Sulforaphane Inhibits Proliferation Of Nasopharyngeal Carcinoma Through MiRNA-124-3p-mediated Downregulation Of STAT3

BackgroundSulforaphane is one of common antioxidants and is rich in cruciferous vegetables such as broccoli and kale. Traditionally SF is used as a chemopreventive agent against cancer initiation and the mechanisms of action of SF has been attributed to its ability in inhibiting phase1metabolism enzymes that convert procarcinogens to carcinogens and inducing phase2metabolism enzymes that promote excretion of carcinogens from the body. Once cancer is initiated, SF can modulate diverse cellular activities, such as inhibiting the growth of cancer cells, inducing apoptosis and cell cycle arrest and suppressing angiogenesis and metastasis by different signaling pathway. Recent studies revealed that SF inhibits cancer stem-like cells (CSCs) via different mechanisms. In colon cancer, SF arrests cell cycle by increasing cyclinA and cyclinB1and induces cell apoptosis through phosphoric acid acyl serine protein membrane eversion, chromatin concentration and other ultrastructural modifications. In addition, SF can also protect cells from DNA methylation by reducing the expression of5hydroxytryptamine receptor in colon cancer. In lung adenocarcinoma, SF can promote cell apoptosis by regulating caspase3, and inhibit tumor occurrence and development in nude mice. In breast cancer, SF inhibits CSCs through suppressing Wnt/β-catenin. In pancreatic cancer, SF targets pancreatic CSCs by NF-kB-induced antiapoptotic signaling. In prostate cancer, SF regulates cell differentiation and self-renewal ability by suppressing the expression of Notch1and Sox2. It also lowers the invasion and metastasis through reducing ALDH1activity. In addition to acting as an antioxidant and antitumor reagent, SF also exhibits antiviral effect. It was reported that SF is effective in the inhibition of EBV reactivation in latent EBV-positive in nasopharyngeal carcinoma cells by suppressing transactivation activity of the EBV immediate-early gene Rta but not Zta.Signal Transducer and Activator of Transcription (STAT) family comprise seven members:STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. They can be activated by growth factors and cytokines via phosphorylation. Acitivation of STAT leads to formation of homologous or heterologous dimmers and translocation into the nucleus where they regulate gene transcription by binding to specific DNA response elements. Among the STAT family, STAT3is the only member that leads to early embryonic lethality when the gene is disrupted. Accumulated evidence demonstrated that STAT3plays critical roles in tumorigenesis in a wide range of human cancers. In addition, the involvement of STAT3in cancer cell growth and invasion has been defined in various human malignancies including NPC. In response to a variety of diverse agents such as cytokines and growth factors, STAT3gets phosphorylated at the Tyr705located in the C-terminal domain and this phosphorylation leads to its activation. In addition, STAT3can also be phosphorylated on the Ser727, which further enhances transcriptional activity of STAT3. SF was reported to inhibit the activity of P-STAT3(Tyr705) in prostate cancer. In pancreatic cancer, SF and benzyl isothiocyanate combination can significantly reduce the expression of STAT3and its phosphorylation level. There are many studies of developing drug targeted STAT3, and the antitumor effects of SF are complex. It is necessary to research the mechanism in NPC.MicroRNAs (miRNAs) are small non-coding RNAs with16-26bases in length. MiRNAs have been reported to regulate a large number of cellular pathways, such as cellular proliferation, differentiation, and apoptosis. MiRNAs exert its functions by binding to the3’-untranslated region of a subset of mRNAs through base pairing, thus resulting in a reduced translation rate or increased degradation of the mRNA. Dysregulation of miRNAs expression levels are associated with various human malignancies. MiRNAs can be classified as onco-miRs or tumor suppressor-miRs based on their biological functions. MiRNA-124acts as a tumor suppressor and downregulation of miR-124were frequently observed in gastric, cervical, and breast cancer. A recent study found that miR-124is absent in all grades and pathologic types of gliomas. Upregulation of miR-124in glioma cancer stem cells inhibited the STAT3pathway. The expression of STAT3in HONE1and CNE1is abnormal. We determine whether SF can reduce the expression of STAT3in HONE1and CNE1cells and miRNAs participate in this process.In this study we investigated the antitumor effect of SF and the underlying mechanisms in NPC. We use HONE1, CNE1, CNE2and Sunel as object, to observe the influence of SF in cell growth, apoptosis and tumor sphere growth and to determine whether SF has effects on CSCs and STAT3. These maybe provide a theoretical basis on prevention and treatment in NPC on the further.Method1. Examination of the effect of SF on cells growth. Cell survival rates after treatment with SF for48hours were measured by MTT assay, and the half inhibitory concentrations (IC50) of the two cell lines were determined.2. SF induces apoptosis of NPC cells. HONE1and CNE1cells were treated with increasing concentrations of SF for48hours followed by apoptosis analysis with