Transcriptional suppression of microRNA-27a contributes to laryngeal cancer differentiation via GSK-3β-involved Wnt/β-catenin pathway

miR-27a regulates cell differentiation in a variety of diseases. However, whether and how miR-27a participates in laryngeal cancer cell differentiation remains unknown. Therefore, we explored role and molecular mechanism of miR-27a in laryngeal cancer differentiation in the study. We found that miR-27a expression was inversely correlated with laryngeal cancer differentiation degree based on the clinical pathological diagnosis of each patient. miR-27 asignificantly rescued differentiation and inhibited β-catenin, LEF1, OCT4 and SOX2 in Wnt/β-catenin pathway in all-trans-retinoic acid (ATRA)-induced laryngeal cancer cells. Bindings of RARα to miR-27a and miR-27a to GSK-3β were confirmed by ChIP and Luciferase reporter assays, respectively. In conclusion, miR-27a is a negative regulator in laryngeal cancer differentiation. RARα-mediated miR-27a transcriptional inactivation releases the inhibition of miR-27a on GSK-3β leading to laryngeal cancer differentiation through GSK-3β-involved Wnt/β-catenin pathway, suggesting that miR-27a is a usefully therapeutic target at least in ATRA-induced laryngeal cancer differentiation.


INTRODUCTION
Abnormal differentiation is commonly found in cancer [1,2]. Studies have shown that cancer is initiated and maintained by a subset of cancer cells, 'the cancer stem cells', which are able to self-renew and differentiate like normal stem cells presenting cell surface markers [3][4][5]. Inducement of cancer cell differentiation has become a promising strategy in modern antineoplastic therapy [6]. Thus, study on molecular mechanism of cancer differentiation is still a hotspot at present.
It is well-known that all-trans-retinoic acid (ATRA) is a powerful agent commonly used in cancer differentiation study [7][8][9][10]. Retinoid-involved signal is often disrupted during carcinogenesis and restoration of the signaling may be a viable option for cancer prevention [11]. Retinoid induces cell differentiation through a lot of signal pathways such as PI3K/Akt and Wnt/β-catenin pathways. In retinoid-inducing differentiation, GSK-3β coordinates the crosstalk between the PI3K/Akt and Wnt/β-catenin pathways. ATRA and retinoic acid receptor (RAR) complex inactivate Akt through PI3K/Akt pathway and prevent GSK-3β from phosphorylation by Akt leading to maintenance of GSK-3β activity [12]. GSK-3β then phosphorylates β-catenin and promotes its degradation mediated by ubiquitin in Wnt/β-catenin pathway [13]. As a result, β-catenin cannot enter nuclear to transactivate the differentiation inhibitors such as OCT4 and SOX2 via TCF/LEF [14][15][16].
In addition, RAR also acts as a transcriptional factor. Studies have shown that RAR regulates transcription of target genes usually together with retinoid X receptor (RXR). For example, GM-CSF/RA-induced RALDH2 transcription in dendritic cells requires binding of cooperative RAR/RXR complex to Sp1 sites of the RALDH2 promoter through p38MAPK-associated pathway [17].

Research Paper
Oncotarget 14709 www.impactjournals.com/oncotarget MicroRNA, a 20-23 nt functional RNA molecule, is involved in some biological processes including embryonic development and mammalian cell differentiation [18]. It has been reported that miR-27a is an important differentiation-associated regulator in a variety of diseases [19][20][21]. However, whether and how miR-27a participates in laryngeal cancer cell differentiation remains unknown.
By prediction, we found that there exists a potential retinoic acid response element (RARE) in the miR-27a promoter whereas GSK-3β is a putative target gene of miR-27a. We wonder miR-27a could regulate differentiation through GSK-3β and RARα-related signaling pathways.

