Mir-31 Ablates Expression of the Hif Regulatory Factor Fih to Activate the Hif Pathway in Head and Neck Carcinoma

MicroRNAs (miRNA) are endogenously expressed noncoding RNAs with important biological and pathological functions that are yet to be fully defined. This study investigated alterations in miRNA expression in head and neck squamous cell carcinoma (HNSCC), the incidence of which is rising throughout the world. Initial screening and subsequent analysis identified a panel of aberrantly expressed miRNAs in HNSCC tissues, with miR-31 among the most markedly upregulated. Ectopic expression of miR-31 increased the oncogenic potential of HNSCC cells under normoxic conditions in cell culture or tumor xenografts. Conversely, blocking miR-31 expression reduced the growth of tumor xenografts. The in silico analysis suggested that miR-31 may target the 3′ un-translated region (UTR) of factor-inhibiting hypoxia-inducible factor (FIH), a hypoxia-inducible factor (HIF) regulatory factor that inhibits the ability of HIF to act as a transcriptional regulator under normoxic conditions. In support of this likelihood, miR-31 expression repressed FIH expression and mutations within the predictive miR-31 target site in the FIH 3′ UTR abrogated FIH repression. Furthermore, miR-31 expression increased HIF transactivation activity. We found that FIH suppressed oncogenic phenotypes under normoxic conditions and that this activity was abrogated by functional mutations. Lastly, increased miR-31 expression was correlated with decreased levels of FIH in tumor tissues. Our findings suggest that miR-31 contributes to the development of HNSCC by impeding FIH to activate HIF under normoxic conditions.


Introduction
Head and neck squamous cell carcinoma (HNSCC) is the fifth most common carcinoma worldwide (1,2).MicroRNAs (miRNA) are 19 to 24 nucleotides noncoding RNAs that regulate the translation and degradation of target mRNAs (3,4).Lines of evidence indicate that miRNA may play important roles in carcinogenesis (5).The abundance of miRNAs and their apparent pluripotent actions suggest that the identification of miRNAs involved in oncogenesis might yield targets or networks that are suitable for therapeutic intervention (3).miR-21 was found to be oncogenic in HNSCC (6)(7)(8).miR-98 regulates HMGA2 expression in HNSCC cells (9).An association has also been identified between miR-211 expression and the vascular invasion of oral carcinoma (10).miRNA expression can also be used as a diagnostic marker of HNSCC (7,11).

Materials and Methods
HNSCC tissues.Resected tissues from 46 HNSCC patients (Supplementary Table S1), who gave informed consent for the use of their tissue, were harvested at surgery.The study was approved by the institutional review board.Tissue specimens were taken from primary HNSCC, along with paired noncancerous matched tissues (NCMT) as well as available neck metastatic lesions (NML).Ten primary tumor tissues were microscopically screened to have >70% of their areas occupied by tumor cells; these were used to analyze a panel of 154 highly conserved miRNAs using quantitative reverse transcription-PCR (qRT-PCR) array (Applied Biosystems).The results of this assay allowed the selection of particular miRNAs to be assessed in tissues retrieved by laser capture microdissection (LCM; ref. 1).
Quantitative PCR.A mirVana PARIS kit (Ambion) was used to isolate miRNA from the total RNA.A Taqman miRNA assay kit and Taqman miRNA assay supplies were used to quantify the expression of mature miRNAs according to the manufacturer's instructions (Applied Biosystems).miRNAspecific qRT-PCR was done in duplicate or triplicate on the ABI Prism 7700 Sequence Detector system (Applied Biosystems) using RNU19 or let-7a as internal controls (28).C t was the number of cycle at which the fluorescence signal passed threshold.ΔC t was the difference of C t values between tested miRNA and internal control (10).ΔΔC t was the difference of ΔC t values between different experimental settings or paired samples. 2 ΔΔCt represents the fold change in miRNA expression.mRNA expression of FIH, ING4, and PHD4 was analyzed using Taqman qRT-PCR analysis (Applied Biosystems).
