miR-365 Promotes Cutaneous Squamous Cell Carcinoma (CSCC) through Targeting Nuclear Factor I/B (NFIB)

Aberrant expression of microRNAs plays vital roles in tumor development and progression. As transcription factors (TFs) are the critical components of signaling cascades, specific targeting effects of microRNAs to specific TFs may determine the role of microRNAs in different cancers. In this study, we identified Nuclear Factor I/B (NFIB) as one of the targets of miR-365 which was previously verified as an onco-miR in cutaneous squamous cell carcinoma (CSCC). Down-regulation of NFIB was a general feature in both CSCC cell lines and tumors from patients which show drastically up-regulated miR-365 expression levels. The siRNA-based knockdown of NFIB mimic the carcinogenic transformation of normal cells by ectopically expression of miR-365 which indicates depletion of NFIB is necessary for miR-365 to exert its pro-carcinogenic function. NFIB may represent a functional barrier targeted by miR-365 to the development of CSCC. Further studies also discovered a conserved feedback regulatory circuitry formed by NFIB and miR-365 in CSCC development which may be potentially utilized as therapeutic target to improve the clinical CSCC treatment.


Introduction
Cutaneous squamous cell carcinoma (also called cutaneous squamouse cell cancers, CSCC) counts about twenty percent of total skin cancers which is more malignant than basal cell cancers because CSCCs tend to grow and spread much faster than the later. CSCCs commonly develop on sun-exposed areas of the body such as the face, ears, neck, lips, and backs of the hands. Thus Ultraviolet (UV) exposure is a major cause and directly contributes to the occurrence of CSCC.
The current understanding of the molecular mechanism of CSCC is mainly related with transcription factors (TFs), e.g. p53, nuclear factor-kappa B (NF-kB) and activator protein-1 (AP-1) [1]. Mutation or aberrant expression of these TFs contributes directly or indirectly to some or all the cancer hallmarks, including insensitivity to antigrowth or apoptotic signals, self-sufficient growth signals, sustained angiogenesis, limitless replicative potential and invasive or metastatic capability which in turn play important roles in tumorigenesis of CSCC. In addition to causing mutations in the tumor suppressor p53 [2,3], UVB radiation could induce NF-kB activation and translocation into nucleus which upregulates IL-6 expression and secretion leading to chronic inflammation. All the above mechanisms will in turn contribute to the development of skin cancer [4,5].
In addition to the transcriptional events regulated by TFs, the post-transcriptional events in cancer development were recently investigated owing to the fast-growing field of microRNAs. microRNAs are 19-25 nt noncoding RNAs (ncRNAs), which regulate mRNA expression level post-transcriptionally by binding to the 39 UTR region of target genes. Recent survey of microRNA expression profile in skin cancers led to the identification of a number of aberrantly-expressed microRNAs and their correlation with clinical and pathological features [6]. However, their functions and the following downstream events remain largely unclear [7].
In our previous work, miR-365 was ranked as one of the highest expressed microRNAs induced by UVB treatment and identified as an onco-miR which was highly expressed in both cells and clinical specimens of CSCC [6,7]. Evidences also supported the role of miR-365 in promoting the development of tumors in nude mice and treatment with antagomiR-365 could inhibit cutaneous tumor formation in vivo [6,7]. The clarified role of miR-365 can help to develop new drug and targeting miR-365-based therapeutic regimen for CSCC. However, the target genes of miR-365 were unclear in CSCC although miR-365 has been shown to targeting cyclin D1, cdc25A, thyroid transcription factor 1 (TTF1) and so on in other cancers [8][9][10][11][12]. The lack of detailed molecular mechanism of miR-365 may delay the process of clinical application of anti-miR-365-based therapy in CSCC.
In this work, we performed bioinformatic analysis to find the target gene of miR-365. Evidences from in vivo and in vitro experiments showed that Nuclear Factor I/B (NFIB) is the target gene of miR-365 in CSCCs. The regulation of NFIB by miR-365 affects the expression of downstream cancer-related effectors, e.g. p53, Bcl-2 and CDK6, which shall be responsible for the development of CSCC.