miR-27a expression is negatively associated with differentiation degree in laryngeal cancer tissues
RT-qPCR result demonstrated that miR-27a was up-regulated in 86% (43 of 50) cases of laryngeal cancer ( Figure 1A) and miR27a average expression was significantly higher in LSCC tissue (4.131 ± 0.537) than that in adjacent normal tissue (1.4201 ± 0.206) ( Figure 1B, P < 0.001), suggesting that miR-27a is involved in laryngeal oncogenesis.
As shown in Figure 1C and Table 1, we found that the higher miR-27a level in laryngeal carcinoma was, the lower the differentiation degree was (poor, 8.016 ± 1.790; moderate, 3.843 ± 0.560; well, 2.136 ± 0.336), which showed significant differences between miR-27a expression and differentiation degree (P < 0.001), indicating that miR-27a is a negative regulator in laryngeal cancer differentiation.

miR-27a resists ATRA-induced laryngeal cancer cell differentiation
In order to explore whether miR-27a takes part in laryngeal cancer cell differentiation, we first detected effect of miR-27a on expression of stem cell markers and differentiation-associated keratinocyte markers in nontreated Hep2 cells. RT-qPCR and Western blotting results showed that miR-27a mimic significantly increased OCT4 and SOX2 expression both at mRNA and protein levels in Hep2 cells compared to the mock, miR-27a inhibitor and mimic NC, respectively. Conversely, miR-27a mimic significantly reduced Involucrin and Keratin10 expression. However, not all markers showed significant difference between miR-27a inhibitor and inhibitor NC (Figure 2A). These results imply that miR-27a potentially represses laryngeal cancer cell differentiation.
We then explored effect of miR-27a on differentiation in ATRA-induced Hep2 cells. As shown in Supplementary Under light microscope, we then found that nontreated Hep2 cells cultured in vitro were shown to have a polygon body with mass nucleocytoplasmic ratio and rapid growth ( Figure 2B, left) whereas long shuttle cell bodies, cell marginal shrivel, small nucleocytoplasmic ratio and slow growth were observed in the ATRAinduced Hep2 cells ( Figure 2B, middle), which could be blocked by miR-27a introduction ( Figure 2B, right). Moreover, significantly lower expression of OCT4 and SOX2 and higher expression of Involucrin and Keratin10 at both mRNA and protein levels were found in Hep2 cells treated with 5 μM RA for 48 h compared to the controls, respectively. Nevertheless, the expression alterations of the four markers could be restored by miR-27a ( Figure 2C). These results suggest a suppressive role of miR-27a in ATRA-induced laryngeal cancer cell differentiation.

Mir-27a directly targets GSK-3β and increases expression of β-catenin and LEF1 in ATRAinduced Hep2 cells
Based on the bioinformatics analysis using different programs, we found a highly-conserved miR-27a targeting sequence in the GSK-3β 3'-untranslated region ( Figure  3A), which suggests that GSK-3β mRNA is a candidate target of miR-27a. Dual luciferase reporter assay result displayed that luciferase activity was significantly reduced in HEK293T and Hep2 cells when GSK-3β 3'UTR wildtype construct was cotransfected with miR-27a mimic compared to the controls ( Figure 3B). Western blot and real-time RT-qPCR results showed that miR-27a significantly decreased GSK-3β expression at protein level but not at mRNA level ( Figure 3C, left and middle), respectively, implying that miR-27a represses GSK-3β expression from post-transcriptional level. Moreover, ATRA significantly increased the GSK3β gene expression, which was restored by miR-27a ( Figure 3C, right). These results indicate that miR-27a directly inhibits GSK-3β in ATRA-induced Hep2 cells.
Whether GSK-3β is a mediator in miR-27aregulated differentiation of laryngeal cancer cells induced by ATRA is also a problem. As indicated in Figure 3D, the GSK-3β gene was significantly knocked down at both mRNA and protein levels in laryngeal cancer cells transfected with si-GSK-3β compared to the control, which means the GSK-3β gene is successfully silenced by its small interference RNA. Similar to miR-27a, GSK-3β kockdown significantly altered differentiation-associated markers in ATRA-induced Hep2 cells ( Figure 3E). These suggest that miR-27a regulates ATRA-induced Hep2 cell differentiation via down-regulating GSK-3β expression. www.impactjournals.com/oncotarget We also assessed influence of miR-27a in expression of β-catenin and LEF1 in Wnt/β-catenin signal in ATRAinduced Hep2 cells. We found that ATRA significantly decreased β-catenin and LEF1 expression compared to the controls, respectively, and such effects of ATRA were significantly rescued by miR-27a ( Figure 3F), indicating that miR-27a increases β-catenin and LEF1 in ATRAinduced Hep2 cells.