Cell culture, reagents, and phenotypic assays.The HNSCC cell lines Fadu, OECM-1, and SAS cells; HT29 CRC cells; and 293FT and phoenix package cells were cultured as previously described (2,29), as were normal human oral keratinocytes (NHOK), which were used as controls (30).Fadu and SAS were tumorigenic, whereas OECM-1 was nontumorigenic in nude mice (31).For hypoxia culture, cells were grown in an incubator (Astec) with 1% O 2 , 5% CO 2 , and 94% N 2 for 24 h.miR-31 antisense locked nucleic acid (2′-O,4′-C-methylene-linked ribonucleotide derivative; LNA) together with control scramble LNA were purchased from Ambion.The inhibitory efficacy of miR-31 LNA in cultivated SAS cells was validated as 120 nmol/L for 24 to 72 h.The dose of miR-21 mimic (Ambion) was determined as 30 nmol/L by pilot studies.VEGF production in aliquots of cell supernatant or tissue lysate was analyzed using a kit (Invitrogen).Analysis of cell viability, proliferation, migration, and anchorage-independent colony formation followed the protocols previously published (2).
Plasmids, transfection, and infection.For exogenous miR-31 expression, pre-mir-31 sequence was cloned into a lentivirus vector, pLV-EF1a-GFP, which contains a green fluorescent protein (GFP) tag (10).An empty vector or a vec-tor carrying the miR-31 insert was cotransfected with helper plasmids into 293FT cells to produce lentiviruses.The stable cell lines were established by lentiviral infection.
To test the targeting activity of miR-31, antisense sequences complementary to miR-31 sequences and the 3′ untranslated region (UTR) of FIH containing predictive miR-31 target sites were cloned into the pCMV-LacZ plasmid at the 3′-end of the lacZ coding sequence; these reporter plasmids were designated miR-31-asR and pCMV-LacZ-FIH3′UTR, respectively.Cotransfection of reporter plasmids with pCMV-Luc into cells was performed using Lipofectamine 2000 (Life Technologies).LacZ gene expression would be repressed if the pre-mir-31 insert was expressed, processed, and served as small interfering RNA against lacZ.Two putative miR-31 target sites residing at nucleotides 117 to 124 and 774 to 780 in the FIH3′UTR predicted by TargetScan 7 and Pictar8 were designated A and B as conservative and nonconservative sites, respectively.Mutant reporter plasmids were obtained from pCMV-LacZ-FIH3′UTR using a site-directed mutagenesis kit (Stratagene).After confirmation of the replacement of the target sequence UCUUGC by AAGCUU, the plasmids were designated A Mut, B Mut, and AB Mut, which had mutations in A, mutations in B, and double mutations in both A and B sites, respectively.pHIF1-luc reporter plasmid (Panomics) was used for the assay of HIF transactivation.
For exogenous FIH expression, two wild-type and two mutant FIH cDNA sequences generated by site-directed mutagenesis were cloned into pBabe-puro vector to produce retroviruses for infection.The stable cells FIH(3′UTR), FIHWt, FIHD201A, and FIH 1-302 cells were established by puromycin selection after retroviral infection.FIH(3′UTR) cells would express wild-type full-length FIH cDNA, including 3′ UTR, whereas FIHWt would express wild-type FIH coding sequence without 3′ UTR.FIH is a dioxygenase, and its activity is Fe(II) dependent (14).FIH D201A cells would express a mutant FIH coding sequence encompassing an aspartic acidto-alanine mutation at residue 201 that disrupts Fe (II) binding and attenuates catalytic activity.FIH 1-302 cells would express a protein with truncation of 303 to 349 residues that prevents the dimerization of FIH molecules at COOH terminal region and attenuates FIH (16,17).Vector alone is a control cell infected with control virus.
