Part ? The Expression And Significance Of FZD5 Gene In Intrahepatic Cholangiocarcinoma Cells The Relationship Betweent Neutrophil To Lymphocyte Ratio Before Preoperative Chemotherapy And Outcomes After The Resection Of Colorectal Liver Metastases

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The expression and significance of FZD5 gene in intrahepatic cholangiocarcinoma cellsBackgroundIntrahepatic cholangiocarcinoma(ICC)is the second most common primary liver cancer.It's a highly malignant cancer.Most ICCs are at advanced stages at diagnosis,and these patients have no chance for radical surgery.Therefore,effective targeted drugs are urgently needed.The activation of Wnt/?-catenin signaling pathway plays an important role in tumor proliferation,invasion and metastasis.Activation of this signaling pathway is very common in ICC,and it is closely related to the biological behavior of ICC.FZD5 protein is one of the components of the Wnt receptor complex.Previous studies have shown that specific antibodies binding to FZD5 and FZD8 can effectively inhibit the growth of certain digestive system tumors,suggesting that it might be a potential treatment target for ICC.However,the effect of FZD5 on the biological behavior of ICC is still unclear.This study intends to clarify the role of FZD5 protein in the development of ICC and provide insights for the treatment of ICC.Methods1.The siRNA was transfected into ICC cells by lipofectamine 2000.We examined FZD5 mRNA and protein expression by real time Q-PCR and Western blot.2.After knockdown of FZD5,the CCK-8 test was performed to detect its proliferation ability;and clone formation assay was performed to detect clone formation ability.3.After knockdown of FZD5,the invasion and migration ability was tested using Transwell chamber.4.After knockdown of FZD5,we detected the expression of cell cycle and EMT-related proteins by Western blot.5.We designed the CRISPR/cas9 knock-out target sites of FZD5 gene for all transcripts,and the vectors were constructed.CRISPR-Cas9-mediated FZD5 knockout RBE cells were then constructed.6.The FCM was performed to detect the cell cycle distribution of FZD5 knockout RBE cells.7.The transcriptome analysis of FZD5 knockout RBE cells was performed based on RNA-Seq technique.8.Public databases was used to explore FZD5 expression,mutations and the relationship between FZD5 expression and prognosis in patients with cholangiocarcinoma9.Public databases was used to explore xpthe relationship between FZD5 gene methylation and FZD5 expression.Results1.After FZD5 siRNA was transfected,the expression of FZD5 and mRNA and protein was significantly inhibited.2.After FZD5 siRNA was transfected,the proliferation ability,the clone formation ability,and invasion/migration ability of ICC cells were inhibited.3.After FZD5 siRNA transfection,the expression of cyclin D1,C-myc and ZEB1,vimentin,snail and N-cadherin was decreased.4.The proportion of cells in G0/G1 phase was increased and that in G2/M phase was decreased in FZD5 knockout RBE cells.5.The tumorigenicity of FZD5 knockout RBE cells in nude mice was significantly reduced.6.A total of 1100 genes were found to be altered after FZD5 gene was knocked out.Differential expressed genes are mainly enriched in cell adhesion molecules,extracellular matrix receptor interactions,and Rap1 pathways.7.The TCGA database showed that there was a difference in FZD5 expression between cholangiocarcinoma tissues and normal tissue.Sequencing data from MSK hospital did not find mutations in the FZD5 gene.8.Changes in FZD5 gene methylation may cause changes in FZD5 expression.ConclusionsThe silencing of the FZD5 gene can inhibit the proliferation and invasion/migration ability of ICC by inhibiting the Wnt/?-catenin signaling pathway,thereby downregulating cell cycle and EMT related proteins.This will help us to find the best treatment strategy for ICC in the future.Part ? The relationship betweent Neutrophil to Lymphocyte Ratio Before Preoperative Chemotherapy and Outcomes After the Resection of Colorectal Liver MetastasesBackgroundThe neutrophil to lymphocyte ratio(NLR)is a marker of inflammation and is associated with poor outcomes.We aimed to evaluate the role of the pretreatment NLR in predicting the outcomes after preoperat ive chemotherapy in patients with colorectal liver metastases(CRLM).MethodsA retrospective review was performed for 183 patients with CRLM.The NLR was measured before chemotherapy,and a receiver operating characteristic(ROC)curve was used to estimate the cutoff value.Logistic regressions were applied to analyze potential predictors of the pathological response.The Cox proportional hazard method was used to analyze survival.ResultsThe pre-chemotherapy NLR was 2.4±1.1,whereas the post-chemotherapy NLR was 2.1±1.6(p<0.001).The pretreatment NLR of 2.3 was a significant predictive marker for the pathological response.The pathologic:al response rates were 67.1%in the patients in NLR?2.3 group and 48.1%in patients in NLR>2.3 group(p=0.01).Multivariate analysis found that low pretreatment NLR(p=0.043),radiological response to chemotherapy(p<0.001),first-line chemotherapy(p=0.001),and targeted therapy(p=0.002)were associated with pathological responses.The median overall survival(OS)and recurrence-free survival(RFS)were worse in the increased NLR cohort than in the low NLR cohort(OS:31.1 vs.43.1 months,p=0.012;RFS:6.5 vs.9.4 months,p=0.06).According to multivariate analyses,a high pretreatment NLR was a significant predictor for both worse OS(HR = 2.43,95%CI=1.49-3.94,p<0.001)and RFS(HR = 1.53,95%CI = 1.08-2.18,p=0.017).ConclusionsAn increased pretreatment NLR was a significant predictor of a poor pathological response and worse prognosis after preoperative chemotherapy The NLR is a simple biomarker for assessing chemotherapy efficacy.