognosis prediction in HNSCC therapy. In this study, we comprehensively investigated the function and putative target of miRNA-451 in vitro.
Methods: The expression of miRNA-451 was detected in HNSCC tissues and cell lines by real-time PCR. Forced expression or inhibition of miRNA-451 was done by transient transfection of mimics or inhibitor of miRNA-451 into indicated cells, respectively. Cell proliferation was evaluated by cell counting and crystal staining. Afterwards, we perform western blot to verify the expression of the miRNA-451 predicted target, c-myc, after miRNA-451 was overexpressed.
Results: We showed that miRNA-451 was downregulated in paired HNSCC tissues as well as in cell lines. And overexpression of miRNA-451 in cells with low endogenous expression of miRNA-451 accelerated proliferation. To the contrast, knockdown of miRNA-451 in cells with high levels of miRNA-451 significantly reduced cell growth rate. Furthermore, we used bioinformatics and cellular methods to predict and prove that c-myc was targeted by miRNA-451, since forced expression of miRNA-451 leaded to decreased c-myc protein expression in HNSCC cells.
Conclusion: Our findings identify miRNA-451 as a potential biomarker and suggest a key role of miRNA-451-c-myc pathway in HNSCC cell transformation, which could represent a novel therapeutic strategy in HNSCC treatment.
Keywords: c-myc, head and neck squamous cell carcinomas, miRNA-451, tumor suppressor
|How to cite this article:|
Wang H, Zhang G, Wu Z, Lu B, Yuan D, Li X, Lu Z. MicoRNA-451 is a novel tumor suppressor via targeting c-myc in head and neck squamous cell carcinomas. J Can Res Ther 2015;11, Suppl S2:216-21
|How to cite this URL:|
Wang H, Zhang G, Wu Z, Lu B, Yuan D, Li X, Lu Z. MicoRNA-451 is a novel tumor suppressor via targeting c-myc in head and neck squamous cell carcinomas. J Can Res Ther [serial online] 2015 [cited 2021 Oct 16];11:216-21. Available from: https://www.cancerjournal.net/text.asp?2015/11/6/216/168189
| > Introduction|| |
Head and neck squamous cell carcinoma (HNSCC) now presents a serious public health problem and it is the 6th most common cancer worldwide. Over 600,000 new patients are diagnosed and about 350,000 individuals die of this disease worldwide each year. Tobacco use and alcohol consumption are among the most important risk factors for HNSCC. During the last few decades, efforts have been paid on developing new strategies for early diagnosis, and the recent advances do have improved the quality of life of HNSCC patients. However, satisfactory survival rates or prognosis have not yet been reached. HNSCC is a multi-step process driven by an accumulation of alterations in oncogenes and tumor suppressor genes. Hence, there is a pressing need to acquire a deeper understanding of HNSCC biology and to find novel biomarkers, which benefit early diagnosis, prognosis prediction, and the establishment of effective therapeutics for HNSCC.
MicroRNAs (miRNAs) are a sort of small noncoding RNA molecules with 19–25 nucleotides in length. They are known to negatively regulate gene expression by base-pairing to the 3'-untranslated region (3'-UTR) of target mRNAs, and further inducing their degradation or inhibiting their translation. The dysregulation and dysfunction of the endogenous miRNAs have been shown to participate in major cellular pathways including differentiation, proliferation, apoptosis, and metastasis in multiple human malignancies., Increasing reports describe that aberrant expression of miRNAs occurred in human HNSCC cell lines as well as in frozen tissue samples.,,, The diagnostic and prognostic values of miRNAs have also been shown in several human cancers including gastric, colorectal, breast, and lung cancers.,,, In addition, HNSCC-specific microRNA expression profiles and key miRNAs known to modulate gene expression in these tumors have been established. Recent studies discussing different applications of these miRNAs as novel biomarkers in HNSCC highlight the future putative diagnostic and therapeutic potentials of this disease.
MicroRNA-451 (miRNA-451) is located on chromosome 17q11.2, a region known to be amplified in certain types of cancers, in close proximity to ERBB2 (17q12)., Emerging evidence supports a tumor suppressor role of miRNA-451 in multiple types of human malignancies, including osteosarcoma, bladder cancer, lung cancer, and colon cancer,,,, primarily via inhibiting cell growth, invasion, and metastasis. However, still little was known about the function, clinical significance, and mechanisms of miRNA-451 in HNSCC. Liu et al.first addressed that miRNA-451 was significantly decreased in nasopharyngeal carcinoma (NPC) cell lines and clinical tissues, and its downregulation was associated with poor survival of NPC patients. They further proved a tumor suppressor activity of miRNA-451 in NPC cells, and migration inhibitory factor was verified as its direct target. miRNA-451 as prognostic molecular signatures in HNSCC was also evaluated in a recent meta-analysis. According to the previous two reports, it needs to be systematically determine the function and underlying mechanisms of miRNA-451 in HNSCC.