RARα contributes to transcriptional silence of miR-27a in ATRA-induced laryngeal cancer cell differentiation.
Interestingly, we found that miR-27a level was significantly lower in ATRA-treated Hep2 cells in a dosedependent manner compared to the controls ( Figure 4A). Therefore, how ATRA downregulates miR-27a expression is the other issue we have to solve in the study.
It is well-known that RARα is an important receptor of ATRA. According to bioinformatics prediction, we found a potential RARE site in the miR-27a gene promoter region ( Figure 4B). By ChIP assay, we obtained a PCR product including RARE site of miR-27a promoter from chromatin fragment precipitated by anti-RARα antibody, which confirms the direct binding of RARα to miR-27a promoter region. Furthermore, RARα exhibited significantly strong binding to RARE located within the miR-27a promoter in ATRA-induced Hep2 cells compared to the controls ( Figure  4C and 4D). Luciferase reporter assay result showed that ATRA significantly decreased transcriptional activity of the miR-27a promoter containing RARE compared to the controls and si-RARα significantly rescued the effect ( Figure 4E), indicating that ATRA/RARα negatively regulate miR-27a transcriptional activity.
Collectively, these results above implied that ATRA promotes RARα binding to miR-27a promoter, leading to miR-27a transcription inhibition.

DISCUSSION
In recent years, miR-27a has been a new star in differentiation research area. Studies have shown that 9.928 ± 2.263 Notes: One-way ANOVOA was used to analyze the correlation between the miR-27a expression and clinical features of each patient. ** and *** indicates P < 0.01 and P < 0.001, respectively. www.impactjournals.com/oncotarget miR-27a is a critical differentiation regulator in various kinds of cells such as myofibroblast [22], embryonic stem cell (ESC) [23], adipocyte [24], osteoblast [25], myoblast [26], erythroid [27] and breast cancer cells [28]. Except for breast cancer, role of miR-27a in other cancer differentiation is seldom reported. In addition, whether miR-27a affects ATAR-induced differentiation is not reported either.
In the study, we found that miR-27a overexpression is negatively associated with differentiation degree in laryngeal cancer tissues and suppresses differentiation of Hep2 cells mediated by ATRA. miR-27a also maintains Oncotarget 14712 www.impactjournals.com/oncotarget OCT4 and SOX2 expression and suppresses Involucrin and Keratin10 expression in ATRA-treated and -untreated Hep2 cells. These findings suggest that miR-27a inhibits laryngeal cancer differentiation.
Studies have shown that ATRA promotes translocation of phosphorylated p38MAPK into nuclear leading to activation of Msk1 whereas phosphorylated mRNA and protein levels of each gene above were measured by RT-qPCR and Western blot, respectively. GAPDH was used for the internal control. Data were expressed as the mean ± SD from at least three independent experiments. *, **, *** and ns represent p < 0.05, p < 0.01, p < 0.001 and no significance, respectively.
Oncotarget 14714 www.impactjournals.com/oncotarget Msk1 recruits transcription factors such as RARα to the promoter region of their targets [29,30]. In the study, we found that RARα directly binds to the miR-27a gene promoter region and decrease its transcription activity in Hep2 cells. We speculate that RARα directly inhibits miR-27a transcription probably via p38MAPK-involved pathway.
In the study, we found that miR-27a itself directly binds to GSK-3β 3′-UTR in both HEK293T and Hep2 cells and inhibits the gene expression in Hep2 cells. Interestingly, ATRA also decreased the expression of miR-27a.Whereas in ATRA-treated Hep2 cells, the RARα, GSK-3β, Involucrin and Keratin10 genes are (E) Effect of RARα on miR-27a transcription activity by Luciferase Reporter assay. GV148-miR-27a wild/mutant promoters, Renilla luciferase plasmids and si-RARα/NC small RNAs were cotransfected into Hep2 cells, respectively. Data were expressed as the mean ± SD from at least three independent experiments. *, **, *** and ns represent p < 0.05, p < 0.01, p < 0.001 and no significance, respectively.
In conclusion, miR-27a plays a suppressive role in laryngeal cancer differentiation. ATRA enhances the suppressive role of RARα on miR-27a promoter and release the inhibition of miR-27a on GSK-3β leading to laryngeal cancer differentiation through GSK-3β-involved Wnt/β-catenin pathway ( Figure 5). We believe that miR-27a is a usefully therapeutic target at least in ATRA-induced laryngeal cancer differentiation.

