| Cholangiocarcinoma(CCA)is a malignant tumor originating from biliary epithelial cells.CCA accounts for 15% of all primary liver malignancies.World widely,the incidence rate of CCA has increased year by year.CCA is highly heterogeneous and can appear anywhere in the biliary tree.According to the anatomical location of CCA,it can be divided into intrahepatic CCA,perihilar CCA,and distal CCA(i CCA,p CCA,and d CCA).The onset of CCA is hidden and is usually asymptomatic at the early stage.Due to lacking effective diagnostic markers,most patients are diagnosed at a late stage.At present,the clinical treatment options for CCA are limited.Surgical treatment is the best choice for early patients,while chemotherapy is mainly used for middle and late patients.The mechanism of CCA is complex.Involved genes include oncogenes,tumor suppressor genes,tumor metastasis-related suppressor genes,and genes regulating adhesion molecules.These genes participate in the occurrence and development of CCA at different stages.With the research progress of genomics and molecular biology,it has been reported that new molecular markers have been found in CCA.Cholestasis and chronic inflammation lead to malignant transformation of bile duct cells,which is a classic pathway for the occurrence and development of CCA.Chronic inflammation can lead to the continuous increase of inflammatory mediators,including interleukin-6and tumor necrosis factor-α,cyclooxygenase-2,and Wnt in the environment exposed to bile duct cells,further leading to the progressive mutation of tumor suppressor gene,proto-oncogene,and DNA mismatch repair gene.On the other hand,cholestasis can inhibit apoptosis and lead to the production and secretion of growth factors stimulated by conjugated bile acids.In this biliary system environment,a variety of proto-oncogenes with low expression are activated and highly expressed,resulting in sequence reaction,destroying the intracellular pathways controlled by proto-oncogenes,and then leading to cell carcinogenesis.Accumulating evidence has indicated the crucial role of micro RNA(mi RNA)in mediating tumor progression.Mir-196-5p is processed from the 5’end arm of the mi R-196 precursor.Reports indicated that mi R-196-5p plays a key regulated role in colorectal cancer,ovarian cancer,and hepatocellular carcinoma,while the expression nor significance of micro RNA-196 in CCA remains unclear.Based on the previous bioinformatics work of our research group,we found that mi R-196-5p was involved in the regulation of CAA invasion and metastasis.We analyzed the expression of mi R-196-5p in clinical CCA combined with the analysis data of the GEO database.To clarify its expression in patients with CCA and its correlation with different clinical characteristics.Then the expression of mir-196-5p in different CCA cell lines was analyzed,and then Hu CCT1 cells were selected to further analyze the regulation mechanism of mi R-196-5p in CCA.Part One The expression of mi R-196-5p in clinical CCA samples and its correlation with clinical characteristics of patientsObjective: to analyze the expression of mi R-196-5p in the GEO datasets and clinical CCA samples.To clarify the expression pattern of mi R-196-5p in CCA patients with different clinical characteristics,and to provide a reference for the clinical diagnosis of mi R-196-5p in CCA.Methods:1.Two public data sets from GEO(GSE140001 and GSE47764)were adopted for analysis.The GSE140001 dataset included 12 patients with d CCA and 3 matched non-neoplastic bile duct epithelial samples.The GSE47764 data set contains 3 human CCA samples and their associated normal bile duct tissues.The relevant data and matrix files were downloaded to compare the final difference genes of the two data sets.2.The CCA tissue samples and matched adjacent bile duct tissue samples studied were obtained from patients who underwent CCA resection in the second hospital of Hebei Medical University from March 2019 to December 2020.A total of 56 pairs of CCA resection sample tissues and matched adjacent bile duct tissues were collected.3.The expression of mi R-196-5p was detected by real-time quantitative PCR(RT-q PCR).4.Statistical analysis: Graph Pad 8.0 software was used for statistical analysis.The measurement data is expressed as mean ± standard deviation(SD);T-test was used to compare the mean of two samples in normal distribution;non-normal distribution data were analyzed by rank-sum