| Background and ObjectivesMicroRNAs (miRNAs or miRs) play important roles in development, cellular differentiation, proliferation, cell-cycle control, and cell death, and have been implicated in a variety of human diseases, including cancer. Despite being one of the original miRNAs discovered, the biological role of miR-3188 and its molecular mechanisms underlying cancer initiation and progression have not been reported.Nasopharyngeal carcinoma (NPC) is a tumor type arising from the epithelial cells that line the nasopharynx. It is common in certain regions of East Asia, with Epstein-Barr virus (EBV) exposure, diet and genetic factors implicated in its etiology. Although relatively rare in the USA, NPC accounts for one third of childhood nasopharyngeal neoplasms. In recent studies, abnormal expression of miRNAs was been broadly implicated in the pathogenesis of NPC. For example, Epstein-Barr virus-encoded microRNA BART1 induces tumour metastasis by regulating PTEN-dependent pathways. In addition, tumor suppressor PDCD4 modulates miR-184-mediated direct suppression of c-MYC and BCL2 blocking cell growth and survival.FOXO1 is a transcription factor and a member of the FOXO subfamily of the Forkhead/winged helix family. The phosphorylation of FOXO 1 by AKT leads to its inactivation after nuclear to cytoplasmic translocation. Previous evidence has supported that FOXO1 functions as tumor suppressor based on its role in regulating cell cycle progression, differentiation, metabolism and survival. Furthermore, decreased FOXO1 expression has been demonstrated in many tumor types, such as Hodgkin lymphoma, breast cancer and alveolar rhabdomyosarcoma. Recent evidence suggested that LMP1 silencing slows cell growth and enhances chemosensitivity through inhibition of AKT signaling pathway and its downstream factor phospho-FOXO1 in EBV-positive nasopharyngeal carcinoma cell line. Elevated levels of phosphorylated AKT also correlated with phospho-FOXO1 in NPC samples. However, the detailed role of FOXO 1 in the suppression of NPC cell growth remains unclear.Here, we examined the relationship between miR-3188, mTOR and FOXO1 in NPC and found an atypical miR-3188-mTOR-pPI3K/AKT-c-JUN feedback loop modulated by FOXO1. This pathway suppresses the proliferation of NPC. Together these results provide a mechanism by which miR-3188 modulates NPC cell growth.Contents and methods1. The effect on cell biology of NPC by miR-3188 and the molecular mechanism(1) We used MTT assay, colony formation, cell cycle analysis, and Edu incorporation assays, xenograft tumor to examine the effect of miR-3188 expression on NPC cells or NP cell growth and definite the function of miR-3188 in NPC cells.(2) To explore the mechanisms by which miR-3188 suppresses NPC cell proliferation, Western blot was used to examine whether the expression of p-PI3K, p-AKT, c-JUN, CCND1, P27, P21 were influenced by miR-3188.2. miR-3188 directly targets mTOR.(1) Through TargetScan and RNAhybrid algorithms, mTOR was predicted to be a direct target of miR-3188;(2) mTOR expression was detected by qPCR in miR-3188-overexpressing or miR-3188-inhibited NPC cells;(3) Western blots of mTOR and p-mTOR in miR-3188-overexpressing or miR-3188-inhibited NPC and NP69 cells;(4) mTOR expression was evaluated by immunohistochemistry in xenografts derived from NPC cells;(5) Luciferase reporter assay was used to determine miR-3188 direct targeting the mTOR3’UTR;(6) MTT assays, EdU incorporation assays and FACS assays of NPC cells were performed after transfection with NC, ectopic mTOR or miR-3188;(7) Western blot of endogenous mTOR, p-mTOR, PI3K, P-PI3K, AKT, P-AKT, CCND1 and c-JUN protein expression levels in HONE1, SUNE1 and 5-8F cells treated with si-mTOR or si-control.3. c-JUN binds the promoter region of human miR-3188 and inhibits its transcription(1) To test the transcriptional regulatory mechanisms of miR-3188 expression, UCSC, PROMO, and TFSEARCH bioinformatics software was utilized to analyze a 3-kb region upstream of the transcription start site (TSS) of