The Effect And Molecular Mechanisms Of FSTL1 In The Invasion And Metastasis Of Colorectal Cancer

Colorectal cancer(CRC)is one of the malignant tumours of digestive tract with high morbidity and mortality in the world.In China,the incidence of CRC is also increasing year by year and average age of onset become younger.The urgent problem of colorectal cancer research is to elucidate the molecular mechanism of the development of CRC,and to predict,diagnose,intervene and treat it.In previous study,we had confirmed that Follistatin-like protein 1(FSTL1)was up-regulated in CRC.And bioinformatics survival analysis revealed that CRC patients with a higher level of FSTL1 expression had a worse clinical outcome.FSTL1 is a secretory glycoprotein with a variety of biological regulation functions.In recent years,relevant reports mainly focus on its role in inflammation,autoimmune diseases,improvement of ischemia reperfusion injury and immunoregulation.In addition,FSTL1 has been reported to have both tumour-promoting and tumour-suppressive characters.However,the role of FSTL1 in CRC remains unclear.Therefore,in order to explore the exact role and specific mechanism of FSTL1 in CRC,We are going to carry out the following study,which including the effect of FSTL1 on the progression of CRC,its transcription regulation and downstream signalling molecular mechanisms,and its role in the tumour microenvironment of CRC.Firstly,we found that the expression of FSTL1 in the tissues and peripheral blood samples of CRC patients was significantly up-regulated by western blotting and ELISA.FSTL1 overexpression was associated with the infiltrating depth,lymph node metastasis of CRC by immunohistochemistry.Then we constructed stable CRC cells with high expression and knockdown of FSTL1,and used experiments in vivo and in vitro proved that enhanced expression of FSTL1 distinctly increased cell migration and invasion in vitro as well as promoting liver metastasis of CRC and shorting the survival time of nude mice in vivo.Moreover,we preliminary explored the role of FSTL1 in the microenvironment of CRC.We carried out the chemotactic assays and found recombinant human cytokine FSTL1(rhFSTL1)had obvious chemotactic effect on normal fibroblasts(NF),and the ability was related to the concentration of rhFSTLl.Using rhFSTL1 to stimulate NF,FSTL1 could induce NF to be activated to cancer associated fibroblasts(CAF)by immunofluorescent confocal laser scanning.Secondly,FSTL1 and TGF-?1 protein expression levels in consecutive paraffin-embedded slice of human CRC tissue were detected by IHC.Positively related between them coincided with bioinformatics prediction.Subsequently,we performed chromatin immunoprecipitation assay(ChIP),dual-Luciferase activity assay and western blotting to confirm that TGF?1-Smad2/3 signalling pathway regulated FSTL1 expression through activating the transcriptional activity of transcription factor Smad3.Finally,we found that FSTL1 promoted CRC progression via activating the focal adhesions signalling and regulating cytoskeleton rearrangement by GSEA,western blotting,FN adhesion assay and immunofluorescence.We identified VIM,as an interactive protein of FSTL1,participated in FSTL1-mediated aggressive phenotype by immunoprecipitation,mass spectrometry and co-localization.In summary,we demonstrated that FSTL1 was up-regulated and had a close relationship with poor outcome in CRC.The overexpression of FSTL1,inducing by TGF?1-Smad2/3 signalling pathway,functionally promoted CRC cell migration,invasion,and metastasis by combining with VIM and activating focal adhesion signalling pathway consequently.FSTL1 could induce normal fibroblasts to be activated to cancer associated fibroblasts in the tumour microenvironment of CRC.Therefore,our data provide evidence for FSTL1 being regarded as a detectable biological marker that reflects malignant degree and evaluates prognosis in CRC.