Immunohistochemistry. FIH and VEGF immunoreactivities in HNSCC, together with available NCMT and NML, were detected by immunohistochemistry following previously reported protocols (1).Incubation of primary antibodies was at 4°C overnight (Supplementary Table S2).Preimmunized mouse IgG was used as a negative control.The immunoreactivity was scored −, +, and ++ when <10%, 10% to 50%, and >50% immunoreactive cells were counted, respectively, in five randomly selected 200× fields.
Tumorigenesis.The analysis of tumorigenesis followed the previously published protocols (10).For blockage of xenographic tumors, 0.5% atelocollagen (Koken Co.) on pepsin treatment to remove antigenecity was complexed with 2.5 μmol/L miR-31 LNA and equivalent scramble controls in a volume of 200 μL.When SAS xenografts grow to ∼0.2 cm 3 , the complexes were injected s.c. to the periphery of tumors (31).
Statistical and bioinformatics analysis.Pair-wise analysis was performed using website-accessible Generalized Association Plots (GAP) program. 9Mann-Whitney test and Fisher's exact test were used to compare the differences among variants.Receiver operating characteristic (ROC) analysis was performed, and the area under curve was used as a measurement for the level of separation between clinical settings.The leave-one-out cross-validation (LOOCV) model was used to compute the accuracy of assays.A P value of <0.05 was considered statistically significant.

Results
Upregulation of miR-31 in HNSCC.The miRNA expression profile of HNSCC was achieved from 10 screened tumors analyzed with qRT-PCR array (Supplementary Table S3).miRNAs having concordant ΔΔC t of >0 or <0 across at least seven screened tumors relative to their paired NCMT were selected for further stratification.The average ΔΔC t was >1 for 22 miRNAs and <−1 for 9 miRNAs.These miRNAs were therefore considered aberrant in the HNSCC samples (Fig. 1A).Six tumors had high correlation in the miRNA aberration.The highest overexpression was found for miR-31, and miR-21 was also markedly upregulated.GAP analysis showed a strong correlation between expression of miR-31 and miR-21, miR-34b and miR-34c, and miR-99a and miR-100 (Fig. 1B).Analysis of the 46 LCM-retrieved samples confirmed that there was an increase in miR-21, miR-31, and miR-371 in 89%, 84%, and 70% HNSCC relative to NCMT, respectively (Fig. 1C).miR-31 was confirmed to be shown the greatest level of overexpression in HNSCC, with a mean ΔΔC t of 3.64.ROC and LOOCV analyses indicated the analytic power (0.82) and accuracy (0.72) using miR-31 expression to dissect NCMT from HNSCC (Fig. 1D).The analytic power was better than that shown by miR-21 expression or miR-371 expression (Supplementary Fig. S1).No significant association was identified between miR-31 expression and clinical parameters.
miR-31 expression increased the oncogenic potential of HNSCC cells.HNSCC cells had higher miR-31 expression relative to NHOK (Supplementary Fig. S2).Blockage of miR-31 expression with miR-31 LNA treatment significantly decreased viability and migration of SAS cells (Fig. 2A).HNSCC cells were infected with lentivirus carrying miR-31.The de-tection of green fluorescence in SAS cells measured infectivity successfully (Fig. 2B, left).SAS-miR-31 cells exhibited upregulation of miR-31 expression by 32-fold relative to SAS-GFP control cells.There was also a remarkable Lac Z repression in SAS-miR-31 cells when transfected with the reporter plasmids miR-31-asR (Fig. 2B, right).This suggests the presence of targeting activity in exogenous miR-31.SAS-miR-31 had significantly greater proliferation, migration, and anchorage-independent growth than controls (Fig. 2C).SAS-miR-31 also showed significantly greater xenographic tumor growth than controls in nude mice (Fig. 2D, left).With the treatment of miR-31 LNA and atelocollagen mixture (32), the tumorigenic potential of SAS cells was attenuated (Fig. 2D, right).