miR-3188. Three c-JUN-binding motifs at -492 to -498,-1628 to-1634 and -2356 to -2362 were identified inside the putative miR-3188 promoter region;(2) Knocking down c-JUN expression by siRNA stimulated miR-3188 expression in SUNE1, HONE1-EBV and 5-8F cells;(3) The EMSA results detected whether the three predicted c-JUN binding sites in the promoter region of miR-3188 were functional;(4) Chromatin immunoprecipitation (ChIP) assays further confirmed that c-JUN protein was recruited to all the three binding sites in the putative miR-3188 promoter in SUNE1 and HONE1-EBV;(5) Luciferase indicate that c-JUN binds to specific promoter TFBS of miR-3188 and inhibits transcription.4. The function and mechanism research of FOXO1 in NPC(1) We used MTT assay, colony formation, cell cycle analysis, and Edu incorporation assays, xenograft tumor to examine the effect of miR-3188 expression on NPC cells or NP cell growth and definite the function of miR-3188 in NPC cells;(2) To explore the mechanisms by which miR-3188 suppresses NPC cell proliferation, Western blot was used to examine whether the expression of p-PI3K, p-AKT, c-JUN, CCND1, P27, P21 were influenced by miR-3188;(3) Immunofluorescence and Immunohistchemistry confirmed reduced expression of c-JUN in FOXO1-overexpressing NPC cells;(4) ChIP assay revealed less c-JUN binding to the miR-3188 promoter in FOXO1-overexpressing NPC cells compared to control cells.5. miR-3188 is induced by FOXO1(1) Using miRNA array to detect differentially expressed miRNAs in cells with miR-3188 overexpressed, and detecting the expression of miR-3188 by qPCR in cells with FOXO1 overexpression or knockdown, which was upregulated the most obvious after FOXO1 overexpression;(2) Reduction of miR-3188 by its specific inhibitor could reverse the growth suppressive effect after ectopic FOXO1 expression in MTT and Edu assays;(3) Western blot analysis showed that treatment with a miR-3188 inhibitor increased expression of p-PI3K, p-AKT, c-JUN and CCND1 but reduced p27 and p21 levels in FOXO1-overexpressing NPC cells;(4) Specific PI3K inhibitor Ly294002 reversed the changes in miR-3188 expression in NPC cells with both FOXO1-overexpression or silencing.6. miR-3188 is associated with pathoclinical features and clinical associations of miR-3188 in NPC.(1). Levels of miR-3188 were detected in eight NPC cell lines and NPCs compared to NP tissues by QPCR;(2). in situ hybridization assay confirmed reduced expression of miR-3188 in NPC tissues compared to NP tissues;(3). Levels of mTOR, C-JUN, FOXO1 were detected in NPCs compared to NP tissues by QPCR;(4). The relationship between miR-3188, mTOR, FOXO1 and c-JUN.Results1. miR-3188 regulates cell growth.(1) miR-3188 expression was elevated in NP69 and SXSW-1489 cells but weakly expressed in NPC cells.(2) Using MTT assay, colony formation, cell cycle analysis, and Edu incorporation assays, we found that overexpressed miR-3188 significantly suppressed cell growth and G1 to S cell cycle transition in HONE1-EBV and SUNE1 cells. Conversely, suppression of miR-3188 markedly restored cell proliferation and induced G1/S transition in NP69 and 5-8F cells.(3) The mice injected with HONE 1-EBV-miR-3188 and SUNE1-miR-3188 cells had smaller tumor burdens and displayed lower expression of Ki67 and PCNA in tumor tissues relative to controls. These results suggested miR-3188 significantly inhibits tumorigenesis in vivo.(4) miR-3188 overexpression downregulated c-JUN and CCND1 but enhanced p27 and p21. miR-3188 inhibitors rescued these decreased levels. Interestingly, miR-3188 knockdown in 5-8F cells exhibited opposite results and miR-3188 mimics could restore levels of these cell cycle regulators. Furthermore, we found levels of p-PI3K and p-AKT were decreased in miR-3188-overexpressing SUNE1 and HONE1-EBV cells yet increased in miR-3188-inhibited 5-8F cells. These results suggest that miR-3188 decreases cell growth by inactivating PI3K/AKT as well as downstream c-JUN and G1/S cell cycle transition signaling.2. miR-3188 directly targets mTOR.