| Part one: WNT4 is a direct target of mi R-497,which is upregulated in CRC samples.Objectives: To study the expression of WNT4 in colorectal cancer and the relationship between WNT4 and micro RNA497.Methods: 1.Micro RNA497 knockout mice were constructed,the colorectal tissues of wild mice(WT)and knockout mice(KO)were subjected to secondgeneration sequencing.WNT4 expression in the colorectal was detected by immunohistochemistry,while the concentration of WNT4 in serum of wild mice(WT)and knockout mice(KO)was detected by ELISA.2.Normal control and micro RNA497mimics(mimics having the same effect as micro RNA497)were transfected into human colorectal cancer cells Lo Vo and HCT 116,respectively,and m RNA and protein expression levels of WNT4 in different groups were detected by real-time PCR and Western blot.The binding relationship between micro RNA497 and WNT4 was verified by the Dual-Luciferase Reporter Assay system.3.The m RNA expression levels of micro RNA497 and WNT4 in human colorectal cancer and paracancer tissues were detected by real-time PCR,while the expression levels of WNT4 in human colorectal cancer and paracancer tissues were detected by immunohistochemistry and Western blot.4.The serum of healthy people and colorectal cancer patients(before and after surgery)were collected,and serum expression level of WNT4 was detected by ELISA.5.Fresh colorectal cancer and paracancer tissues were collected.1 m L DME/F12medium(no serum)was cultured overnight,and the superplasmid was collected.Then the secretion of WNT4 was detected by ELISA.Results: 1.The results of Real-time PCR and Western blot confirmed the successful construction of micro RNA497 knockout mice.The Second generation sequencing of colorectal tissues of wild mice(WT)and micro RNA497 knockout mice(KO)showed that WNT4 expression was increased in the knockout mice,and immunohistochemistry also showed that WNT4 expression was increased.Moreover,the concentration of WNT4 in the serum of mi R-497 KO mice was significantly higher than that in WT mice(P=0.0244).2.After transfection with mi R-497 mimics,we observed the decreased expression of WNT4 at both the m RNA and protein levels in Lo Vo and HCT 116 CRC cells.Moreover,the results of Dual-Luciferase Reporter Assay system confirmed that WNT4 was a target gene of micro RNA497.3.All 10 CRC tissues exhibited significant downregulation of mi R-497,while WNT4 expression was upregulated in 9 CRC tissues.An inverse correlation between mi R-497 and the WNT4 expression levels was also seen in the CRC samples(P < 0.001,R2 = 0.7117).The results of Western blot and immunohistochemical showed that the expression of WNT4 was increased in colorectal cancer tissues compared with normal adjacent tissues.4.We found elevated serum levels of WNT4 in patients with CRC compared to healthy donors(P<0.01)and an evident decline after radical surgery in 21/23(91.30%)of CRC patients.WNT4 expression was closely associated with TNM T stage(55.94 pg/m L vs.88.08 pg/m L;P = 0.0293),tumor metastasis(71.73 pg/m L vs.111.1 pg/m L;P = 0.0428),TNM stage(66.35 pg/m L vs.94.57 pg/m L;P = 0.0464)and tumor size(59.38 pg/m L vs.96.60 pg/m L;P = 0.0087).Remarkably,WNT4 showed a better diagnosis performance than CA199 and CEA in our samples.5.The results showed that the WNT4 levels in conditioned medium from fresh tumors were significantly higher than those from adjacent tissues(P<0.05).Conclusion: 1.WNT4 expression was significantly upregulated in the colorectal tissues of mi R-497 KO mice,compared to the WT mice.2.WNT4 is a direct target of micro RNA497.3.WNT4 is upregulated in CRC samples,which is negatively correlated with the expression of micro RNA497.4.The results showed elevated serum levels of WNT4 in patients with CRC compared to healthy donors and an evident decline after radical surgery in CRC patients.WNT4 expression was closely associated with TNM T stage,tumor