NFIB is Predicted to be the Direct Target of miR-365
As higher expression of miR-365 in CSCC tumors and cells has been verified in the previous study [7], the direct downstream targets of miR-365 become more important for understanding the oncogenic roles performed by miR-365. To gain insights into the miR-365-mediated events in the development of CSCC, we performed target gene screening using web-based algorithms. Among the down-regulated genes, NFIB caught our attention because it was consistently predicted to be a direct target of miR-365 by three well-cited algorithms, TargetScan [13], miRanda [14], miRDB [15]. Moreover, as a known transcription factor, its role in cutaneous carcinogenesis is unclear.

NFIB is Down-regulated in CSCC Cells and Tumors
To probe into the possible involvement of NFIB in CSCC development and progression, we began by examining the expression levels of NFIB in CSCC cell lines, A431, HSC-1 and Tca8113, which were compared with normal control (NC), HaCaT cells. Results of western blot showed that the expression of NFIB was down-regulated in all the CSCC cell lines compared with the normal HaCaT cells ( Figure 1A). We also checked the miR-365 expression in HaCaT and CSCC cell lines which is inversely correlated with NFIB expression in those cell lines ( Figure 1A). To extend this study from cell lines to patient tumors, we first collected and verified miR-365 was overexpressed in clinical patient CSCC tissue samples (Fig. 1B) which is consistent with the previous study [7]. Then NFIB expression in both mRNA and protein levels were then examined in the above tumor samples. As predicted, NFIB expression levels were also inversely correlated with miR-365 in patient tumors ( Figure 1C). Collectively, the above results suggest that NFIB is down-regulated in CSCC cell lines and primary tumors in response to the upregulation of miR-365.

miR-365 Directly Targets NFIB through Binding to its 39UTR Region
To investigate whether the down-regulation of NFIB is due to direct targeting by miR-365, a search for miR-365 binding sites within the NFIB 39UTR revealed that miR-365 was predicted to hybridize to two evolutionarily conserved sites among vertebrate species ( Figure S1A in File S1). Perfect matches exist between the seed regions of miR-365 and the 39 UTR of NFIB, suggesting that miR-365 can directly repress NFIB expression which were verified by cloning the fragments of the NFIB 39UTR regions encompassing the target sites to the downstream of the firefly luciferase gene. Cotransfection of miR-365 with each of the two wild-type reporters caused similar repression of the two luciferase reporters (Figure 2A). Such targeting effects were specific to miR-365 binding because the reporter activity was less affected when transfections were repeated with mutant miR-365 binding sites in the NFIB 39UTR ( Figure 2A).

Inhibition of miR-365 Up-regulates NFIB Expression
To address whether NFIB expression was functionally related with the expression of miR-365 in CSCC cells and tumors, we predicted that miR-365 binding to NFIB 39UTR would lead to the repression of NFIB. Indeed, ectopic expression of miR-365 by transfection of miR-365 mimic led to decreased expression of NFIB and knocked down of miR-365 by antagomiR-365 could upregulate the expression of NFIB in normal HaCaT cells (Figure 2 B, left panel for NFIB expression and right panel for miR-365 expression). As the downregulation of NFIB in CSCC tumor cells has been shown in Figure 1, we thus treated CSCC cell lines with miR-365 inhibitor and found that NFIB expression was significantly rescued in two different CSCC cell lines, A431 and HSC-1 ( Figure 2C). Together, the above results indicated that NFIB is definitely one of the targets of miR-365 in both normal and CSCC cells and knockdown of miR-365 could alleviate the repression and upregulate the expression of NFIB.