test;P <0.05 was statistically significant.Results:1.Four mi RNAs were significantly up-regulated in the public data sets GSE140001 and GSE47764,including mi R-196-5p.2.Compared with normal bile duct tissue,the expression of mi R-196-5p in CCA samples was obviously up-regulated.According to the median expression value of mi R-196-5p.Patients were classified as high expression group and low expression group.The expression of mi R-196-5p had no correlation with the gender or age of CCA patients.In patients with tumor diameter ≥ 5cm,metastasis,and TNM stage of III-IV,mi R-196-5p was highly expressed and positively correlated with these clinical profiles.Conclusions: mi R-196-5p has a carcinogenic effect in CCA and is positively correlated with tumor size,tumor metastasis,and TMN stage.It may be used as a new biomarker for the early diagnosis and prognosis of CCA.Part Two The mechanism of mi R-196-5p promoting proliferation and migration of cholangiocarcinoma cellsObjective: the expression of mi R-196-5p is positively correlated with tumor size and metastasis of CCA.The expression and biological function analysis of mir-196-5p in the CCA cell line can lay a foundation for further mechanism research.Methods:1.The expression of mi R-196-5p in CCA cell lines Hu CCT1,QBC939,Huh-28,and human intrahepatic bile duct epithelial cells HIBEC were analyzed.According to the expression of mi R-196-5p,Hu CCT1 was selected for further study.2.After the constructed vector plasmid was transfected into hucct1 cells,the effects of down-regulated mi R-196-5p expression on the proliferation,invasion,and migration of CCA cells were analyzed by CCK8,Edu method,cell clone formation experiment,cell migration and Transwell assay.Results: The expression of mi R-196-5p was up-regulated in the CCA cell line.After inhibiting mi R-196-5p,the proliferation,migration,and invasion of CCA cells were inhibited.Conclusion: Mir-196-5p is up-regulated in CCA and plays a regulatory role through promoting tumor cell invasion and migration Part Three The mechanism of mi R-196-5p promoting proliferation and migration of cholangiocarcinoma cellsObjective: the expression of mi R-196-5p is positively correlated with tumor size and metastasis of CCA.Therefore,we speculate that mi R-196-5p may play a carcinogenic role by regulating cell proliferation and migration in CCA.This part study aims to further explore the pathway and regulation mechanism of mi R-196-5p in this process.Methods:1.Literature search was performed,and bioinformatics analysis tools were used to analyze the potential targets of mi R-196-5p.2.The regulatory effect of mi R-196-5p on the target gene HAND1 was analyzed by the dual-luciferase reporter gene assay.3.The correlation between the expression of HAND1 in CCA clinical tissue samples and the clinical characteristics of patients.4.Through constructing up-regulated / down-regulated expression plasmids [including HAND1 overexpression plasmid(HAND1 OE),β-catenin interference sequence(si-β-catenin),and negative control plasmid],the regulatory effect of mi R-196-5p on target genes and related pathways was verified,and the effect of regulatory pathways on the CCA cell function was analyzed.5.The position changes of key proteins of the mi R-196-5p targeted pathway in the cytoplasm and cell nucleus were analyzed and verified by immunocytochemical staining.Results:1.Bioinformatics analysis showed that HAND1 was the target of mi R-196b-5p.2.Dual-luciferase reporter gene assay verified that mi R-196-5p combined with the 3 ’UTR of the HAND1 gene to inhibit its expression.3.The rescue assay confirmed that silencing HAND1 or β-catenin could reverse the enhancement of cell proliferation and migration caused by the up-regulation of mi R-196-5p.4.Western blot analysis showed that down-regulation of mi R-196-5p significantly inhibited the expression of cell proliferation marker Ki67 and mesenchymal marker N-cadherin,silencing HAND1(HAND1-KO)or silencing β-catenin(si-β-catenin)reversed the regulatory effect of mi R-196-5p on the above proteins.5.The cellular immunochemical analysis showed that silencing HAND1 or silenced β-catenin inhibited the activity of the Wnt signaling pathway when catenin was transferred from nucleus to cell membrane.Conclusion: mi R-196-5p promotes proliferation,migration,and invasion of through activating HAND1/ Wnt / β-catenin axis. |
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