Inhibition Of G-CSF Expression Downregulates The Signaling Pathway Of G-CSFR And Suppresses The Malignant Behaviors Of Murine Mammary Carcinoma Cells

IntroductionGranulocyte colony-stimulating factor(G-CSF),which is mainly secreted by monocytes/macrophages,endothelial cells and fibroblasts in response to many stimuli,is a polypeptide growth factor that plays important roles in granulopoieisis and regulating the function of neutrophil.G-CSF exerts its biologic activities by binding to the G-CSF receptor(G-CSFR)that is predominantly expressed on the surface of neutrophils throughout all stages of maturation.Recombinant human G-CSF(rhG-CSF)is widely used to treat congenital neutropenia or chemotherapy-induced severe neutropenia and to mobilize hematopoietic stem and progenitor cells(HSPCs)from bone marrow to the blood for transplantation.Recent studies have reported that many tumors produce G-CSF;G-CSF might be involved in cancer development and progression.Long-term alterations in gene and microRNA expression profiles have been detected in hematopoietic progenitor cells from healthy donors who have received G-CSF for HSPCs mobilization and collection;G-CSF treatment has been implicated in driving myeloid malignancy development.G-CSF promotes prostate cancer development by regulating tumor neurogenesis.G-CSF contributes to aggressive phenotypes of non-small-cell lung cancer cells by promoting epithelial-mesenchymal cell transition(EMT).Functional G-CSFR has been reported to be expressed on high-grade ovarian tumors and ovarian cancer cell lines;G-CSF stimulation on G-CSFR-positive ovarian cancer cells can drive migration and survival.Breast cancer is a common cancer among women of all races and one of the leading causes of death in women worldwide.Increased G-CSF expression is associated with a metastatic phenotype in breast cancer cells and poor prognosis in breast cancer patients.However,the mechanisms by which G-CSF mediates growth and metastasis of breast cancer cells are not completely known.To acquire a greater insight into the pathophysiological role of G-CSF in breast cancer progression,we mediated the deletion of G-CSF in two murine mammary carcinoma cell lines using CRISPR/Cas9 gene editing.We investigated the effects of G-CSF knockout on malignant properties of murine mammary carcinoma cells and explored the implicated mechanisms.This study not only indicates that G-CSF plays an active and important role in breast tumor growth and metastasis,but also may provide a therapeutic target to restrict breast tumor spread.ObjectiveTo investigate the in vitro and in vivo effects of knockout of G-CSF on malignant properties of murine mammary carinoma cells and explore the possible mechanisms.Methods1.G-CSF gene sequence data was consulted at Genebank.Then Oligos which can recognize the splice sites was designed and synthesized.Construct the vectors through plasmid construction and Lenti-virus packaging.2.Lentivirus with Cas9,sgRNA and puromysin-resistant infected 4T1 and 4T07 cells.The transfected cells were selected and enriched by applying puromycin(4μg/ml)in culture medium.Target cells were selected by monoclonal cultivation and spreading cultivation was conducted.The concentration of G-CSF in supernatant was measured by CBA assay.3.Breast tumor models were established and serum of tumor-bearing mice was collected after 30 days of tumorigenesis.The concentration of G-CSF in serumt was measured by CBA assay.4.SRB method was used to measure proliferation capacity in 96 well plate.The cancer cells were planted in 96 well plate(3000 cells/well)and cultured for 72 h.Cells were stained by SRB for 15minutes after fixed with 10%TCA.150 μl Tris-Hcl buffer per well was added and the OD value was measured at 560nm in microplate.5 · The cancer cells in the logarithmic phase were digested with trypsin to monoplast suspension.The cloning formation assay was performed in 6 well plate(1000 cells/well)for 14 days.The clones formed were stained by crystal violet for 30 minutes after fixed with 5%glutaraldehyde.The clones were counted and photographed.6.The cancer cells in the logarithmic phase were digested with trypsin to monoplast suspension and then cultured in soft agar(10000cells/well).After cultured at 37℃ for 21 days,the clones were photographed by inverted microscope.7.The cells were plated at 2×105 cells per well in 96 well plate,and the monolayer was scratched with a 200 μl pipette.The scratch area was photographed at 0 h,24 h and the wound healing percentage at 24 h was analyzed statistically.8.3×104 cells were suspended in 100 μl serum-free RPMI1640 medium and seeded into the upper chamber of each insert.The lower chamber was filled with 700 μl RPMI1640 containing 20%FBS to induce cell migration.The chamber was incubated at 37℃ for 8 h and then fixed with 5%glutaraldehyde,stained by crystal violet.Cells in the upper surface of membrane were removed.The images were obtained by inverted microscope and the migrated cells were counted in 5 different view fields.9.The cells were cultured in serum-free medium leading to apoptosis.The apoptotic ratio was measured by flow cytometric analysis of Annexin V/PI stained cells after 36h.10.The cells were cultured in plate incubated with poly-HEMA for inducing anoikis.The TUNEL assay was used to measure apoptotic index by flow cytometry after 36 h.11.Subcutaneous orthotopic animal model of breast cancer was established and the tumors werevmonitored to plot growth curve every 3 days after tumor formed.The tumors were photographed and weighed after excised from sacrificed mice.The lungs were fixed in Bouin’s solution for 24 h and then photographed.The lung metastatic nodules were counted according to metastasis level.12.The model of experimental lung metastasis was established by i.v.injection of 4T1 cells.The mice were sacrificed after 14 days and the lung was excised.The lungs were fixed in Bouin’s solution for 24 h and then photographed.The lung metastasis nodules were counted according to metastasis level.13.G-CSFR expression was assessed by flow cytometric analysis.The levels of p-JAK2,p-Stat3,Stat3,p-ERK1/2,ERK1/2,p-Akt,Akt,Bcl-2 and cyclinD1 were determined by Western blot analysis.Results1.The lenti-vector was constructed successfully and the G-CSF expression was inhibited by CRISPR/Cas9 editing system both in vitro and in vivo.2.Inhibition of G-CSF expression suppresses the malignant behaviors of murine mammary carcinoma cells.The proliferative,anti-apoptosis,anti-anoikis capacity and migration were inhibited after inhibition of G-CSF expression.3.Inhibition of G-CSF expression represses the activation of proliferative and metastasis signaling pathways.The protein levels of p-JAK2,p-Stat3,p-ERK1/2,p-Akt were decreased while Stat3,ERK1/2,Akt protein levels remained unchanged.Also,proliferation and apoptosis-related factors,cyclinDl and Bcl-2 protein levels of G-CSF knockout murine mammary carcinoma cells were decreased compared to control cells.ConclusionCRISPR/Cas9-mediated knockout of G-CSF suppresses the malignant behaviors of 4T1 and 4T07 murine mammary carcinoma cells by downregulating the signaling pathway of G-CSFR.Our data suggest that G-CSF is critical in modulating the malignant properties of murine mammary carcinoma cells.