The online version of this article (https://doi.org/10.1186/s12943-018-0916-8) contains supplementary material, which is available to authorized users.
Head and neck squamous cell carcinoma
Ingenuity pathway analysis
Long non-coding RNAs
Laryngeal squamous cell carcinoma
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with about 650,000 new cases and 200,000 deaths annually [ 1]. Recently, there have been substantial improvements in multimodal approaches including surgery, chemotherapy, and radiotherapy. However, the 5-year overall survival (OS) rate has not increased significantly and the mortality rate has not decreased dramatically [ 2]. Thus, it is urgently needed to search for molecular biomarkers in the diagnosis and treatment of HNSCC.
Long non-coding RNAs (lncRNAs) are transcripts of length more than 200 nucleotides without protein-coding potential, regulating gene expression at the transcriptional, posttranscriptional, and epigenetic levels [ 3]. It is promising to investigate the functions and molecular mechanisms of dysregulated lncRNAs in HNSCC tumorigenesis and progression. MIR31HG (NCBI No: NR 027054) is a recently discovered long non-coding RNA, length of 2166 bp; its transcription is regulated by methylation of the promoter region [ 4]. Reportedly, MIR31HG plays oncogenic role and its overexpression can serve as a poor prognosis marker in several cancers, including oral cancer, breast cancer, and pancreatic ductal adenocarcinoma [ 5– 7]. Accumulating study has revealed that MIR31HG can promote cancer initiation, progression, and metastasis by multiple mechanisms. MIR31HG inhibits the oncogene-induced cell senescence phenotype by regulating transcription of tumor suppressor p16 (INK4A) [ 8]. MIR31HG knockdown suppresses the capacity for proliferation, migration, and invasion of ESCC cells by targeting Furin and MMP1 [ 9]. Another study identified MIR31HG as a hypoxia-inducible lncRNA that formed a complex with HIF1A via direct binding and facilitating the recruitment of HIF1A and p300 cofactor for driving the progression of oral cancer [ 5]. However, the molecular mechanism, aggressive features, and diverse clinical prognosis of MIR31HG in HNSCC have not been fully understood. In our previous study, we screened lncRNA and mRNA expression profiles in LSCC tissues and found MIR31HG was positively correlated with HIF1A that plays an oncogenic role in LSCC [ 10]. To determine whether lncRNA MIR31HG served as a poor prognostic factor and targeted HIF1A to facilitate cell proliferation and tumorigenesis in human HNSCC, we analyzed the correlation between MIR31HG, HIF1A, and p21 expression and clinical prognosis. We conducted in vitro and vivo functional experiments and investigated the putative downstream pathway. The current results indicated that MIR31HG overexpression or co-expression of HIF1A-positive and p21-negative was correlated with the aggressive clinicopathological traits and served as a poor prognostic factor for LSCC patients. Moreover, MIR31HG facilitated cell proliferation and tumorigenesis via HIF1A and p21 by promoting the cell-cycle progression in HNSCC.
We performed qRT-PCR to test the relative expression of MIR31HG and HIF1A in 60 pairs of LSCC cancer tissues and corresponding adjacent normal tissues. The results showed that MIR31HG and HIF1A were overexpressed in LSCC tissues (Additional file 1: Figure S1a, P < 0.05). qRT-PCR was also performed to test the expression of MIR31HG gene in plasma. It found that the expression of MIR31HG was higher in the early-stage and advanced LSCC plasma than that in the vocal polyp plasma (Additional file 1: Figure S1b, P < 0.05). To further investigate the relationship between MIR31HG expression level and clinicopathological traits, we divided the 60 patients into high- and low-MIR31HG expression groups according to the patients’ overall median MIR31HG expression level. The MIR31HG overexpression was significantly correlated with advanced T category and poor lymph node metastasis (Additional file 1: Table S1). Kaplan–Meier analysis showed significantly better OS and recurrence-free survival (RFS) rates in patients with lower MIR31HG expression than in patients with higher MIR31HG expression (Fig. 1a). However, no significant difference was observed between patients with lower and higher HIF1A expression (Fig. 1b). Additionally, the univariate and multivariate analysis identified high MIR31HG expression as an independent prognostic factor for LSCC patients (Additional file 1: Table S2). Thus, these data indicated that MIR31HG expression may exert a critical role in LSCC progression and metastasis and serve as a novel prognostic factor for LSCC patients. Compared with the study in oral cancer [ 5], our study, by contrast, used a larger tissue samples and plasma samples to validate that MIR31HG was a prognostic biomarker for HNSCC.
We further performed immunochemistry to test the level of HIF1A and p21 proteins in LSCC. The results showed that HIF1A was found primarily in the cytosol, whereas p21 was predominantly expressed in the nucleus (Additional file 1: Figure S2). The positive rate of HIF1A expression in LSCC was higher than that in vocal polyp. Oppositely, the positive rate of p21 in LSCC was lower (Additional file 1: Table S3–4, P < 0.05). Furthermore, the expression of HIF1A or p21 in LSCC was closely associated with the clinical T stage, lymph node metastasis, and tumor differentiation (P < 0.05). Spearman analysis indicated that HIF1A expression was inversely associated with that of p21 in LSCC tissues ( P < 0.01, Additional file 1: Table S5). Kaplan–Meier analysis showed that patients with HIF1A-positive and p21-negative expression had poorer OS and RFS rate ( P < 0.05, Fig. 1c). Moreover, multivariate Cox regression analysis showed that co-expression of HIF1A-positive and p21-negative was identified as a significant independent factor related to recurrence (Additional file 1: Table S6).