In this study, we first examined the expression of miRNA-451 in 50 pairs of human primary HNSCC tissues compared with matched noncancerous controls, as well as in a series of human HNSCC cell lines, compared with normal cell line by using quantitative real-time polymerase chain reaction (RT-PCR). Then, we investigated the cell growth rates after the forced expression of exogenetic miRNA-451 mimics or inhibitors. The results showed that cell proliferation was markedly limited by miRNA-451 overexpression in vitro. Furthermore, we demonstrated that tumor suppressor role of miRNA-451 in HNSCC cells might be mediated by targeting the proto-oncogene c-myc.
| > Materials and Methods|| |
Clinical samples and patients
Tissue samples and paired noncancerous matched samples were harvested during surgery from 50 patients with HNSCC at TheFirst Affiliated Hospital of Xinxiang Medical College, from 2012 to 2014. All the samples were quickly frozen in liquid nitrogen after surgery and stored at −80°C. Written informed consent was received in advance for all the patients selected in this study.
Human normal oral keratinocyte cell (NOK), human dysplastic oral keratinocyte cell (DOK), and HNSCC cell lines KB, HN5, HN13, FaDu, HEp-2, cancer cell lines-27 (CAL27), squamous cell carcinoma-4 (SCC-4), and Tca8113 were ordered from the Cell Line Resource Center, Shanghai Institute of Biochemistry and Cell Biology, The Chinese Academy of Sciences (Shanghai, China) or ATCC. All cells were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO BRL, NY, USA), penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37°C in a humidified 5% CO2 atmosphere.
MicroRNA mimics and inhibitors
Both the miRNA-451 mimics and inhibitors were purchased by GenePharma (Shanghai, China). The cells were transfected with mimics or inhibitors using Lipofectamine ™ 2000 as described below.
Transfections were performed using Lipofectamine ™ 2000 (Invitrogen) as per the manufacturer's instructions. Forty-eight hours posttransfection, cells were harvested for further tests.
Real-time polymerase chain reaction analysis
MicroRNAs were extracted by Trizol from the tissues of HNSCC patients. RT-PCRs were performed using an ABI7500 RT-PCR system (Applied Biosystems, Carlsbad, CA) and the SYBR Premix Ex Taq reagent kit (Takara Bio Inc., Shiga, Japan) using Ct quantization method. Ct was the number of PCR cycles at which the fluorescence signal exceeded the threshold. △Ct was the difference in Ct values between the control (U6B) and test microRNAs (miRNA-451) in each sample. △△Ct was the difference in △Ct values between the experimental samples and paired control samples. △△Ct represents the fold change in microRNA expression.
KB and HEp-2 cells were transfected with control or miRNA-451 mimics and control or miRNA-451 inhibitor for 48 h. After that, cells were trypsinized and seeded into 12-well plates at a density of 105 cells. Then cells were counted by blood cell counting chamber for the next 3 days. Counting tests were repeated at least 3 times independently.
Crystal violet staining
KB and HEp-2 cells were transfected with control or miRNA-451 mimics and control or miRNA-451 inhibitor. Forty-eight hours later, cells were fixed in 4% paraformaldehyde for 20 min. After wash, the cells were stained with crystal violet solution for 30 min. The plates were aspirated, washed, and allowed to air dry. Finally, the plates were scanned with a high resolution for photograph.
Western blot analysis
In brief, for Western blot analysis, the cell lysate was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis in 9% acrylamide gels and transferred onto nitrocellulose membranes. After blocking, blots were incubated with c-myc and β-actin mAb (Santa Cruz Biotechnology Inc., Santa Cruz, CA). β-actin was used to normalize protein loading. A total of 40 μg cell lysate was loaded in each lane for Western blot analysis
The target genes of miRNA-451 were predicted by using three bioinformatics algorithms: miRanda (microrna.org), PicTar (4-way), and TargetScans.