miR-31 targets FIH.TargetScan and/or Pictar systems predicted hypoxia-associated genes FIH, HIF-3α, ING4, and PHD4, to be putative targets of miR-31.Downregulation of FIH mRNA expression and protein expression was found in SAS-miR-31 cells compared with controls.Conversely, upregulation of FIH mRNA expression and protein by ∼50% on miR-31 LNA treatment relative to controls was found (Fig. 3A).Preliminary assays excluded HIF-3α, ING4, and PHD4 as miR-31 targets (Supplementary Fig. S5).To prove the repression of FIH by miR-31, the pCMV-LacZ-FIH3′UTR reporter was used.LacZ activity was repressed more in SAS-miR-31 than in controls; however, the repression reverted on treatment with miR-31 LNA (Fig. 3B).Conservative and nonconservative predicted targets for miR-31 in the 3′ UTR of FIH was designated as the A site and the B site, respectively.The mutant reporter plasmids A Mut, B Mut, and AB Mut were generated.In SAS-miR-31 cells, the A site mutation resulted in a considerable reversion of the Lac Z repression compared with the B site mutation (Fig. 3C).Mutations in both A and B sites resulted in a complete reversion of LacZ repression.These findings suggest that the binding of miR-31 to the conservative site in 3′ UTR may repress FIH expression.For exogenous FIH expression, SAS-miR-31 cells and SAS-GFP cells were further infected with retroviruses to achieve FIH(3′UTR) and FIHWt subclones.The former expressed FIH coding sequence together with 3′ UTR, and the latter expressed only FIH coding sequence.These subclones exhibited an increase in FIH expression relative to control subclones (Fig. 3D, top).The FIH expression in FIH (3′UTR) and FIHWt subclones from SAS-miR-31 cells was lower than corresponding subclones from SAS-GFP cells.Similar changes were also present in OECM-1 cells (Supplementary Fig. S3H).Furthermore, FIH expression is higher in FIHWt subclones than FIH(3′UTR) subclones in both SAS-miR-31 cells and OECM-1-miR-31 cells (Fig. 3D, bottom).These findings validated the presence of a miR-31 target in the 3′UTR of FIH.Sequence complimentarity was found between miR-21 and FIH 3′ UTR using RNA22 10 program.The treatment with miR-21 mimic upregulated miR-21 expression and slightly repressed the FIH expression in SAS and OECM-1 cells (Supplementary Fig. S6).
FIH expression decreased the oncogenic potential of HNSCC cells in normoxia.Exogenous FIH expression was associated with decreased proliferation, migration, and anchorage-independent colony formation of SAS-GFP cells under normoxia culture conditions, but not in hypoxia culture (Fig. 4A).Exogenous FIH expression and hypoxia culture had only limited influence on the growth, but exogenous FIH expression significantly decreased migration and anchorage-independent colony formation of OECM-1-GFP cells in normoxia culture (Supplementary Fig. S3I-K).In hypoxic culture, the suppressive function of FIH(3′UTR) was remarkably attenuated.Because FIH function seemed inactivated in the hypoxia state, FIH D201A mutant subcone and FIH 1-302 truncaton subclone were established in SAS cells to insight the effects of FIH activity in normoxia (16,17).The expression of the exogenous mutant FIH and the truncated FIH was shown by Western blot analysis (Fig. 4B).The suppressive effect of wild-type FIH on anchorage-independent growth (Fig. 4C) and tumorigenesis (Fig. 4D) was significantly reversed by the mutation or the truncation.Thus, FIH activity is suppressive to SAS cells in normoxia.
