| Backgroud and ObjectionForkhead box (FOX) K1 is a member of the FOX transcription factor superfamily,which is associated with several cancers. Our previous findings showed that FOXK1 enhanced the occurrence and development of colorectal cancer. However, whether FOXK1 expression contributes to gastric cancer (GC) development and progression remains unknown. We analyzed the FOXK1 promoter using Promo software and found several binding sequence transcription factors, including c-jun. As an identigied oncogene,c-jun is the main downstream molecule of classic JNK pathway,which involves in the proliferration,migration and invasion of many kinds of cancer including GC. This issue was targeted to investigate the effects of FOXK1 on GC,and find the relationship between c-jun and the upstream promoter sequences of FOXK1, in the hope of being used in tumor biological target therapy.Methods and materialMaterialHuman gastric cancer cell lines AGS,SGC7901,MGC803,BGC823,MKN45, MKN28. Mouse anti-human c-jun,vimentin moloclonal antibody, Rabbit anti-human FOXK1,E-cadherin polyclonal antibody. Dual luciferase reporter gene detection kit, Chromatin Imumunopprecipitation (CHIP) Assay Kit. Immunohistochemistry assay kit. Western blot associated reagents.cck-8 assay kit.EdU assay kit.Transwell chamber.Methods1. FOXK1 enhanced the proliferation, invasion and metastasis of GC1.1 The expression of FOXKI in human GC tissueAn additional 10 pairs of fresh gastric cancer tissues and thecorresponding non-tumor mucosa samples were acquired and were immediately stored in liquid nitrogen after resection. Western blot analysis were performed to examine the protein levels of FOXK1. An additional 20 pairs of paraffin-embedded tissues were obtained from GC patients.Immunohistochemical staining was performed to detect the expression of FOXK1 in gastric cancer specimens and the adjacent normal gastric tissues.1.2 The influence of FOXK1 on proliferation of GC.To establish stable cell lines,MKN28 was transfected with pcDNA 3.1 vector and pcDNA3.1-FOXK1.48 hours later, cells were cultured in complete medium supplemented with 1000mg/ml geneticin (G418) to select monoclonal cells. The expression of FOXK1 in stable cell lines was verified by western blot. cck-8 and EdU test were performed to investigate the proliferation of GC cells.1.3 The influence of FOXK1 on migration and invasion of GC.MKN28-FOXK1 and MKN28-Vector plated in 6-well plates with 100% confluence were wounded with a pipette tip at time 0. Photographs were taken at every 12 hours to assess cell migration. Plate MKN28-FOXK1 or MKN28-Vector in transwell chamber coated with matrigel. There were media without FBS in chamber and complete medium out of chamber. After culturing for 24 hours, cells dyeing with crystal violet were counted to assess the influence of FOXK1 overexpression on invasion.2. The influence of FOXK1 on EMT of GC.2.1 FOXK1 overexpression enhanced EMT of GCWe examined the cell morphology and compared cells phenotype between MKN28-FOXK1 and MKN28-Vector.Western blot was performed to detect the expression of E-cadherin and Vimentin in cells.2.2 FOXK1 was involved in TGF-β1 induced EMT on GCDifferent concertrations of TGF-β1 were added into cells, and then incubated cells for additional 48 hours before protein extraction. Cells were also treated with a concertrations of 1ng/ml TGF-β1 and protein were gathered after cultured 0,12,48 hours. Cells transfected with FOXK1 siRNA for 24 hours were treated with TGF-β1 and protein was harvested 48 hours later. Western blot was performed to detect FOXK1, E-cadherin and Vimentin expression. Transwell invasion chambers were used to investigate the invasion ability of TGF-β1 treated cells.3. FOXKl is a direct transcriptional activation target of c-jun3.1 c-jun could bind to the FOXK1 promoter and activate transcriptionWe inserted the promoter sequences of FOXK1 into pGL3-basic to construct plasmid pGL3-FOXKlpromoter,which contain different binding sites of c-jun. Co-transfect these pGL3-FOXKlpromoter and c-jun plasmid into MKN28 to program dual luciferase reporter gene detection assay. To obtain more evidence of c-jun binding to FOXK1 promoter, chip was also used to investigate the binding site of c-jun in FOXK1 promoter.3.2 c-jun transactivated the FOXK1 promoter by binding to the-362 to-355 regionSite-directed mutagenesis of potential c-jun binding sites was carried out in the pGL3-FOXKl promoter plasmid. All mutations were verified by sequencing.Luciferase activity was compared between the wild type and mutation type. Different amounts of c-jun plasmid were co-transfect into MKN28 with pGL3-FOXKlpromoter to investigate whether the expression level of c-jun can affect the FOXK1 promoter activation.4. FOXK1 and c-jun expression are positively correlated in GC and predicted poor prognosis in GC patients4.1 FOXK1 expression was consistent with c-jun in GC cellsProtein of GC cell lines was extracted for western blot to detect the expression of FOXK1 and c-jun. Immunofluorescent assay was performed to detect the distribution of FOXK1 and c-jun in