Expression Of AF1q In Colorectal Cancer Tissues And Its Effect In Cell Proliferation And Metastasis

Object:To study the biological function of AF1q in human CRC,and to explore the relationship between AF1q and colorectal cancer.Method:The mRNA expression level of AF1q was first explored in 38 pairs of CRC specimens and patient-matched normal tissues by quantitative RT-PCR.Next,we examined AF1q protein expression in 96 CRC specimens and their matched normal tissues by immunohistochemical staining(IHC).Then,we conducted bioinformatics analysis.Level 3 HiSeq RNASeq data was downloaded from the The Cancer Genome Atlas(TCGA)website(Zhu et al.2009)for 380 cancer samples,R package survival was used to analysis the relationship between AF1q expression and survival from the TCGA and other CRC cohorts.In addition,AF1q expression was evaluated in five CRC cell lines(SW480,SW48,KM12,SW620,and LoVo)and one normal intestinal epithelial cell line(NCM460).Results:First,AF1q mRNA expression level was higher in the tumor tissues than in normal colorectal tissues.Of all 96 paired samples,54 CRC tissues(56.3%)and 28 normal tissues(29.2%)were found to be positive for AF1q expression(P<0.05).Immunostained AF1q protein was mainly located in the cell cytoplasm.We also found that AF1q upregulation was significantly related to lymph node metastasis(P = 0.0159)and late TNM stage.Second,The results showed that AF1q upregulation indicated poor overall survival and poor disease-free survival.Third,both mRNA and protein levels of AF1q in the five CRC cell lines(SW480,SW48,KM 12,SW620,and LoVo)were higher than those of NCM460.Conclusion:AF1q is overexpressed in CRC and AF1q upregulation was significantly related to lymph node metastasis,late TNM stage,and poor prognosis in CRC patients.Object:To study the function of AF1q on colorectal cancer cell(CRC)proliferation,apoptosis,and metastasis.Methods:1.Three AF1q shRNAs(sh#1,sh#2,and sh#3)were designed and transfected into SW620 and LoVo cells with high levels of AF1q expression.sh#2 showed the best efficiency in silencing AF1q expression.Therefore,sh#2 was selected to establish SW620/AF1q-shRNA and LoVo/AF1q-shRNA cells with stable AF1q downregulation.Meanwhile,AF1q expression in CRC cells was upregulated by transfecting the AF1q-expressing vector into SW48 and SW480 cells having low AF1q expression levels.The transfection effects were determined by Western Blot.2.CCK-8 was used to conduct a cell proliferation assay according to the manufacture's instructions.Cells were harvested and labeled with Annexin V and propidium iodide,apoptotic cells were analysed by using Flow Cytometer.3.The effects of AF1q on CRC cell invasion,migration,and metastasis were detected by Wound-healing and transwell assays.We further explored the effects of AF1q in xenograft model.Result:1.SW620/AF1q-shRNA and LoVo/AF1q-shRNA cells with stable AF1q downregulation were established.SW48/AF1q and the SW480/AF1q cells with stable AF1q upregulation were established.2.AF1q downregulation significantly suppressed CRC cell proliferation,while AF1q upregulation increased CRC cell proliferation.In the apoptosis test,the number of apoptotic cells was significantly higher in SW620/AF1q-shRNA and LoVo/AF1q-shRNA groups than in SW620/nc-shRNA and LoVo/nc-shRNA groups.By using colony-formation assay,we found both SW48/AF1q and the SW480/AF1q cells had enhanced colony-formation ability than their control groups.3.AF1q downregulation suppressed CRC cell wound-healing ability,while AF1q upregulation increased CRC cell wound-healing ability.In addition,AF1q knockdown significantly inhibited CRC cell migration and invasion ability,while AF1q overexpression showed the opposite effects.We further explored the effects of AF1q in xenograft model.In vivo xenograft assays showed that the tumors generated from SW620/AF1q-shRNA cells were significantly smaller than tumors from the SW620/nc-shRNA cells.Tumors formed from SW620/AFlq-shRNA cells proliferated more slowly than tumors generated from the SW620/nc-shRNA cells.In CRC liver metastasis model,SW620/nc-shRNA and SW620/AFlq-shRNA clones were injected into the subcapsular region of spleen in nude mice.Four weeks later,all mice were sacrificed and their livers investigated.As a result,80%(4/5)of the SW620/nc-shRNA mice underwent liver metastases,while 40%(2/5)of SW620/AFlq-shRNA mice did.The number of liver metastatic nodules significantly decreased when AF1q was downregulated.Conclusion:AF1q promotes CRC cell proliferation,migration,and invasion in vitro.AF1q inhibits CRC cell apoptosis in vitro.AF1q downregulation inhibits CRC tumor growth and liver metastasis in vivo.Object:To explore the exact mechanisms of AF1q in promoting colorectal cancer progression and metastasis.Method:Firstly,the expression of EMT markers(a-catenin.E-cadherin,N-cadherin and ZO-1)were analysed by Western blot analysis after knockdown and overexpression of AF1q in CRC cells.Then,we investigated whether AKT phosphorylation is associated with AF1q in CRC cells.Next,we treated SW48/AF1q cells with a selective AKT inhibitor,SH-6,to further confirm the function of AF1q.Results:1.AF1q knockdown increased the expression levels of epithelial cell markers(?-catenin and E-cadherin)and decreased the expression levels of mesenchymal cell markers(N-cadherin and ZO-1),while AF1q overexpression significantly promoted the EMT phenotype.Taken together,our results suggested that AF1q increased CRC cell migration and invasion via the induction of EMT.2.AF1q significantly increased the phosphorylation of AKT at Thr308.but not Ser473.3.The cell proliferation,wound-healing,migration and invasion ability of SW48/AF1q cells was suppressed after cultured with 10?M SH-6.Furthermore,SH-6 also reversed the EMT of CRC cells.Conclusion:AF1q significantly increased the phosphorylation of AKT at Thr308,which induced the phenotype change of ENT,and affected CRC cells proliferation,migration,and invasion.Inhibition of AKT phosphorylation may reverse the function caused by AF1q.