We performed MTT assay and constructed HNSCC tumor xenograft model to evaluate the effect of MIR31HG on cell proliferation in vitro and vivo. First, we constructed three MIR31HG knockdown (KD) shRNAs to downregulate the expression of MIR31HG in FaDu and Cal-27 cells (Additional file 1: Figure S3). The KD2 shRNA decreased MIR31HG expression by 81% in FaDu cells and 83% in Cal-27 cells, indicating the high efficiency and stability of the transfection (Fig. 2a). Subsequently, the MTT assay displayed that MIR31HG KD significantly inhibited the growth of cancer cells (Fig. 2b). Then, we constructed MIR31HG overexpression (OE) lentivirus to upregulate the expression of MIR31HG in Cal-27 cells. The MIR31HG OE lentivirus increased MIR31HG expression by 13.667 times (Fig. 2c). The MTT assay displayed that MIR31HG OE promoted the growth of cancer cells (Fig. 2d). Moreover, we established a subcutaneously implanted tumor model in the nude mice by stably transfecting the FaDu cells with MIR31HG KD or NC lentivirus. After 4 weeks, the tumor volume in the MIR31HG KD group was remarkably smaller than that in the NC group and the tumor weight in the MIR31HG KD group was significantly lighter than that in the NC group (Fig. 2e-g). The Ki67 staining was decreased in xenografts of MIR31HG KD group compared with NC group. These results suggested that MIR31HG could promote HNSCC cells proliferation in vitro and vivo.
To elucidate the mechanism of MIR31HG underlying cell proliferation, we evaluated the cell cycle, cell apoptosis, and the differential gene expression in MIR31HG KD cells. PI-FACS analysis revealed that MIR31HG KD promoted cell cycle arrest at G1 or S phase (Fig. 3a). Interestingly, Annexin V-APC-FACS cell apoptosis analysis showed an increased number of apoptotic cells in the MIR31HG KD group (Fig. 3b). Furthermore, we found 345 differentially expressed mRNAs after MIR31HG shRNA transfected in FaDu cells, including 180 upregulated mRNAs and 165 downregulated mRNAs (Fig. 3c). The hierarchical clustering showed that differentially expressed mRNAs perfectly distinguished the MIR31HG KD cells from the NC cells. The ingenuity pathway analysis showed the differential expressed genes were significantly related to cell cycle (Fig. 3d). Western blot further validated that MIR31HG KD decreased the expression of HIF1A and CCND1 but increased expression of p21 (Fig. 3e).These results suggested that MIR31HG might target HIF1A and p21 to regulate the cell cycle progression and apoptosis in HNSCC (see materials and methods in Additional file 2). Different from the study in oral cancer [ 5], our study found another action mechanism of MIR31HG and HIF1A.
In this study, for the first time, we showed that lncRNA MIR31HG was specifically overexpressed in HNSCC and found that the overexpression of MIR31HG or the co-expression of HIF1A-positive and p21-negative served as a poor prognostic factors for LSCC patients. LncRNA MIR31HG facilitated HNSCC cell proliferation and tumorigenesis via HIF1A and p21 by promoting cell-cycle progression and inhibiting cell apoptosis. Thus, the results implicated the key role of MIR31HG in HNSCC progression and identified MIR31HG as a prognostic predictor and putative therapeutic target in HNSCC.
We thank all individuals who take part in this research.
This work was supported by Beijing Natural Science Foundation (7184196), National Natural Science Foundation of China (81473499), Beijing Municipal Administration of Hospitals’ Ascent Plan (DFL20180202), and the Priming Scientific Research Foundation for the Junior Researcher in Beijing Tongren Hospital, Capital Medical University (2017-YJJ-ZZL-001).
All data generated or analyzed during this study are included in this published article and its supplementary information files.
The study protocol was reviewed and approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University. The study was undertaken in accordance with the ethical standards of the World Medical Association Declaration of Helsinki. The experimental animal protocols complied with IACUC (Institutional Animal Care and Use Committee) regulations and were approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University, Beijing, China.
All authors have read and approved the final manuscript, and consent to the publication of the manuscript in Molecular Cancer.
The authors declare that they have no competing interests.
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Additional file 1: Figure S1. Relative gene expression of MIR31HG in LSCC cancer tissues and plasma. Figure S2. IHC of HIF1A and p21 proteins in LSCC tissues. Figure S3. MIR31HG KD lentivirus transfected Fadu and Cal-27 cells. Table S1. Association between MIR31HG and clinicopathological characteristics of LSCC patients. Table S2 Multivariate analyses of factors associated with OS in LSCC patients. Table S3. Relationship of HIF1A expression and clinicopathological parameters in LSCC patients. Table S4. Relationship of p21 expression and clinicopathological parameters in LSCC patients. Table S5. The correlation of HIF1A with p21 expression in LSCC. Table S6. Multivariate analyses for recurrence free survival of the postoperative LSCC by COX regression. (RAR 8552 kb)
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