miR-31 transactivated HIF activity in normoxia.Under normoxia, SAS-miR-31 cells and OECM-1-miR-31 cells exhibited a HIF-1α and HIF-2α expression level that is equal to the controls (Fig. 5A, top).SAS-GFP cells carrying exogenous FIH variants also had similar levels of HIF-1α and HIF-2α expression.SAS-miR-31 had higher HIF transactivation activity than controls, and this could be decreased by miR-31 LNA (Fig. 5A, bottom).The expression of HIF-1α and transactivation of HIF activity in SAS-miR-31 and controls were induced under hypoxia culture (Fig. 5B, top); however, exogenous miR-31 expression had no effect on HIF activation in hypoxia (Fig. 5B, bottom).In normoxia, SAS-miR-31 showed higher proliferation, migration, and anchorage-independent growth than controls, but this was not the case in hypoxia culture (Fig. 5C, a-c).VEGF production in SAS-miR-31 was higher than in the controls during normoxia, but VEGF production in SAS-miR-31 and the controls were similar under hypoxia (Fig. 5C, d).VEGF production in OECM-1-miR-31 was also higher than control cells during normoxia (Supplementary Fig. S2G).SAS-miR-31 xenografts showed higher VEGF production than SAS-GFP tumors, whereas SAS xenografts carrying exogenous FIH expression showed lower VEGF production (Fig. 5D).
Exogenous miR-31 expression decreased both cytosolic and nuclear FIH in SAS cells.Exogenous FIH expression resulted in a greater increase in cytosolic FIH compared with nuclear FIH; and exogenous miR-31 expression seemed to decrease nuclear FIH more remarkably than cytosolic FIH in SAS cells (Supplementary Fig. S7).Exogenous miR-31 decreased FIH expression and exogenous FIH increased FIH expression in SAS xenografs (Supplementary Fig. S8).
Decreased FIH and increased VEGF during head and neck carcinogenesis.FIH immunoreactivity was detected in the lower half of the epithelium in NCMT.This displayed heterogeneity in terms of cellular localization including the cytosol, membrane, and nucleus (Fig. 6A, a, b and B, a).Intensive cytosolic FIH was found in all HNSCC (Fig. 6A, a, c  and B, a, b).In tissue pairs available for comparison, the FIH immunoreactivity was remarkably attenuated in the NML in relation to their HNSCC counterparts (Fig. 6B, b,  c).A statistically significant difference in FIH immunoreactivity was found between HNSCC and NML (Supplementary Table S4).Nuclear FIH was obvious in the superbasal cell layer of the NCMT samples, and it seemed stronger than the cytosolic FIH (indent in Fig. 6A, d).Only 24% of HNSCC samples were scored as having nuclear FIH immunoreactivity in >10% cells.A statistically significant decrease in nuclear FIH from NCMT samples to HNSCC samples was noted (Supplementary Table S4).A reverse association lying between miR-31 expression and nuclear FIH immunoreactivity was also noted in HNSCC (Supplementary Table S5).
There was 18% NCMT (Fig. 6C, a) and >60% HNSCC (Fig. 6C, b) or NML (Fig. 6C, c) having cytosolic VEGF immunoreactivity in >50% cells.A statistically significant increase of VEGF immunoreactivity from NCMT to HNSCC was noted (Supplementary Table S6).A trend for reverse association lying between nuclear FIH immunoreactivity and VEGF immunoreactivity was found in HNSCC samples.A reverse association lying between nuclear FIH immunoreactivity and VEGF immunoreactivity was noted in NML and the overall tumor tissues consisting of HNSCC and NML (Supplementary Table S7).

Discussion
We identified significant upregulation of miR-31 in HNSCC tumors and cell lines, which is similar to the situation with several important human cancers (7,(21)(22)(23)(24).However, only expression studies were carried out during the above research, and no study up to the present has ever addressed the regulation and function of miR-31 in HNSCC, CRC, or HCC (7,(21)(22)(23)(24).Our functional characterization verified the oncogenic roles of miR-31 in HNSCC, and this result was supported by the enhancement of in vitro oncogenicity and tumorigenesis after overexpression in cell lines.The oncogenic roles of miR-31 were also substantiated by in vitro or in vivo clues from miR-31 blockage.It seems that the increase in tumor induction was limited when miR-31 was overexpressed in SAS and OECM-1 cells.Because the endogenous expression of miR-31 has been found to be high in HNSCC tissues and cells, the exogenously expressed miR-31 might allow only limited further enhancement of tumor growth.We provide evidence that the oncogenic potential of miR-31 is abrogated in hypoxia status.As a consequence, SAS or OECM-1 xenografts are postulated to contain a high fraction of hypoxic cells and limited miR-31 functionality.Our study also confirmed the upregulation of miR-21 and miR-371 in HNSCC (6)(7)(8).Although the dissection power of miR-21 expression did not exceed that of miR-31 in distinguishing benign from malignant status, miR-21 should still play key roles in HNSCC and other malignancies, because it targets crucial suppressors (4,5,8,33).