Patient tissues and cell lines
Tissue specimens including tumor tissues and matched non-tumor tissues from 50 laryngeal squamous cell carcinoma (LSCC) patients were collected after they gave informed consent. Verification of the specimens was performed by a pathologist and the samples were immediately frozen at −80˚C after been removed from the patients. Human laryngeal cancer cells Hep2 and human embryonic kidneycells HEK293T were obtained from the KeyGEN BioTECH Company of Jiangsu Province and were maintained in RPMI 1640 (GIBCO, LA, CA) with 10% new-born calf serum (Hyclone, Logan, USA) and DMEM (GIBCO, LA, CA) with 10% fetal bovine serum (Hyclone, Logan, USA), 100 units/ml penicillin and 100μg/ml streptomycin in a humidified atmosphere at 37°C in 5% CO 2 .

Gene transfection
Cell-based experiments were carried out by transfection of 20nM miRNA duplex (GenePharma, Shanghai, China), non-relative control RNA duplex (NC duplex, GenePharma) and small interfering RNA (siRNA, GenePharma) into the Hep2 cells using Lipofectamine™ 3000 (Invitrogen, USA) in accordance with the manufacturer's procedure. Sequences of the corresponding small non-coding RNAs were shown in Table 2.

RNA isolation and RT-qPCR
Total RNA was extracted from the specimens and the cells using Trizol (Takara, Dalian, China) according to the manufacturer's instructions. Concentrations of total RNA were measured by reading the absorbance at OD260/280 nm.
RT-qPCR was carried out using the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). First-strand cDNAs for mRNA and miRNA were obtained using the Reverse Transcription Kit (Takara, Dalian, China) and TransScript miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) in accordance with the manufacturer's procedure, respectively. Quantitative PCR was performed using SYBR ® Premix Ex Taq™ II (Takara) according to the manufacturer's instructions. GAPDH mRNA and endogenous U6 small nuclear RNA (snRNA) levels were assayed for normalization, respectively. 2 -ΔΔCt method was  Table 2.

Cell viability assay
Hep2 cells were transferred to a 96-well plate at a density of 2-3 × 10 3 cells per well in serum-free culture conditions, treated by different concentrations of ATRA (Sigma-Aldrich, St. Louis, MO, USA) and cultured for 1, 2, 3 or 4 days, respectively. Absorbance at 450 nm was measured using Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA) after incubation of the cells with 10 μl CCK8 (KeyGEN, Jiangsu, China) for 4 h at 37˚C.

Cell staining and morphology
Hep2 cells were incubated for 48 hours at 37°C in a 5% CO2 incubator in 6-well plates after ATRA treatment. Cells were then fixed with methanol, stained with hematoxylin and eosin, and subjected to light microscopic inspection and photographed.