(1) Through TargetScan and RNAhybrid algorithms, mTOR was predicted to be a direct target of miR-3188.(2) Overexpression of miR-3188 downregulated mTOR mRNA and protein levels as well as p-mTOR levels in HONE1-EBV and SUNE1 cells. Conversely, miR-3188 downregulation elevated mTOR and p-mTOR levels in 5-8F and NP69 cells.(3) Consistent with in vitro results, immunohistochemistry of xenografts generated from HONE1-EBV-3188 and SUNE1-3188 cells revealed a marked reduction in mTOR expression.(4) Simlarly, cotransfection miR-3188 mimics significantly decreased mTOR luciferase reporter activity (lanes 1 and 2; P<0.05) while miR-3188 inhibitor had the opposite effect (lanes 3 and 4; P<0.05). These effects on luciferase activity were abrogated when cotransfected with mutated mTOR reporter (lanes 5 and 6, P= 0.078). Collectively, these data suggest that miR-3188 exerts its effects in NPC through direct suppression of mTOR.3. mTOR overexpression rescues the function of miR-3188.(1) Transiently transfecting mTOR into miR-3188 overexpressing NPC cells enhanced cell proliferation by MTT and EdU incorporation assays as well as promoted G1 to S cell cycle transition.(2) Furthermore, we found that mTOR overexpression induced expression of c-JUN and CCND1 but reduced p27 and p21. These results indicate that mTOR overexpression can overcome NPC cell growth suppression induced by miR-3188.(3) Subsequently, we found that levels of mTOR, p-mTOR, p-PI3K, p-AKT, CCND1, and c-JUN were significantly decreased while p27 and p21 were elevated after mTOR siRNA treatment. These results were consistent with miR-3188 overexpression, suggesting that mTOR is a direct target of miR-3188 responsible for suppressing cell growth.4. c-JUN binds the promoter region of human miR-3188.(1) Three c-JUN-binding motifs at -492 to-498,-1628 to-1634 and -2356 to-2362 were identified inside the putative miR-3188 promoter region. These 3 transcription factor-binding sites (TFBSs) were named A, B and C.(2) qPCR analysis indicated that miR-3188 expression was markedly increased in all lines after c-JUN knockdown, suggesting that c-JUN is an upstream regulator of miR-3188.(3) EMSA experiment showed a shift band was formed when the probe of DIG-ddUTP labeled c-JUN was incubated with the nuclear protein extracted from SUNE1 and HONE1-EBV cells (Lane 2 and 8), whereas the band was nearly gone when unlabelled oligonucleo tides of c-JUN were added as binding competition (Lane 6 and 12). Bands were not affected when mutated A, B or C was added to compete with DIG-ddUTP-labeled A, B or C in SUNE1 cell and HONE1-EBV cell (Lane 3-5, Lane 9-11). The EMSA results demonstrate that the three predicted c-JUN binding sites in the promoter region of miR-3188 were functional.(4) Chromatin immunoprecipitation (ChIP) assays further confirmed that c-JUN protein was recruited to all the three binding sites in the putative miR-3188 promoter in SUNE1 and HONE1-EBV.(5) Furthermore, a reduction of the wildtype miR-3188 promoter luciferase activity was observed upon upregulation of c-JUN in the HEK293T, SUNE1 and HONE1-EBV cell lines. A similar effect was observed when sites A and B, sites A and C, sites B and C, and sites A, B and C were mutated respectively in 293 T, SUNE1 and HONE1-EBV cells (P< 0.05). These data indicate that c-JUN binds to specific promoter TFBS of miR-3188 and inhibits transcription.5. FOXO1 inactivates the PI3K/AKT/c-JUN pathway.(1) To evaluate its functional significance on cell proliferation, we used a lentiviral vector overexpress FOXO1 in HONE1-EBV, SUNE1 and 5-8F cell lines. Significant upregulation was confirmed for each line, which markedly inhibited cell growth and cell cycle G1/S transition in NPC cells by MTT, colony formation, flow cytometry, and EdU incorporation assays. Further, we used siRNA to knock down FOXO1 and found siFOXO1s could reverse the cell growth suppresion mediated by ectopic expression.