Hypoxia is linked to epithelial-mesenchymal transition, metastasis, and therapy resistance among neoplasms (12).Using blockers, an exogenous expression system, reporters, and mutant constructs, we were able to verify that miR-31 targets the conservative complementary sequence in the 3′ UTR of FIH and transactivates HIF under normoxia.This resulted in an increase in VEGF and other factors that could be contributive to the HNSCC pathogenesis (11,12).Although miR-17-92 cluster and miR-210 were shown involved in the regulation of hypoxia (34)(35)(36), this is the first study that identifies a miRNA, which is overexpressed in HNSCC, as being involved in the indirect activation of HIF through targeting FIH during normoxia.Several HNSCCs were found to have extremely low miR-31 expression.Loss of heterozygosity at 9p21 frequently altered in HNSCC could be the underlying genomic event (37).Preliminary analyses showed that miR-21 repressed FIH, whereas further analyses are required to clarify FIH as a target of miR-21.Because miR-21 expression was correlated with miR-31 expression in HNSCC, their synergistism for HNSCC development should be unraveled.It was known that HIF activation occurs during hypoxia and that this usually occurs at a late stage of neoplastic process.Because the downregulation of miR-31 occurs in metastatic breast carcinoma (27) and tumor metastasis occurs mainly in deeper tissue regions probably having oxygen deficiency (38), wherein miR-31 could be unable to activate HIF to augment the aggressiveness of HNSCC, different mechanisms regulated by FIH in hypoxic tumor regions must be further investigated.Whether miR-21 plays more prominent roles for HNSCC metastasis than miR-31 is also an interesting issue to be addressed (8).
FIH suppressed HNSCC during normoxia, and the suppression was abrogated in hypoxic culture.The findings were concordant with the fact that miR-31 enhanced oncogenicity only when oxygen supply is sufficient.This study also provided functional clues by inactivating FIH using mutant constructs, which specified the requirement of FIH activity for the tumor suppression in normoxia (16,17).Nuclear FIH was prominent in the superbasal layers of NCMT, whereas it was significantly decreased in HNSCC.Nuclear FIH expression was opposite to the miR-31 expression in HNSCC.Moreover, a reverse association between nuclear FIH expression and VEGF expression was also found in tumor tissues.These lines of tissue evidence together with the functional clues provided above indicated a miR-31-FIH-HIF cascade involved in HNSCC development in normoxia state.Because a reverse association between nuclear FIH expression and VEGF expression was also noted in metastatic lesions, it suggested that the oxygen level in these tissues could be sufficient for nuclear FIH to hydroxylate HIF.The high abundance of FIH protein, particularly cytosolic FIH, was found in HNSCC tissues and cell lines and in other types of cancers (39).In HNSCC cells with exogenous FIH expression, the FIH is localized mainly in cytosol with only a small fraction of the FIH being distributed to the nucleus or onto the membrane (40).In addition, the high cytosolic FIH levels seem to define a poor prognosis or progression for breast carcinoma, nonsmall cell lung carcinoma, and pancreatic endocrine tumors (18)(19)(20).The high abundance of FIH in the cytosol of HNSCC cells might act as a reservoir and allows it to act as a direct oxygen sensor.Alternatively, because FIH also modulates Notch and IκBα by hydroxylation (14,15,39,40), the stability or activity of Notch and IκBα being regulated by cytosolic FIH for pathogenesis requires further stratification using specific constructs.