metastasis,TNM stage and tumor size.5.WNT4 can be highly secreted by colorectal cancer tissues.Part two: The mechanism of WNT4 on colorectal cancer cells.Objectives: To investigate the effect of WNT4 on colorectal cancer cells.Methods: 1.Top-flash&fop-flash plasmids were transfected with human colorectal cancer cells Lo Vo and HCT 116 to detect whether exogenous recombinant WNT4 protein could activate the classical WNT pathway.We observed the β-catenin,AXIN2,E-cadherin,ZO-1,nuclear β-catenin and nuclear AXIN2 expression changs induced by WNT4 protein at different times in Lo Vo and HCT 116 cell lines by Western blot.The expression of β-catenin and AXIN2 were also detected by immunofluorescence.2.The cultured Lo Vo and HCT 116 human colorectal cancer cell lines were divided into three groups: DMSO,ICG-001(25 μM),ICG-001(25 μM)+WNT4 protein(100 ng / m L).The inhibition of WNT pathway was detected by TOP/FOP-Flash reporter assays and Western blot.3.The migration and invasion ability of the cells was detected in the Lo Vo and HCT 116 cell lines treated with WNT4 protein.4.We observed the β-catenin,AXIN2,E-cadherin,ZO-1,nuclear β-catenin and nuclear AXIN2 expression changs in the Lo Vo and HCT 116 cell lines transfected with Normal control、WNT4-si RNA1 and WNT4-si RNA2 by Western blot.5.TOP/FOP-Flash reporter assays were uesd to detect whether overexpression of WNT4 could activate the classical WNT pathway.6.Cells were injected subcutaneously into the dorsal flank of each nude mouse to generate a xenograft model.7.Cells were injected into the tail vein of each nude mouse for establishing the tumor xenograft model.Results: 1.Exogenous recombinant WNT4 protein can activate the WNT classical pathways in human colorectal cancer cells Lo Vo and HCT 116.After treatment with exogenous WNT4 protein(100 ng/m L)for 0,0.5,1,2,6,and 24 h in CRC tumor cells,we found that the total β–catenin and AXIN2 in cells were increased,while the levels of E-cadherin and ZO-1 were decreased.AXIN2 is a downstream target gene of β–catenin.The level of β-catenin in the nucleus also increased,while AXIN2 was highest at one hour of treatment.The fluorescence microscopy also demonstrated that WNT4 raised β–catenin and AXIN2 accumulation and remarkably induced nuclear localization of β–catenin and AXIN2.2.The cultured colorectal cancer cells were divided into three groups: DMSO,ICG-001(25 μM),ICG-001(25 μM)+ WNT4 protein(100 ng/m L).The results showed that 25 μM ICG-001 completely inhibited activation of β-catenin signaling caused by WNT4,suggesting that WNT4 has its effect through β-cateninmediated transcription activity.3.The migration and invasion of CRC cells was enhanced after WNT4 protein(100 ng/m L)pretreatment.4.The colorectal cancer cells were transfected with normal control,WNT4-si RNA1 and WNT4-si RNA2,and then cultured for 48 h.The results of QPCR confirmed that the m RNA level of WNT4 was knocked down.We found that the total β–catenin and AXIN2 in these cells decreased notably,while the levels of E-cad and ZO-1 increased.Meanwhile,the level of β-catenin and AXIN2 in the nucleus also decreased,indicating that lowered nuclear translocation of β-catenin thus inhibits the WNT4/catenin signal pathway.5.Next,we overexpressed WNT4(WNT4-HA)and found that the TCF/LEF transcription activity is significantly enhanced.6.We performed an in vivo tumor cell metastasis assay,in which 1× 106 Lo Vo cells,transfected with negative control sh RNA(Scramble)or WNT4-sh RNA,were injected into the caudal vein of nude mice.The ratio of liver weight to body weight and the number of liver metastases in the Scramble group are much higher than the WNT4-sh RNA group.7.Sh WNT4-Lovo-and HAWNT4-SW480-transfected cells were injected subcutaneously into the dorsal flank of each nude mouse at a concentration of 1 × 107 cells/200 μL.Results showed that tumors developed from control cells were larger and heavier