AntagomiR-365-regulated NFIB Expression Inhibits Tumorigenesis in vitro and Abolishes the Tumor Growth in vivo
To further elucidate the mechanism of NFIB down-regulation in CSCC tumors, we detected the expression of cancer-related genes. p53 is a well known tumor suppressor [16] while Bcl-2 and CDK6 are recognized as pro-carcinogenic factors [17,18]. Western blot detection in A431 CSCC cells clearly showed that p53 was up-regulated together with NFIB while both Bcl-2 and CDK6 were significantly repressed after treatment of antag-omiR-365 ( Figure 3A). To firmly reach such conclusion, we thus challenged CSCC tumors formed by injecting A431 cells into nude mice with antagomiR-365 treatment. Tumors growth were greatly suppressed by antagomiR-365 treatment ( Figure 3B & 3C) as previous study [7]. We next checked the expression levels of miR-365 and the above mentioned cancer-related genes in tumors. In response to the drastically downregulation of miR-365 in xenografts treated by antagomiR-365, the NFIB and p53 were both upregulated while Bcl-2 and CDK6 decreased ( Figure 3D). IHC staining showed that NFIB and p53 were highly up-regulated while Bcl-2 and CDK6 were greatly suppressed in antagomiR-365-treated group than control group (Fig. 3E). Together, the above results might indicate Antagomir-365-regulated NFIB performs tumor-suppressive role in CSCC.
The Pro-carcinogenic Role of miR-365 is Performed through Targeting NFIB and NFIB can Inversely Regulate the Expression of miR-365 to form a Regulator Circuit To address whether NFIB plays roles in miR-365 pro-carcinogenic network in CSCC, we knocked down the expression of NFIB by siRNA oligos and checked the downstream responses of carcinogenic regulators. Knockdown assays showed that NFIB can be strongly downregulated by two of the three siRNA oligos targeting NFIB, siNFIB_2 and siNFIB_3 ( Figure 4A). Next, the detection of the downstream cancer-related effectors showed that p53 was significantly suppressed while Bcl-2 and CDK6 were highly up-regulated in both RNA and protein levels using the above two effective siRNAs against NFIB ( Figure 4B). Such results were consistent with the expression changes of the above carcinogenic regulators after ectopic expression of miR-365 which proved that NFIB is a critical target when miR-365 performs its pro-carcinogenic function.
Many microRNAs can form a feedback regulatory circuit with their targets, e.g. miR-29 with its targets, YY1 and Rybp [19,20]. To explore this hypothesis, we also detected miR-365 expression after knockdown of NFIB by the above two effective siRNA oligos. As expected, down-regulation of NFIB could strongly up-regulate miR-365 expression ( Figure 4C) in two different CSCC cell lines.