Annexin V Apoptosis Detection kit. Cells were collected, washed, and stained with Annexin V and PI as recommended by the manufacture’s instruction. The apoptotic cells were detected by fluorescence activated cell sorting (FACS).3. SF suppresses tumor sphere growth of NPC cells. Stem/progenitor cells are enriched in tumorsphere of many cancers based on the unique ability of stem/progenitor cells to grow and form spheres in serum-free medium. HONE1and CNE1cells were cultured in tumorsphere forming medium. Cells were cultured in a density of1000-1500cells/ml. The tumor spheres formed5days from HONE1and CNE1were treated with different concentration of SF.14days after treatment, the number of tumor sphere was counted and diameter of tumor sphere was measured in microscope.4. The influence of SF on CSCs in NPC cells. HONE1, CNE1, CNE2and Sunel were treated with different concentration of SF (0,5,10,20μmol/L) for48hours. The expression of P-catenin, Nanog, c-Myc, Oct3/4and Sox2were examined by Western blot analysis.5. The expression of STAT3protein in NPC treated with SF. HONE1, CNE1, CNE2and Sune1cells were treated with different concentrations of SF (0,5,10,20μmol/L) for48hours. The expression of STAT3protein and its level of dephosphorylation were examined by Western blot analysis. HONE1and CNE1cells were treated with10μmol/L SF or DMSO for48hours. The expression of STAT3protein and its level of dephosphorylation were examined by immunofluorescence assay. 6. The influence of SF in HONE1and CNE1on STAT3, p-catenin and Nanog under5ng EGF. There are5groups:1, Control (DMSO);2, DMSO+5ng EGF;3,5ng EGF+5μM SF;4,5ng EGF+10μM SF;5,5ng EGF+10μM SF. After48hours, Cells were collected and lysed. The lysates were subjected to SDS-PAGE and expression of STAT3, P-catenin and Nanog were detected by Western blot analysis.7. Regulation of STAT3by miR-124-3p. Three different miRNA databases were searched for miRNAs targeting STAT3. Six of them were selected for the next experiment. To identify miRNAs regulated by SF, HONE1and CNE1cells were treated with three different concentrations of SF or DMSO for48hours. Total RNA was extracted, and reversely transcripted with specific primers expression levels of miRNAs were measured by fluorescence quantitative polymerase chain reaction (Q-PCR). The miRNA-124-3p was most significantly upregulated in both HONE1and CNE1cells after treatment with SF. Bioinformatics analysis revealed that STAT3is a target gene of miR-124-3p and a previous study reported that the expressions of STAT3were regulated by miRNA-124-3p directly.8. SF reduced the STAT3expression via miRNA-124-3p in HONE1and CNE1. HONE1and CNE1cells were transfected with micrornas-124-3p mimic or inhibitor in the following combinations:1, Control;2, miRNA-124-3p mimic;3, miRNA-124-3p inhibitor;4, SF (10μmol/L) and5, miRNA-124-3p inhibitor+SF (10μmol/L). After48hours, Cells were collected and lysed. The lysates were subjected to SDS-PAGE and expression of STAT3was detected by Western blot analysis.Results1. SF inhibits the growth of HONE1and CNE1with IC50values9.51μmol/L and16.86μmol/L, respectively. HONE1cells are more sensitive to SF.2. The apoptosis of HONE1and CNE1treated with various concentrations of SF were detected by FACS. The results showed that SF induces apoptosis in HONE1and CNE1cells. The apoptosis rate was increased with the increasing concentrations SF. The apoptosis rates of HONE1and CNE1treated with0,5,10or20μmol/L SF after48hours were0.9%,1.2%,2.2%,6.4%and0.9%,1.0%,1.3%,5.0%, respectively. The results showed that SF induces apoptosis in HONE1and CNE1cells.3. SF suppresses the growth of tumor sphere from HONE1and CNE1. The volume of tumor sphere was reversely correlated with the concentration of SF.4. The expression of β-catenin, Nanog, c-Myc, Oct3/4and Sox2in NPC cells were decreased by SF in a dose-dependent manner. The results showed that SF inhitited CSCs in HONE1, CNE1, CNE2and Sunel.5. Western blot experiments showed that the expressions of STAT3were decreased in NPC cells treated with SF for48hours. The expression levels of STAT3were decreased by SF in a dose-dependent manner. Immunofluorescence test display10μmol/L SF decreased STAT3, p-STAT3(Tyr705) and p-STAT3(Ser727) expression in HONE1and CNE1compared with control.6. Under the condition of5ng EGF exciting, SF can also decreased the expression of STAT3, β-catenin and Nanog in NPC cells.7. SF decreased the expression of several miRNAs and the expression of miRNA-124-3p was most significantly upregulated. Treatment with5,10or20μmol/L SF increased expression of miRNA-124-3p2,3.5,4folds in HONE1and1.5,1.5,3.5folds in CNE1, respectively.8. Western blot analysis showed that miRNA-124-3p reduced the expression of STAT3to the level equivalent to the effect of10μmol/L SF treatment for48hours, whereas treatment with miRNA-124-3p inhibitor blocked the inhibitory effect of SF on STAT3expression.Conlusion 1. SF inhibits cell growth, induces apoptosis and suppresses growth of tumor sphere from CNE1and HONE1cells.2. SF decreases the expression of P-catenin、Nanog、c-Myc、Oct3/4and Sox2in NPC cells.3. SF reduces the expression of STAT3, p-STAT3in HONE1and CNE1.4. SF decreases the expression of STAT3, β-catenin and Nanog in NPC cells under5ng EGF exciting.5. SF inhibits STAT3expression through upregulation of miRNA-124-3p.