Data are presented as mean ± standard deviation of at least triplicate experiments. All the results were analyzed with SAS version 9.2 (http://www.sas.com/en_us/home.html). The two-tailed Student's t-test was performed in the analysis of differential miRNA-451 expression between tumor and normal tissues. P < 0.05 was considered as statistically significant.
| > Results|| |
Expression of miRNA-451 in head and neck squamous cell carcinoma clinical samples and cell lines
To determine the expression of mature miRNA-451 in HNSCC, we first examined its level in 50 pairs of HNSCC clinical samples by RT-PCR. As shown in [Figure 1]a, the expression of miRNA-451 in the 50 samples of HNSCC tumors was 0.01143 ± 0.001531 (mean ± standard error of the mean [SEM]), whereas its expression in the paired normal tissues was 0.02366 ± 0.002430 (mean ± SEM, P = 0.0016). The results indicated that miRNA-451 expression was significantly decreased in clinical HNSCC samples, which agrees with the former reports of miRNA-451 expression in other types of cancers.,,, We, next similarly detected the expression of miRNA-451 in the available HNSCC cell lines. A NOK cell line, abbreviated as NOK was used as the control cell line in our experiments. We found a slightly decreased expression of miRNA-451 in DOK cells, indicating its putative roles in the dysplasia pathogenesis of oral keratinocytes. Importantly, we found a consistent downregulation of miRNA-451 in three lines of oral-derived (KB, HN5, and HN13), one line of pharynx-derived (FaDu), one line of laryngeal-derived (HEp-2), and three lines of tongue-derived (CAL27, SCC-4, and Tca8113) HNSCC cell lines [Figure 1]b. These findings strongly suggested an accordant role of miRNA-451 in the various subtypes of HNSCC cells, which further indicated that miRNA-451 functions as a tumor suppressor in HNSCC cells.
|Figure 1: miR-451 is downregulated in head and neck squamous cell carcinoma tissues and cell lines. (a) Real-time polymerase chain reaction analysis was performed to detect the expression of miR-451 in 50 cases of head and neck squamous cell carcinoma. Normal: Noncancerous matched tissues; tumor: Head and neck squamous cell carcinoma. miR-451 expression levels were calculated by the 2−△Ct method and normalized to U6 small nuclear RNA. The plot shows that the miR-451 expression reduced head and neck squamous cell carcinoma tissues. (b) Relative expression of miR-451 in eight head and neck squamous cell carcinoma cell lines, normal oral keratinocyte cell line, and dysplastic oral keratinocyte cell line|
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miRNA-451 inhibits cell proliferation
To explore the functional effects of miRNA-451 in HNSCC cells, we performed gain-of-function and loss-of-function studies using miRNA transfection of KB cells (with low endogenous miRNA-451 expression, oral-derived) and HEp-2 cells (with high low endogenous miRNA-451 expression, laryngeal-derived), respectively. The results showed in [Figure 2]a and [Figure 2]d illustrated the effective transfections of miRNA-451 mimics or inhibitors in the indicated cell lines. The cell counting assay demonstrated that cell proliferation was significantly inhibited in miRNA-451-mimics transfectants in comparison with the scramble transfectant cells [Figure 2]b. Conversely, inhibition of miRNA-451 induced a notable acceleration of cell growth rate than the corresponding scramble transfectants [Figure 2]e. In addition, as shown in [Figure 2]c and [Figure 2]f, we found that miRNA-451 significantly reduced the cell viabilities of HNSCC cells by crystal violate staining. These results suggested that miRNA-451 inhibits cell proliferation, which supports the tumor suppressing activities in HNSCC cells.