than tumors from WNT4-sh RNA cells,while tumors developed from WNT4-vector cells were smaller and lighter than those developed from WNT4-HA cells.However,no significant difference was observed between the tumors from mice in the two groups,which indicated that WNT4 only has a slight effect on tumor proliferation.Conclusion: 1.WNT4 can activate the classic WNT pathways in human colorectal cancer cells.2.Knockdown of WNT4 in two colorectal cancer cells inhibited the classical WNT pathway.3.Knockdown of WNT4 can inhibit cell metastasis in nude mice.Part three: The mechanism of WNT4 on fibroblasts in the stroma of colorectal cancer.Objectives: To investigate the role of WNT4 in fibroblasts in the stroma of colorectal cancer.Methods: 1.Immunofluorescence was performed on the subcutaneous nodules in the second part to detect the expression of α-SMA.2.To investigate whether WNT4 can activate fibroblasts,normal fibroblasts(NF)and cancer-associated fibroblasts were isolated and identified from the tumor and adjacent tissues of CRC patients by an enzymatic dissociation method.The specific cell surface markers CD31,CD45,and CD329 were used to confirm the absence of endothelial,immune,and epithelial cell contamination by flow cytometry analysis.3.A suspension coculture of CAFs and tumor cells with complete medium was established in ultra-low attachment plates.The ability of typical heterospheroids formation in Lo Vo Scramble and Lo Vo sh-wnt4 groups,SW480 wnt4-vector and SW480 wnt4-ha groups was compared.WNT4 protein was added to the growth medium(without FBS)of normal fibroblasts(NF)at a final concentration of 0(control)or 400 ng/m L for 24 h.Stronger gel contraction was observed after the NF#1 and NF#2 treatments with WNT4.4.We also tested the related markers of CAFs,fibronectin(FN)and α-SMA,by immunofluorescence and western blotting to verify the recruitment and contraction ability of WNT4 towards fibroblasts.5.Exogenous protein WNT4 was added to normal fibroblasts to detect the expression of amento-catenin in the nucleus by immunofluorescence.6.Normal fibroblasts were divided into four groups: DMSO,DMSO+WNT4,ICG-001 and ICG-001+WNT4.Protein expression levels of α-SMA and fibronectin were detected by Western blot and immunofluorescence.The ability of typical heterospheroids formation was compared.The ability of contraction of collagen gels was observed.Results: 1.We found that infiltrating α-SMA-positive CAFs are more abundant in the samples from mice injected with the Lo Vo-Scramble-and SW480-WNT4-HAtransfected cells,compared to those from mice injected with the Lo Vo-sh WNT4 and SW480-WNT4-vectors,respectively.2.The separated cells were identified as fibroblasts by flow cytometry.3.We established a suspension coculture of CAFs and tumor cells with complete medium in ultra-low attachment plates.Intriguingly,fewer and smaller typical heterospheroids were formed in the WNT4-sh RNA groups,supporting the suggestion that WNT4 was related to the adhesive capacity of CRC cells and had the ability to recruit fibroblasts.Moreover,in the WNT4-HA group,more heterospheroids were formed,further confirming our hypothesis.Stronger gel contraction was observed after the NF#1 and NF#2 treatments with WNT4,indicating increased extracellular matrix remodeling.4.Increased expression of FN and α-SMA were observed after the NFs were stimulated with WNT4(400 ng,24 h).5.β-catenin translocation from the cytoplasm to the nucleus was observed in case of the NF#1 and NF#2 treatments(400 ng/ml WNT4,24 h).6.Normal fibroblasts were divided into DMSO,DMSO + WNT4,ICG-001,ICG + WNT4 four groups.ICG-001 completely blocked elevation of α-SMA and fibronectin at a concentration of 10 μM.Additionally,the effects of the WNT4 protein on heterospheroid formation and gel contraction ability were also completely inhibited by ICG-001.Conclusion: WNT4 promotes fibroblast recruitment and activation. |
