Functions And Mechanisms Of FAT1 In Regulating Proliferation And Metastasis Of CC Cells

IntroductionCervical cancer(CC)is one of the most common gynaecological cancers.According to global cancer statistics in 2018,the incidence and mortality of CC ranks fourth among women,with an incidence rate of 6.6% and mortality rate 7.5%.Meanwhile,CC ranked second in the number of new cancer cases among women,indicating that CC is growing rapidly on a global scale.About 311,000 women died of CC each year.There were more than 30,000 women die of CC in China each year,the incidence and mortality rate of CC is significantly higher than Western countries.According to statistics,the survival rate of Phase I patients of the International Federation of Obstetrics and Gynecology(FIGO)is about 80-98%,but when CC metastasizes,the patient survival rate is significantly reduced to 50%.At the same time,although many patients receive great benefits after surgery or chemoradiotherapy,the recurrence rate and metastasis rate of CC remain high(about 29% to 38%)and are the leading cause of death.The invasion and metastasis of CC are associated with many signaling pathways and molecules,but the current research is in the initial stage,and the specific regulatory mechanisms remain unclear.Therefore,studying the mechanism of invasion and metastasis of CC has important practical significance.The human FAT1 gene is a member of the Fat cadherin family and is a homologous gene of Drosophila ft2.Our previous study found that the overall mutation rate of FAT1 in CC was 8.8%.The Fat cadherin family is mainly involved in tissue growth,development,cell plane polarity and cell migration.Analysis of human tissues showed that FAT1 mRNA expression levels were relatively low in most epithelial cells.Abnormal FAT1 deficiency may be associated with a range of hereditary diseases,such as 4q-syndromes,bipolar disorder,and the like.At the same time,abnormalities in development-related genes are often associated with tumor pathogenesis.Studies have found that FAT1 expression levels vary in human cancer cells.Interestingly,FAT1 acts as a tumor suppressor or oncogene in a variety of tumors through different pathways such as Hippo,Wnt and MAPK/ERK signaling pathways.FAT1 is generally considered to be a tumor suppressor gene in epithelial tumors,including oral cancer and head and neck squamous cell carcinoma.In contrast,FAT1 is considered to be an oncogene in melanoma,acute myeloid leukemia and acute lymphocytic leukemia.Current research suggests that FAT1 is associated with the development of multiple malignancies.However,the role and mechanism of FAT1 in CC is still unclear.In view of the fact that there is no research report on the expression level and function of FAT1 in CC,we intend to collect CC samples,detect the expression level of FAT1,and then explore the effect of FAT1 on the proliferation and migration of CC cells and explore the mechanism of FAT1 in CC whether it acts as a tumor suppressor gene or oncogene.These studies would provide a new strategy for reducing the recurrence or metastasis of CC patients,and provide a new way for gene-targeted therapy of CC.Methods1.Detecting the mRNA and protein expression of FAT1 in CC tissues and adjacent normal cervical tissues by qRT-PCR and immunohistochemistry.The collected specimens of CC tissues and paracancerous tissues were collected from hospitalized patients with CC who were treated in the gynecology department of the First Affiliated Hospital of the Army Medical University from January 2016 to July 2017.The expression of FAT1 mRNA and protein in cancer tissues and adjacent non-tumor tissues was detected by qRT-PCR and immunohistochemistry.The relationship between FAT1 expression and clinicopathological features was also explored.2.To construct the small interfering RNA(siRNA)targeting human FAT1 gene and transfect it into Hela and C33 A cell lines to observe the gene silencing effect of FAT1 on CC cells.To construct three siRNA oligos and a negative control(NC),and to screen out one siRNA fragment with the highest interference efficiency.After transfected to two cervical cancer cell lines,CCK-8 will be used to measure the OD value at 450 nm at 0,12,24,and 48 hours which is used to observe the changes in the proliferation of cervical cancer cells.HeLa and C33 a cell migration and invasion will be determined by transwell assay.3.To construct a plasmid for the overexpression of FAT1 gene and transfect it into Hela and C33 A cell lines to observe the effect of FAT1 gene overexpression on proliferation,migration and invasion of cervical cancer cells.Because the FAT1 gene is too long to be manipulated,a FAT1 truncated cDNA fragment containing the N-terminus,two cadherin regions,five EGFP repeat regions,the transmembrane region and the intracellular domain was established as described by a published paper,which was named FATI-TRUNC.Thus,to construct the overexpression vector pcDNA3.1-FAT1 of the FAT1 gene.After transfected to the two cervical cancer cell lines with the overexpression vector,CCK-8,Transwell migration and invasion assay were used to observe the effects of FAT1 expression on proliferation,migration and invasion of cervical cancer cells.4.To examine epithelial-mesenchymal transition(EMT)-associated marker levels to observe the effect of FAT1 on EMT of CC.After silencing or overexpressing FAT1,the expression changes of EMT-related genes(mesenchymal marker such as vimentin(VIM)and epithelial epithelial marker E-cadherin(CDH1))were detected.And whether this regulatory mechanism can be reversed by overexpression of ?