Fstl1 Promotes Glioma Growth Through The BMP4/Smad1/5/8 Signaling Pathway

Research background:Glioblastoma multiforme(GBM)is the most common and deadly brain tumor in adults coming from glial cells.It is characterized as high recurrence rate and high mortality.Its uncontrolled proliferation and insensitivity to chemotherapeutic agents are the main reasons for the rapid progression of glioma,short survival time and difficult to be cured.Proliferation,which enable tumor growth even regrowth,is a key aspect of the clinical progression of malignant gliomas.Despite of advances in standard therapy,including diagnostic methods and treatment strategies,the prognosis for patients with GBM remains poor.Up till the present moment,the treatment of glioma is mainly based on surgery,postoperative radiotherapy and chemotherapy.However,current treatment regimens do not significantly inhibit tumor recurrence and improve patient prognosis.Tumor proliferation is mainly caused by inactivation of tumor suppressor genes and abnormal activation of oncogenes.Therefore,a more comprehensive understanding of the molecular mechanism of tumor proliferation may provide an effective therapeutic strategy for latter treatment of glioma.Follistatin-like protein 1(Fstl1)is a secreted glycoprotein,whose structure is similar to that of follistatin,suggesting that it plays a role in TGF-? superfamily-induced inhibition..Therefore,the protein encoded by Fstl1 is also named follistatin-related polypeptide(FRP).Many studies have shown that Fstl1 plays an important role in the development of ovarian cancer,cervical cancer,lung cancer,pancreatic cancer and prostate cancer.Previous studies show that increased Fstl1 expression was correlated with glioma grade and overall survival in different grade gliomas.Fstl1 expression was significantly increased in high grade gliomas compared with that in lower grade gliomas and was associated with glioma prognosis.BMP4 is reported to inhibit the growth of glioma cells by activating the SMAD1/5/8 signaling pathway in glioma tissue.Our study found that Fstl1 competitively binds BMP4 with BMPR and thereby affects the BMP4/Smad1/5/8 signaling pathway to attect the growth of glioma cells.Vivo experiments also confirmed that Fstl1 promoted the growth of gliomas.All studies above suggest that Fstl1 may act as a cancer-promoting gene and our research could provide new targets for the treatment of malignant brain tumors.Methods:1.Colony formation and CCK8 assay were used to examined cell viability in LN229 ? P-GBM2 ? U87 and U251 glioma cells treated.Then,cell flow cytometry was used to access cell cycles in glioma cells.2.We next performed WB analysis to investigate the expression levels of p-Smad1/5/8?Smad1/5/8?p21 following Fstl1 treatment,and q RT-PCR for their m RNA level.Furthermore,we used coimmunoprecipitation(Co-IP)and immunoprecipitation to validate the relationship between Fstl1,BMP4 and BMPR2.3.Nude mice were first intracranially xenotransplanted with luciferase-expressing U87 glioma cells and primary cell both treated or not.After tumor formation,After the start of treatment,tumor growth was monitored by a live animal bioluminescence imaging system every week.We also performed Immunohistochemistry analysis of p-Smad1/5/8 and p21 levels in human glioma cells.Results:1.Fstl1 overexpression promoted cell proliferation and cell cycle progression.2.Fstl1 knockdown suppressed glioma cell proliferation and cell cycle progression.Tumor growth is inhibited in vivo as well.3.Fstl1 impairs the association between BMP4 and BMPR2 by competitively binding with BMP44.Tumors with low levels of Fstl1 tends to express high levels of p-Smad1/5/8 and p21,whereas those with high Fstl1 levels tends to express low levels of p-Smad1/5/8 and p21Conclusions:1.Fstl1 promotes cell proliferation and growth.2.Fstl1 impairs the association between BMP4 and BMPR2 by competitively binding with BMP4,and eventually promotes malignant biological behavior of glioma cells.