cells.4.2 FOXK1 and c-jun were expressed at high levels in cancer tissues with positively correlationA total of 90 patients with gastric carcinoma who underwent subtotal astrectomy at the Department of Gastrointestinal Surgery, Nanfang Hospital (Guangzhou, China) were enrolled in this study. The paraffin-embedded tissues obtained from these patients were used for the construction of tissue microarrays(Shanghai Biochip Co., Ltd. Shanghai, China). Immunohistochemical staining was performed to detect the expression of FOXK1 in gastric cancer specimens and the adjacent normal gastric tissues. The average score for each sample evaluated by two observers was considered as the final IHC score.The expression difference between cancer and normal tissues and correlation between FOXK1 and c-jun were estimated according to the scores of the staining.4.3 The clinical relevance of FOXK1 and c-jun expression in GC.Chi-square test was performed to evaluate the relation between proteins expression and clinicopathological features.4.4 The effect of FOXK1 and c-jun expression on GC prognosisSurvival curves were estimated using the Kaplan-Meier method. A multivariate Cox proportional hazards model was performed to identify the independent factors of survival based on the variables selected in univariate analysis.5. C-jun promotes GC development and progression by regulating FOXK1.5.1 Knockdown of c-jun inhibited the proliferation of GCScr-RNA and c-jun-siRNA were transfected to MKN28-FOXK1. The expression of c-jun in MKN28-FOXK1 was verified by western blot, cck-8 and EdU test were performed to investigate the proliferation of GC cells.5.2 Knockdown of c-jun inhibited the migration and invasion of GCMKN28-FOXK1 transfected with scr-RNA or c-jun-siRNA plated in 6-well plates with 100% confluence were wounded with a pipette tip at time 0. Photographs were taken at every 12 hours to assess cell migration. Transwell chamber coated with matrigel and cells were plated in it.After culturing for 24 hours, cells were counted to assess the effect of c-jun knockdown on invasion.5.3 Knockdown of c-jun inhibited FOXK1 induced EMTThe morphology of MKN28-FOXK1 transfected with scr-RNA or c-jun siRNA was observed in white light microscope. Immunofluorescent assay and western blot was performed to investigate changes in EMT-related protein and Erk,AKT pathway.6. The influence of c-jun on the ability of invasion and metastasis induced by FOXK1 in vivo6.1 The establishment of the subcutaneous tumor modelA c-jun RNAi lentiviral vector was constructed, GFP-lentiviral vector was used as a negative control.MKN28-Vector,MKN28-FOXK1 and MKN28-FOXK1-c-jun shRNA were injected into nude mouse subcutaneous of 5 to 6 weeks.35days later, we compared the tumor size and the consecutive tissues of tumor were stained with Ki-67 and CD105 by IHC.6.2 The establishment of the lung tumor metastasized by tail vein injectionMice were injected with 4×105/pcDNA3.1, pcDNA3.1-FOXK1 and pcDNA3.1-FOXKl-c-jun shRNA lentivirus cells per mouse through tail vein. Forty days later, the mice were sacrificed and the lungs were harvested and photographed. Tissue sections were attained with traditional method and HE and IHC staining was performed.Results1. FOXK1 enhanced the proliferation, invasion and metastasis of GC1.1 The expression of FOXK1 in human GC tissueStrong FOXK1-positive signals were present in the cancer cell nuclei. Positive signals were not found in the epithelial cells of normal gastric glands1.2 The influence of FOXK1 on proliferation of GC.The cck-8 and EdU incorporation assay revealed that cell proliferation were significantly higher than that in the control vector cells.1.3 The influence of FOXK1 on migration and invasion of GC.Wound-healing assay and invasion experiment indicated that overexpression of FOXK1 enhanced the ability of GC invasion and metastasis.2. The influence of FOXK1 on EMT of GC.2.1 FOXK1 overexpression enhanced EMT of GCFOXK1 overexpression in the cells induced the loss of cobblestone-like phenotype; In contrast, empty vector transfectants displayed a round or flat morphology. Upregulation of mesenchymal markers (vimentin) and downregulation of an epithelial marker (E-cadherin) were observed by Western blot and immunofluorescence analysis after the stable expression of FOXK1.2.2 FOXK1 was involved in TGF-β1 induced EMT on GCTGFβ1 induced EMT in GC and meanwhile FOXK1 expression was increased in a time-and dose-dependent manner by TGF-β-c treatment.Consistently, FOXK1 knockdown altered the expression of EMT markers; E-cadherin expression was significantly upregulated, and vimentin expression evidently decreased.3. FOXK1 is a direct transcriptional activation target of c-jun3.1 c-jun could bind to the FOXK1 promoter and activate transcriptionWe analyzed the FOXK1 promoter using Promo software and found three binding sites of c-jun.Luciferase reporter and ChIP assays showed that c-jun could directly bind to FOXK1 promoter.3.2 c-jun transactivated the FOXK1 promoter by binding to the-362 to-355 regionSite mutation found that the specific binding site was