-catenin protein.5.To explore whether FAT1 regulates the biological behavior of cervical cancer cells through Wnt/?-catenin signaling pathway.To detect the total expression of ?-catenin protein and the expression of phosphorylated ?-catenin protein after silencing or overexpression of FAT1 by Western blot,and dectect the expression levels of downstream target genes(TCF-4,c-Myc,MMP14)in Wnt pathway.The co-immunoprecipitation assay will be used to detect whether FAT1 can bind to ?-catenin.After overexpression of ?-catenin,we will observe whether the effect of FAT1 on cervical cancer cells can be reversed by overexpression of ?-catenin.To detect the expression changes of EMT-related genes(interstitial characteristic genes such as vimentin(VIM)and epithelial characteristic gene E-cadherin(CDH1)),and to observe whether EMT of cervical cancer is also through Wnt signaling pathway,and Whether this regulatory mechanism can be reversed by overexpression of ?-catenin protein.Results1.In 40 cases of cervical cancer tissues and adjacent tissues,the expression level of FAT1 in cervical cancer tissues was significantly lower than that in adjacent non-tomor tissues(p<0.05).At the same time,statistical analysis showed that the expression level of FAT1 protein in cervical cancer was related to lymphatic metastasis and vascular invasion.2.The FAT1 siRNA was successfully constructed and screened out the siRNA fragment with the highest interference efficiency.After silencing the expression of FAT1 gene,the proliferation rate of Hela and C33 A cells increased significantly in vitro.At the same time,the invasion and metastasis ability of the two cervical cancer cell lines increased too.3.The FAT1 overexpression plasmid pcDNA3.1-FAT1 was successfully constructed.After overexpression of FAT1 gene,the proliferation rate of Hela and C33 A cells decreased significantly,the ability of colony formation decreased,and the ability of invasion and metastasis also decreased significantly.4.After overexpression of FAT1,expression of EMT-related mesenchymal marker vimentin and transcription factor Twist decreased,while the expression of epithelial marker E-cadherin increased.Silencing FAT1 results conversly.5.Knockdown of the FAT1 gene increased total ?-catenin levels in cervical cancer cells,while phosphorylated ?-catenin(p-?-catenin)levels decreased.Meanwhile,knockdown of FAT1 increased the expression of the downstream target genes of the Wnt/?-catenin pathway in cervical cancer cells.In contrast,overexpression of the FAT1 gene downregulated the total ?-catenin levels,while increased levels of phosphorylated ?-catenin(p-?-catenin),and decreased expression of downstream target genes of the Wnt/?-catenin pathway.Co-immunoprecipitation showed that the endogenous FAT1 could bind to ?-catenin.6.Overexpression of ?-catenin and FAT1 showed that the expression of ?-catenin,c-Myc,TCF-4,MMP-14,twist and vimentin in pcDNA3.1-?-catenin/Adv.Flag-FAT1 group were higher than those of pcDNA3.1-vector/Adv.Flag-FAT1 group,pcDNA3.1-?-catenin/ Adv.Flag-FAT1 group had higher cell proliferation efficiency,invasion and migration ability than pcDNA3.1 –vector/Adv.Flag-FAT1 group,suggesting that ? –catenin could partially reverse the effect of FAT1 in Wnt pathway.These results suggested that FAT1 regulate EMT via Wnt/?-catenin pathway,and ?-catenin could partially reverse the effect of FAT1 on EMT.Conclusion1.The expression of FAT1 was significantly decreased in cervical cancer,and the expression of FAT1 was associated with lymphatic metastasis and vascular invasion.Abnormal expression of FAT1 is associated with the malignant biological behavior of cervical cancer and may be involved in the development of cervical cancer.2.In vitro experiments suggested that overexpression of FAT1 could inhibit proliferation,invasion and migration of cervical cancer cells through the Wnt/?-catenin pathway.Knockdown FAT1 expression by RNA interference promoted proliferation,invasion and migration of cervical cancer cells via Wnt/?-catenin signaling pathway.We also found that FAT1 inhibited the phosphorylation and degradation of ?-catenin by binding to ?-catenin,thereby inhibiting the nuclear translocation effect and inhibiting the Wnt/?-catenin signaling pathway.We proved that FAT1 has inhibitory effect on cervical cancer cells and has certain clinical significance.3.FAT1 can regulate cervical cancer EMT through Wnt/?-catenin pathway,suggesting that FAT1 may be involved in the process of cervical cancer metastasis,which may provide a new target for the treatment of cervical cancer recurrence and metastasis.