(2) To further confirm the growth-suppressive effect of FOXO1, we performed in vivo tumorigenesis experiment in nude mice. Tumor volumes and growth rates were significantly decreased in tumors derived from FOXO1-overexpressing HONE1-EBV and SUNE1 cells. These tumors also exhibited a reduction in Ki67 and PCNA expression by immunohistochemistry. These results suggest that FOXO1 exerts a significant inhibitory effect on tumorigenesis in vivo.(3) Overexpression of FOXO1 significantly reduced the levels of p-PI3K, p-AKT, mTOR and p-mTOR. Furthermore, we found that ectopic FOXO1 reduced expression of c-JUN and CCND1 but upregulated p21 and p27. Interestingly, the opposite results were observed after siRNA-mediated suppression of ectopic FOXO1. Further, the specific PI3K inhibitor Ly294002 significantly reversed the expression of p-PI3K, p-AKT, mTOR, p-mTOR, c-JUN, CCND1, p21 and p27.(4) We used immunofluorescence to confirm reduced expression of c-JUN in FOXO1-overexpressing NPC cells. This was also confirmed by immunohistochemistry of FOXO1-overexpressing tumor tissues derived from NPC mouse models. Finally, ChIP assay revealed less c-JUN binding to the miR-3188 promoter in FOXO1-overexpressing NPC cells compared to control cells.All the results suggest that FOXO1 regulates NPC cell proliferation and cell cycle progression through the PI3K/AKT/c-JUN pathway.6. miR-3188 is induced by FOXO1 through PI3K/AKT/c-JUN.(1) miR-3188 was confirmed as a positive modulator of FOXO1 via qRT-PCR in NPC cells treated with Mock, FOXO1 or both FOXO1 and siFOXO1.(2) Reduction of miR-3188 by its specific inhibitor could reverse the growth suppressive effect after ectopic FOXO1 expression in MTT and Edu assays.(3) Western blot analysis showed that treatment with a miR-3188 inhibitor increased expression of p-PI3K, p-AKT, c-JUN and CCND1 but reduced p27 and p21 levels in FOXO1-overexpressing NPC cells. These results indicate that miR-3188 is induced by FOXO1 and suppresses NPC cell growth.(4) Specific PI3K inhibitor Ly294002 reversed the changes in miR-3188 expression in NPC cells with both FOXO1-overexpression or silencing. This suggests that FOXO1 positively regulated the expression of miR-3188 through PI3K/AKT pathway. Taken together, these results support that miR-3188 expression is induced by FOXO1 through PI3K/AKT/c-JUN signaling.7. miR-3188 is associated with pathoclinical features.(1) Levels of miR-3188 were significantly decreased in eight NPC cell lines and NPCs compared to NP tissues by qPCR analysis (P= 0.00037, P= 0.00033, respectively).(2) Further, in situ hybridization assay confirmed reduced expression of miR-3188 in NPC tissues compared to NP tissues.(3) We did not find a significant association between miR-3188 expression level and patient age, sex, clinical stage (Ⅰ-Ⅱ versus Ⅲ-Ⅳ), lymph node metastasis (N classification; NO-N1 versus N2-N3) or distant metastasis stage (M classification) in the 142 NPC cases. However, we observed that reduced miR-3188 expression was negatively correlated with tumor size (T classification; P= 0.011).(4)Subsequently, we found that NPC patients with high miR-3188 expression had longer survival times than those of patients with low miR-3188 levels (P= 0.009).8. Clinical associations of miR-3188 in NPC.(1) mTOR and c-JUN expression were significantly higher in NPC than in NP samples (P= 0.0282, P<0.0001, respectively) while FOXO1 expression was significantly lower in NPC samples (P<0.0001).(2) miR-3188 expression was positively correlated with FOXO1 expression (P= 0.0326) but negatively associated with mTOR (P= 0.0288) and c-JUN expression (P = 0.0006) in the same NPC specimens.Conclusions1. miR-3188 inhibits NPC cell growth through PI3K/AKT pathway;2. miR-3188 directly targets mTOR;3. mTOR overexpression rescues miR-3188-mediated cell growth suppression;4. c-JUN binds the promoter region of human miR-3188;5. miR-3188 is induced by FOXO1 through PI3K/AKT/c-JUN and FOXO1 inhibits NPC cell proliferation;6. Downregulated miR-3188 is an unfavorable factor in NPC and is positively correlated with FOXO1 but negatively with mTOR and c-JUN. |
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