in-362 to-355 region;and the relative luciferase activity was enhanced by c-jun in a dose-dependent manner.4. FOXK1 and c-jun expression are positively correlated in GC and predicted poor prognosis in GC patients4.1 FOXK1 expression was consistent with c-jun in GC cellsTwo-color immunofluorescence assay showed that the endogenous c-jun and FOXK1 proteins were co-localized to the nucleus of MKN28 cells;Western blot analysis showed c-jun was expressed at a relatively high level in AGS, BGC823 and MKN28 cells and at a relatively low level in SGC7901 and MKN45 cells. The FOXK1 expression pattern was very similar to that of c-jun in 5 of the above 6 cell lines, but not in MGC8034.2 FOXK1 and c-jun were expressed at high levels in cancer tissues with positively correlationBoth FOXK1 and c-jun were expressed at higher levels in cancer tissues than in adjacent normal gastric mucosa tissues;A significant correlation between c-jun and FOXK1 (R= 0.621) in primary GC was observed.4.3 The clinical relevance of FOXK1 and c-jun expression in GC.FOXK1 and c-jun expression were significantly correlated with tumor differentiation, AJCC (American Joint Committee on Cancer) stage, lymph node metastasis and serosal invasion; however, the expression levels were not correlated with gender, age or tumor size.4.4 The effect of FOXK1 and c-jun expression on GC prognosisHigh positive expression of each protein was correlated with poor outcome; Doubly positive cases (expressing both proteins) exhibited the worst prognosis.AJCC stage and FOXK1 expression but not c-jun expression was considered independent risk predictors for poor overall survival.5. C-jun promotes GC development and progression by regulating FOXK1.5.1 Knockdown of c-jun inhibited the proliferation of GCC-jun downregulation decreased the FOXK1-mediated proliferation of MKN28 cells as shown using a WST-1 assay and EdU incorporation assay.5.2 Knockdown of c-jun inhibited the migration and invasion of GCC-jun knockdown in FOXK1-overexpressing cells led to a decrease in the migratory and invasion potentials of FOXK1-overexpressing cells in vitro.5.3 Knockdown of c-jun inhibited FOXK1 induced EMTC-jun knockdown in FOXK1-overexpressing MKN28 cells led to EMT reversion;The phosphorylation levels of AKT and ERK1/2 were downregulated after c-jun knockdown in FOXK1-overexpressing cells compared with FOXK-overexpressing cells; The expression levels of the typical EMT epithelial markers E-cadherin and-catenin were upregulated after c-jun knockdown in FOXK1-overexpressing cells, in contrast, the mesenchymal markers MMP-2, MMP-9, vimentin, and Snail were downregulated.6. The influence of c-jun on the ability of invasion and metastasis induced by FOXK1 in vivo6.1 The establishment of the subcutaneous tumor modelXenografted FOXK1-overexpressing cells rapidly proliferated in mice compared with vector-expressing cells. However, xenografted tumor growth was suppressed in mice that were injected with FOXK1-overexpressing cells in which c-jun was downregulated compared to FOXK1-overexpressing cells with normal c-jun levels; FOXK1 stable-transfectant group exhibited a significantly increased proliferation rate and tumor vessel density compared to the vector group, whereas c-jun knockdown inhibited the growth rate and tumor vessel density in the FOXK1-overexpressing group.6.2 The establishment of the lung tumor metastasized by tail vein injectionFOXK1-overexpressing MKN28 cells but not control MKN28 cells formed a variety of large metastatic nodules in the lung. Compared with FOXK1-overexpressing cells, c-jun downregulation in FOXK1-overexpressing cells exhibited a significant reduction in visible lung tumors;The overexpression of FOXK1 resulted in a significant loss of E-cadherin, whereas downregulation of c-jun in FOXK1-overexpressing cells caused an increase in E-cadherin.Conclusions1. FOXK1 exhibited a high expression level in GC and facilitates the malignant biological behavior of GC cells;FOXK1 was involved in TGFβinduced EMT.2. FOXK1 is a direct transcriptional activation target of c-jun and the binding site is on-362-3555’upstream of FOXK1 gene.3. c-jun and FOXK1 were expressed at higher levels in cancer tissues than in normal gastric tissues; A strong positive correlation existed between FOXK1 and c-jun expressions in GC; Co-expression of FOXK1 and c-jun correlates with a poor prognosis in human GC; FOXK1 expression was considered independent risk predictors for poor overall survival.4. As a upstream regulatory molecules of FOXK1,c-jun knockdown in FOXK1-overexpressing cells led to a decrease in th eproliferation, migratory and invasion potentials.5. C-jun/FOXK1 is essential for the induction of tumorigenesis and metastasis in vivo.In conclusion, FOXK1 is markedly overexpressed in GC and enhances tumorigenicity and tumor growth in vivo. Moreover, we identified FOXK1 as a direct target of c-jun in GC. FOXK1/c-jun promotes the proliferation, migration and invasion, and metastasis of GC cells and might represent a novel target for treating GC... |
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