|Figure 2: miR-451 inhibits cell proliferation in head and neck squamous cell carcinoma cell lines in vitro. Total levels of miR-451 in (a) KB cells transfected with the synthetic mimics of miR-451 or scrambled oligos, (d) HEp-2 cells transfected with an inhibitor of miR-451 or scrambled oligos. Real-time polymerase chain reaction was carried out to detect the expression of miR-451. U6B was used as an internal control. Cell counting assay over a 4-day period (b and e) and crystal violate staining method (c and f) were performed to access cell growth rates. Data are expressed as the mean ± standard error of the mean of the experiments performed in triplicate. *P < 0.05, **P < 0.01. The results showed that the forced expression of miR-451 suppressed cell proliferation, while the inhibition of miR-451 had the opposite effect|
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miRNA-451 targets c-myc
We next set to seek the underlying mechanisms responsible for the growth inhibitory effects of miRNA-451 in HNSCC cells. As miRNA mainly functions through the targeting downstream genes, we computationally predicted the targets of miRNA-451 by miRanda, PicTar, and TargetScans. Among the putative targets, c-myc is regarded as an important proto-oncogene and is known to promote cell proliferation and survival. As shown in [Figure 3]a, the 3'-UTR of c-myc gene (1891–1912) might base pair with miRNA-451, suggesting cooperative binding and biological effective interactions. Meanwhile, the phenomenon of miRNA-451 targeting murine c-myc has been reported in a previous paper. Recently, Chen et al. also proved that by targeting c-myc, miRNA-451 could induce epithelial-mesenchymal transition in docetaxel-resistant lung cancer cells. We then monitored the extent to which miRNA-451 influenced the expression of endogenous c-myc levels. In KB cells, c-myc protein was specifically downregulated by 80% after miRNA-451 transfection [Figure 3]b, [left panel]. In HEp-2 cells, inhibition of miRNA-451 had the opposite effect [Figure 3]b, [right panel]. Hence, in line with the previous reports, our findings also proved that miRNA-451 targets c-myc in HNSCC cells.
|Figure 3: C-myc might be a potential miR-451 target. (a) Computationally predicted miRNA-mRNA interactions between miR-451 and the 3'-untranslated region of c-myc (1891–1912) by miRanda, PicTar, and TargetScans. (b) KB cells and HEp-2 cells were transfected with miR-451 mimics or inhibitor with control scrambles. Forty-eight hours later, cells were harvested for Western blotting. Protein level of c-myc was tested. β-actin was used as loading control|
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| > Discussion|| |
Despite many advances in HNSCC treatment, the overall and disease-free survival rates of HNSCC patients remain poor. Therefore, it is necessary to employ new concepts or strategies to improve the treatment outcome. Meanwhile, the molecular mechanisms of the unlimited proliferation of HNSCC cells have not yet been fully elucidated at present. We identified here miRNA-451 as a novel tumor suppressor in HNSCC that inhibited cell proliferation in vitro. c-myc is a typical oncogene, which is necessary for the rapid proliferation of cancer cells. In our study, we found that mR-451 might target c-myc to control cell growth in HNSCC cells. Although there is limited evidence, we still provided key clues of the tumor suppressor role of miRNA-451 in HNSCC cells for the 1st time.
Emerging evidence had pointed out that miRNAs contribute to cancer initiation and progression. It is known that miRNA-451 was downregulated in osteosarcoma, bladder cancer, lung cancer, and colon cancer.,,, Its tumor suppressor role was interpreted in these malignancies by the abilities to inhibit cell growth, invasion, and metastasis. These findings suggest that it would also be a candidate of tumor suppressive miRNA in HNSCC cells. In this study, we validated the downregulation of miRNA-451in HNSCC clinical samples. Importantly, the identical decrease of miRNA-451 in various originations of HNSCC cell lines strongly indicates its tumor suppressor role in HNSCC, no matter the different cell types. Hence, we might provide here a potential vital biomarker in the overall HNSCC diagnosis. Forced expression of miRNA-451 by synthetic mimics transfection remarkably caused growth arrest in vitro and vice versa. These data suggested that miRNA-451had a tumor suppressive function, especially contributing to limited cell proliferation in HNSCC.
The targets of miRNA-451 have been discussed in the former studies, which include c-myc,, liver receptor homolog-1, chemokine (C-X-C motif) ligand 16, and activating transcription factor 2. In our study, we showed that miRNA-451 targeted c-myc and leaded to its downregulation at protein levels. Therefore, we concluded that the growth inhibitory effect of miRNA-451 in HNSCC cells might be explained by the reduced c-myc protein expression. However, direct evidence is lacking. Besides, a single miRNA is capable of targeting several downstream genes to participate in the biological processes. The fine evaluations of other target genes, as well as novel cancer pathways regulated by miRNA-451, is important for our understanding of HNSCC tumorigenesis in future.
Furthermore, still little is known about the reason why there is a decreased expression of miRNA-451 in multiple cancer cells and whether epigenetic control mechanisms are involved in or not. It will be of great significance to explore it in the follow-up cancer research.
In general, our results demonstrated a tumor-suppressing role of miRNA-451 which took part in HNSCC cell malignant proliferation through regulating c-myc expression at the 1st time. Elucidation of tumor-suppressing function of miRNA-451 in HNSCC represents a novel biomarker and positive candidates for HNSCC therapeutic reconstitution.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]