This study identified that miR-31 contributes to HNSCC development through HIF activation in tumor regions, wherein oxygen level are above the threshold for FIH activity.miR-31 expression was known to repress several target genes to inhibit the metastasis of breast carcinomas (27); thereby, the identification of other miR-31 targets and pluripotent actions of miR-31 involved in different neoplastic stage of tumorigenesis will be important.Further understanding of the pathogenetic significance of other aberrantly expressed miRNAs identified in this study may eventually contribute to diagnosis and interception of HNSCC (11).

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Figure 1 .
Figure 1.miRNA expression profile.A, 31 differentially expressed miRNAs with an average ΔΔC t of >1 or <−1 in tumor cells compared with normal tissue.Mean ± SEM of the ΔΔC t from 10 screened tumor tissues.Green, upregulated; red, downregulated.B, GAP analysis.Left, unsupervised hierarchical algorithm of miRNA expression of patients; right, association plot analysis using miRNA expression profile indicated an association of expression profile in several clusters of miRNA marked by dark red.C, scatter dot plot of the expression of miR-31, miR-21, and miR-371 in 46 LCM-retrieved tissues.Horizontal lines, mean values.D, ROC and LOOCV analysis of miR-31.It revealed a sensitivity of 0.76 and a specificity of 0.85 using miR-31 expression to distinguish NCMT from HNSCC.

Figure 2 .
Figure 2. Phenotypic effect of miR-31.A, blockage of miR-31 in SAS cells.miR-31 expression was decreased with miR-31 LNA treatment for 24 h.The treatment for 48 h also decreased cell viability and migration.B, left, green fluorescence in SAS-miR-31 cells and SAS-GFP cells; right, reporter assay.It showed the targeting of miR-31 to antisense sequence in reporter plasmid miR-31-asR, which resulted in a repression of β-galactosidase activity in SAS-miR-31 cells compared with controls.C, exogenous miR-31 expression increased proliferation (left), migration, and anchorage-independent colony formation (right) in SAS cells.D, tumorigenesis.Exogenous miR-31 expression increased the growth of SAS xenografts (left), whereas the blockage of endogenous miR-31 expression using miR-31 LNA complexed with atelocllagen decreased the tumorigenic potential of SAS cells (right).Mean ± SEM from triplicate or quadruplicate analysis or from at least five mice.*, P < 0.05; **, P < 0.01; ***, P < 0.001; Mann-Whitney analysis.

Figure 3 .
Figure 3. miR-31 targets FIH.A, FIH expression in SAS cells with exogenous miR-31 expression or after treatment with miR-31 LNA.Top, Western blot analysis; bottom, quantification of FIH mRNA expression and FIH protein expression.B and C, reporter assays.B, SAS-miR-31 cells transfected with reporter plasmid pCMV-LacZ-FIH3′UTR exhibited a remarkable decrease in β-galactosidase activity comparing with controls.The repression was reverted after treatment with miR-31 LNA.C, SAS-miR-31 cells were transfected with pCMV-LacZ-FIH3′UTR and mutant reporters.The repression of β-galactosidase activity was not seen after transfection with A Mut and AB Mut.The results suggested that the A sequence may be the crucial miR-31 target of FIH.Mean ± SEM from triplicate or quadruplicate analysis.*, P < 0.05; **, P < 0.01; ***, P < 0.001.NS, not significant.Mann-Whitney analysis.D, Western blot analysis of FIH expression in various subclones.Numbers below the figures are the normalized FIH expression values.

Figure 4 .
Figure 4. FIH suppresses the oncogenicity in normoxia.A, the phenotypes of SAS cells.a-c, assays for proliferation, migration, and anchorage-independent colony formation, respectively.N, normoxia; H, hypoxia.B-D, SAS-GFP cells.B, Western blots analysis of FIH expression in various subclones.An arrow indicates the truncated FIH.C and D, anchorage-independent colony formation and tumorigenesis, respectively, for various subclones.Mean ± SEM from triplicate or quadruplicate analysis or five nude mice.*, P < 0.05; **, P < 0.01.Mann-Whitney analysis.