The Role Of Transcription Factor FOXG1 In Colorectal Cancer Invasion And Metastasis

?Background? Colorectal cancer(CRC)is one of the most common malignant tumors of digestive tract worldwide,and greatly threatens human well-being.Over the past years,advances in diagnostic techniques,chemoradiotheraputic agents,operative management and molecular targeted anti-tumor drugs have decreased the mortality of CRC.However,the prognosis of CRC is still not optimistic.The poor prognosis of CRC might be attribute to the low rate of early diagnosis so that many patients were detected in the late stage and miss the best period of treatment.The recurrence and metastasis of CRC is the main cause of death,and is also an important factor affecting the prognosis of patients with CRC.Therefore,it is critical to investigate the molecular mechanisms of metastatic CRC(m CRC),which is essential to identify novel biomarkers for m CRC diagnosis and prognosis,and may bring great hope for survival of CRC patients.The activation of transcription factors(TFs)plays an important role in tumor metastasis,but the molecular mechanism is still not clear.In recent years,the FOX family have been reported to make a difference in tumor initiation and progression.Our previousstudy has identified that FOXG1 as a key molecular in the downstream of the Txl-2b-Ran axis,which regulates the progression of CRC,using high-throughput TFs chip.Through reviewing the literatures,we found that FOXG1 expresses highly in tumor tissues and may influence the malignant phenotypes of cancer,such as maligant proliferation,anti-apoptosis and metastasis.However,it is rarely reported in CRC.?Aims? 1.To detect the expression of FOXG1 in CRC tissues and cell lines;2.To investigate the role of FOXG1 in malignant biological behavior of CRC cells.?Methods? 1.Immunohistochemical(IHC)staining was performed to detect the expression of FOXG1 in CRC specimens and the paired normal colorectal tissues;2.Western blot and q RT-PCR were used to detect the expression in immortalized human intestinal epithelium cell HIEC and a variety of CRC cell lines HCT116?Caco-2?SW1463?DLD-1?HCT8?RKO?Lo Vo and HT29;3.Immunofluorescence staining was carried out to examine the subcellular localization of FOXG1 in human intestinal epithelium cell HIEC and CRC cell lines HCT116,HCT8;4.Lentiviral conducts were infected into HCT8 and HCT116 cells for gain-of-function and loss-of-function models;and Western blot,q RT-PCR and green fluorescence were used to ascertain the efficiency of virus infection;5.Transwell assay was employed to determine the role of FOXG1 in migration and invasion of CRC cell lines in vitro;6.Flow cytometry was used to detect the role of FOXG1 in cell cycle of CRC cell lines in vitro;7.CCK-8 assay was used to detect the role of FOXG1 in proliferation phenotype of CRC cell lines in vitro;8.In vivo metastasis assays were performed to examine the role of FOXG1 by injection of CRC cells through the tail vein.Bioluminescence imaging was used to detect the metastasis of CRC cells in vivo.HE staining was used to confirm metastatic lesion in the lung of nude mice.?Results? 1.FOXG1 was significantly upregulated in CRC tissues in comparison to paired normal tissues on colorectal tissue microarray with 196 cases of CRC patients;2.Western blot and q RT-PCR showed that the expression of FOXG1 was significantly increased in CRC cell lines as compared with HIEC both at m RNA and protein levels;3.Immunofluorescence staining revealed that FOXG1 was located primarily in the nucleus of cells and a significant difference in FOXG1 expression between CRC cell lines and HIEC;4.By lentiviral infection and subsequent puromycin screening,we successfully constructed FOXG1 gain-of-function and loss-function models respectively in HCT8 and HCT116 cells,which was then verified by Western blot and q RT-PCR;5.Transwell assay in vitro demonstrated that overexpression of FOXG1 promoted migration and invasion whereas silencing of FOXG1 suppressed migration and invasion of CRC cells;6.Either up-regulation or down-reglation of FOXG1 displayed visible effect on the cell cycle and proliferation of CRC cells as showed in flow cytometry and CCK-8 assays;7.The bioluminescence imaging and HE staining demonstrated that up-regulation of FOXG1 promoted the metastasis of CRC cells in vivo.In contrast,the down-regulation of FOXG1 suppressed its metastasis.?Conclusions? 1.FOXG1 is upregulated in CRC tissues and cell lines compared with their normal controls.2.Over-expression of FOXG1 promotes the invasion and metastasis of CRC cells both in vitro and in vivo;whereas down-regulation of FOXG1 inhibits the invasion and metastasis.The current study elucidates the important role of FOXG1 in CRC metastasis and indicate that FOXG1 may be a metastatic-promoter in CRC.