Expression Of MLF1IP Gene In Cholangiocarcinoma And Its Effect On The Biological Behavior Of Cancer Cell Line RBE

[Background&Objective]Cholangiocarcinoma is a malignant tumor which originally arises from the bileduct epithelial cells. The resection rate and prognosis is extremely poor because of itspowerful ability of invasion and metastasis ability.There is still lack ofcomprehensive treatment and early diagnosis method of cholangiocarcinoma now.Study the mechanism of invasion and metastasis in genetic level and seek for genetreatment method to improve the curative effect of cholangiocarcinoma has importantsignificance.Our group has long been engaged in research of cholangiocarcinoma, We studiedthe cholangiocarcinoma associated gene Fascin and found that Fascin abnormallyhigh expression in cholangiocarcinoma and associated with invasion and metastasis ofcholangiocarcinoma..By RNA interfering technology and tumorigenic experiments innude mice, we demonstrated that the down regulating of Fascin expression by RNAinterference technology can restrain the growth of the bile duct cell carcinoma andreduce the invasion and proliferation. We also identified that fascin induced EMTdeveloping in bile duct carcinoma and promoted tumour invasion and metastasis,which is related to Wnt/β-catenin signaling pathways. In order to further explore thesignaling pathways of Fascin and their downstream gene. We select RNA interferenceknockdown fascin in bile duct cancer as a model to analyze differentially expressedgenes by Affymetrix microarray.Through analysis of differentially expressed genes bythe Pathway and Gene Ontology analysis,we further screen MLF1IP may play a targetgenes role for Fascin. We will study MLF1IP for further in this experiment.The myelodysplasia/myeloid leukemia factor1-interacting protein (MLF1IP) islocated in chromosome4q35.1, and its encoded protein has multiple classic structureand function regions. Hanissian found that MLF1IP genes have abnormally high expression in glioma. Other research has shown that MLF1IP can promote theoccurrence of polycythemia vera.Now, there is no research reports about MLF1IPassociated with bile duct cancer, and its role and mechanism in the occurrence anddevelopment of cholangiocarcinoma is not clear.Based on our preliminary studies andthe unique biological function of MLF1IP, we conclude that MLF1IP may play animportant role in the occurrence and development of cholangiocarcinoma, MLF1IPmay be a target gene of Fascin.We employed Real-time fluorescence quantitative polymerase chain reactiontechnique (Real-time PCR) to initial detect MLF1IP expression in resectionspecimens and paracancerous tissues of cholangiocarcinoma, observing whetherMLF1IP upregulate in bile duct carcinomas or not. At the same time, we apply thelentivirus-mediated RNA interference technology to silent MLF1IP gene expression inbile duct cancer RBE cell line, and use MTT method to detect the changes ofproliferation after RNA interference knockdown MLF1IP expression and Preliminaryinvestigat the function of MLF1IP in bile duct cancer. Lay the foundation for furtherimprovement of the MLF1IP genetic studies and clarify its role in the development ofcholangiocarcinoma.[Methods]1. Apply Real-time PCR method to detect MLF1IP expression in10cholangiocarcinoma tissues and paracancerous tissues collected from patients atthe people’s hospital of Hunan Province from June to December in2013. Analyzethe expressional difference between two tissues and explore the relationship ofMLF1IP and cholangiocarcinoma.2. Design for siRNA sequences of MLF1IP genes and constructs the recombinantshRNA shuttle plasmid and lentivirus packaging. Human bile duct carcinomaRBE cells were divided into experimental group and control group，Respectivelytransfected into MLF1IP-shRNA and shRNA lentiviral vector. Direct observationby fluorescence microscope to evaluate the transfection efficiency. DetectMLF1IP mRNA expression in each group by Real-timePCR to evaluate the interference efficiency.3. Compared the proliferation ability of bile duct cells in the experimental group andcontrol group by MTT.To study MLF1IP affect the biology behavior on bile ductcancer.[Results]1. In10cases of patients with cholangiocarcinoma tissues and paracancerous tissue,MLF1IP mRNA relative expression amount was5.03±1.96in thecholangiocarcinoma group,while in the paracancerous group,the relativeexpression amount was1.40±1.07.The expression of MLF1IP in cancer tissuescompared to the paracancerous tissues was significantly higher, and the differencehas statistically significant (P=0.000).2. Successfully construct MLF1IP-shRNA lentiviral plasmid, DNA sequencingresults show that the recombinant plasmid DNA sequence in accordance with theexpected.The recombinant plasmid MLF1IP-shRNA and two auxiliary packagingplasmids were transfected into293T cells for virus packaging.After60H, greenfluorescent protein expressed peaked and cells grew well which suggested thatlentiviral packaged success. The virus titer was measured by fluorescence afterconcentrated, results showed that the titer of the virus was3×108. After thepackaged lentivirus infected RBE cells respectively, we can observe that cellsinfection efficiency can reach above80%. To detect the expression inexperimental group and control group of MLF1IP gene by Real-time PCR, theresults showed that the experimental group MLF1IP mRNA relative expressionwas0.15±0.02, and in the control group,MLF1IP mRNA expression was1.01±0.12. Expression of MLF1IP in experimental group was significantly lowerthan that of the control group, the difference has statistically significant (P=0.005).The knockdown efficiency was85%.3. Check OD value by MTT assay, draw the growth curve for each group, the resultssuggested that growth curve of cells in the experimental group run smoothly, thegrowth was significantly decreased compared to the control group, the third day, fourth day and fifth day, compared with the control group, the OD value in theexperimental group was significantly reduced (p<0.05). Based on the OD value infifth day, in the experimental group, the inhibition ratio is13.30times lower thanthe control group. The results suggested the bile duct cancer cell proliferationactivity was significantly inhibited when targeted inhibited the expression ofMLF1IP in RBE cells.[Conclusion]1. MLF1IP expression in cholangiocarcinoma tissue is significantly higher than theparacancerous tissues, suggesting that MLF1IP may play a promoting role in thedevelopment of cholangiocarcinoma.2. Successfully construct MLF1IP-shRNA lentiviral vector, package lentiviral andtransfect into RBE cells effectively. Downregulate the expression of MLF1IP genein RBE cells, construct a stably low expressing MLF1IP the bile duct cancer cellsline. Provide a tool for the further study of MLF1IP gene in bile duct carcinoma.3. RNA interference silencing MLF1IP expression in RBE cells leads to thesignificantly decreased of the proliferation in bile duct cancer.