Discussion
In this study, we identified a target gene of miR-365, NFIB, which may represent a barrier to the development of CSCC. Down-regulation of NFIB was a general feature in both CSCC cell lines and tumors from patients which show drastically upregulated miR-365 expression levels ( Figure 1). Knocking down of NFIB by siRNA oligos simulated ectopically expression of miR-365 in normal cells which indicates NFIB is the downstream target of miR-365. Depletion of NFIB is necessary for miR-365 exerting its pro-carcinogenic function.
miR-365 has been identified in colon cancer [11], lung cancer [10,12], pancreatic cancer [9] and gastric cancer [8]. The role of miR-365 in the development and progression of cancers is debating. While it was regarded as an tumor suppressor in some cancers [8,11], definite evidences from different studies supported the oncogenic role of miR-365 in many other cancers including skin cancer [7] and pancreatic cancer [9]. The different conclusions might be established due to the differences of dominant molecular mechanism involving the different target gene-mediated signaling pathways in different cancers. The root causes for the development of specific cancers are drastically different. The role of miR-365 in a specific type of cancer is potentially determined by whether the key component of a specific signaling pathway is the target gene of miR-365. In both gastric and colon cancers, miR-365 targeted Cyclin D1 (CCND1) to inhibit cell cycle progression to repress tumorigenesis [8,11]. However, in pancreatic cancer, miR-365 promote the resistance to Gemcitabine, a standard chemotherapeutic agent for pancreatic cancer, by directly targeted adaptor protein Src Homology 2 Domain Containing 1 (SHC1) and apoptosis-promoting protein BAX [9]. In CSCC, miR-365 was overexpressed in both cells and clinical specimens. Ecotopic expression of miR-365 in normal skin cells could induce subcutaneous tumors in vivo. Antagomir-365 treatment inhibited cutaneous tumor formation in vivo, along with G1 phase arrest and apoptosis of cancer cells. These results showed that miR-365 acts as an oncogene in CSCC [7]. In breast cancer, miR-365 was identified as one of nine miRNAs that were up-regulated greater than two folds in primary breast cancer compared with normal adjacent tumor tissues (NATs) [21]. These evidences strongly supported the up-regulation of miR-365 is necessary and responsible for those malignancies. microRNAs exert their functions through target genes, especially transcription factors (TF), e.g. miR-29 which acts as a tumor suppressor by targeting oncogene YY1 [19]. In this study, NFIB is identified as the functional target of miR-365. NFIB is a member of the NFI gene family in vertebrates with versatile transcriptional activities [22,23]. NFIB functions to regulate more than 100 genes in organs like the brain, lung, liver and intestine [24], and it regulates cell proliferation and differentiation in lung maturation [25]. In human promyelocytic leukemia cell line HL-60, highly up-regulated expression of miR-21 can target NFIB and also NFIB negatively regulated miR-21 expression. Thus NFIB interacts with miR-21 and form a double-negative feedback loop for the survival of leukemia HL-60 [26]. However, NFIB can also act as oncogene in some other cancers. In Small cell lung cancer (SCLC) and triple negative breast cancer, NFIB was highly expressed than normal tissues and repressed apoptosis to promote cell proliferation [27,28]. The above studies indicate NFIB may perform distinct roles in different cancers.
In this study, originally, NFIB expression is lower in both CSCC cells and patient tumor samples. Down-regulation of NFIB by miR-365 overexpression led to carcinogenic transformation in normal skin cells [7] and enhanced tumorigenesis in vivo as demonstrated in this study. Knockdown of NFIB by RNA interference mimics the phenotype and transcriptional responses of carcinogenic regulators which places NFIB as the functional downstream target of miR-365 in miR-365-mediated the procarcinogenic pathway. An interesting discovery is that NFIB can inversely regulate miR-365 expression and thus they form a regulatory circuit to manipulate the normal and carcinogenic development of skin cells ( Figure 4D), just like miR-29 and YY1 [19].
Here we shall point out that miR-365 may also target other genes, e.g. the above mentioned CCND1. We checked CCND1 expression in CSCC cell lines as well as clinical samples which is also inversely correlated with the higher expression of miR-365 in CSCC cells and tumors ( Figure S2A-B in File S1) compared with normal cells and skin samples. And further analysis indicated that CCND1 was negatively regulated by miR-365 ( Figure S2C in File S1) like the targeting mechanism in gastric and colon cancers [8,11]. And knockdown of anti-carcinogenic NFIB in CSCC cells led to the upregulation of CCND1 ( Figure S2D in File S1) which is consistence with the enhanced expression of CDK6. The CCND1 and CDK6 can work in the same complex to promote G1 phase progression in cell cycle and contribute to CSCC progression. The discrepancy that the oncogenic CCND1 can be targeted by procarcigenic miR-365 which still promotes CSCC progression may due to the multi-targeting nature of miR-365. The NFIB-mediated tumor suppressive pathway may play critical roles in normal skin cells and depletion of NFIB by high level expression of miR-365 in CSCC tumors may disrupted the dominant anti-carcinogenic pathway which contributes the progression of CSCC tumors.
Collectively, our findings discovered NFIB as a novel target of miR-365, which may function in repressing carcinogenic transformation in normal skin tissues. Repression of p53 and upregulation of CDK6 and Bcl-2 by knockdown of NFIB indicates that NFIB may mediate the G1 arrest and the following apoptosis in malignant CSCC cells. Also, our results not only discovered a conserved feedback regulatory circuitry formed by NFIB and miR-365 in CSCC development ( Figure 4D), but also showed that this circuitry is potentially utilized as therapeutic target to improve the clinical CSCC treatment.

Ethics Statement
This study was approved by the Institutional Review Board of Nanfang Hospital affiliated to Southern Medical University, and all patients provided written informed consent for the use of surgical samples. All animals were treated according to standard guidelines for the use and care of laboratory animals.

Cell Culture and Tumor Samples
CSCC lines A431, Tca8113 (China Center for Type Culture Collection and Cell Bank of the Chinese Academy of Sciences, is inversely correlated with miR-365 levels. In this figure, the expression of miR-365 was examined by qRT-PCR and normalized to U6 snRNA expression. The expression of NFIB protein or mRNA in CSCC tumors compared with normal skin tissue was detected by western blot using as a loading control or qRT-PCR normalized to GAPDH expression. The P value (,0.01) shows significant inverse correlation between the levels of miR-365 and NFIB. doi:10.1371/journal.pone.0100620.g001 miR-365 Targets NFIB in Cutaneous Squamous Cell Carcinoma PLOS ONE | www.plosone.org

Luciferase Assay
For luciferase experiments, cells were grown in 12-well plates and transfection, including luciferase constructs and oligos, were performed when confluence reaches 70,80% using Lipofectine 2000 (Life Technologies) according to the manufacture' manual. 48 hrs after transfection, cell lysates were harvested and luciferase activities were assayed using Dual-Luciferase Reporter Assay System (Promega) and monitored by Varioskan Flash Spectral Scanning Multimode Microplate Reader (Thermo Scientific).

microRNA-FISH
The formalin-fixed paraffin-embedded CSCC sections were used for miRNA hybridization. Specimen was deparaffinized and hydrated, followed by treatment with proteinase K and refixation in 4% paraformaldehyde. After washing with phosphate-buffered saline and air-drying, the sections were hybridized with the fluorescein isothiocyanate-labeled LNA-miR-365 probe. Nuclei were routinely stained using DAPI.

Treatment of Antagomir-365 in vitro and in vivo
Antagomirs were synthesized by RiboBio Co. (Guangzhou, China), and the sequences were 59-APSUPSUPSAPSCGGG-GAUUUUUAGGAAUPSAPSChol-39 (antagomir-365). The A431 cell line was seeded in antibiotic-free media in 6-well plates (2610 5 cells/well) and treated for 24 h with antagomir-365 at a final concentration of 100 nM, or with an equal volume of phosphate-buffered saline, when the cells were at 50-60% of confluence. The A431 cell line (2610 7 cells) was subcutaneously injected into the right back flank of 5-week-old BALB/c-nu mice. After 1 week, when the tumors reached an average volume of 150 mm 3 , antagomir-365 (25 nM of antagomir-365 diluted in 100 ml phosphate-buffered saline; n = 7), control without antagomir (n = 3), was injected intratumorally three times per week for 2 weeks. Tumor diameters were measured every 2 days.

Statistical Analysis
Data were presented as mean 6 standard deviation (SD). The groups were compared by one-way analysis of variance using SNK-q test or by the t-test with two-tailed P value. Survival data were presented as Kaplan-Meier plots and were analyzed using a log-rank (Mantel-Haenszel) method. The significance level was P,0.05.

Supporting Information
File S1 Contains the following files: Figure S1. miR-365 targets prediction and sequence information. (A) Two of the predicted miR-365 binding sites in the 39UTR region of NFIB. (B) List of miR-365 targets predicted by TargetScan. (C) Sequence and miRBase ID of human miR-365. Figure S2