| The dynamic balances between cells and their surrounding extracellular matrix(ECM)precisely regulate cell activities including cellular proliferation,differentiation,apoptosis,invasion and metastasis in vivo to maintain the tissue homeostasis.However,the process of carcinogenesis and cancer progression in vivo caused by gene mutation constantly breaks the precise balances in vicious circles,which further leads to the gene expression and cell proliferation out of control.In this manuscript,we found that the gene expression level of Kif11(Kinesin family member 11)was positively related to breast cancer progression by the tissue microarray analyses of breast cancer progression.In order to further analyze the correlatios between Kif11 expression level and breast cancer progression,bioinformatics analyses were used in the first part of this manuscript to analysis the differentially expressed genes,signal pathways and hub genes of breast cancer progression.Three cancer progression databases were used to analysis the correlation between Kif11 gene expression levels and prognosis of breast cancer patient.The results showed that breast cancer progression was associated with pathways in cancer(hsa05200: Pathways in cancer),focal adhesion(hsa04510: Focal adhesion),cytokine-cytokine receptor interaction(hsa04060: Cytokine-cytokine receptor interaction),choline metabolism in cancer(hsa05231: Choline metabolism in cancer)and PPAR signaling pathway(hsa03320: PPAR signaling pathway),which were regulted by the hub genes including EGFR(Epidermal growth factor receptor),ESR1(Estrogen receptor 1),JUN(Jun proto-oncogene,AP-1 transcription factor subunit),IGF1(Insulin like growth factor 1),ERBB2(Erb-b2 receptor tyrosine kinase 2),FOS(Fos proto-oncogene,AP-1 transcription factor),TOP2A(Topoisomerase DNA II alpha),AR(Androgen receptor),KIT(KIT proto-oncogene receptor tyrosine kinase)and CD34(CD34 molecule).The expression levels of these above hub genes in breast cancer tissues were significantly correlated with that of Kif11 gene(p < 0.05),except ESR1 and AR.According to the results of cancer progression database,the Kif11 expression level was significantly correlated with cancer stages,subtypes and menopause of breast cancer patients(p < 0.05).In the second part of this manuscript,147 pairs of retrospective paraffin samples and 50 pairs of fresh samples were collected and the correlations between Kif11 expression level and prognosis of breast cancer patients were analyzed by fluorescence quantitative PCR,Western blot and immunohistochemical staining.The correlations between Kif11 expression level and breast cancer progression were further analyzed by siRNA transfection.The results showed that the expression levels of Kif11 gene and protein in breast cancer tissues were significantly higher than that in adjacent tissues(p < 0.05).The expression level of Kif11 protein and TNM stage were independent risk factors for the 5-year overall survival rate of breast cancer patients(p < 0.05).The proliferation,invasion,ECM remodeling,epithelial to mesenchy mal transition(EMT)and resistance to 5-Fu treatments of breast cancer cell lines were significantly inhibited by the down-regulation of Kif11 expression level by siRNA transfection.And the expression levels of hub genes including EGFR,ERBB2,TOP2 A,KIT and CD34 were significantly regulated with the expression pattern of Kif11 genes(p < 0.05).These above results indicated that Kif11 expression level was significantly related to the progression of breast cancer and Kif11 protein could be used as a prognostic marker for breast cancer patients.In order to further analysis the correlations between Kif11 expression level and breast cancer progression in 3D(three-dimensional)culture system,ECM of fresh breast cancer tissues and adjacent tissues were prepard with 1% lauryl ether sulphate solution(SLES)in the third part of this manuscript.Histological stainings(Hematoxylin-eosin staining,DPAI staining,Van Gieson staining,Alican and nuclearfast red staing)and quantification of biochemical content(DNA,collagen,elastin and glycosaminoglycan)were used to optimize the preparation system of ECM.And the results showed that 1% SLES solution could adequately remove the nuclear structure and DNA components of breast cancer tissues and corresponding adjacent tissues.At the same time,collagen,elastin and glycosaminoglycan in ECM were well retained.Accroding to the above results,the treatment of 1% SLES solution for 6 hours was selected as the ECM preparation system for breast cancer tissues and the treatment of1% SLES solution for 12 hours was selected as the ECM preparation system for adjacent tissues.Based on ECM of breast cancer tissues and adjacent tissues,different 3D culture systems were reconstructed in vitro to simulate the progress of breast cancer progression in vivo.Histological stainings and immunohistochemical stainings were used to analysis the effect of ECM on the proliferation of breast cancer cell lines in3 D culture system.Fluorescence quantitative PCR and Western blot were used to analysis the effect of ECM on the expression levels of Kif11 gene and protein in breast cancer cell lines.The expression levels of gene related with ECM remodeling process,EMT transcription factors,hub genes of breast cancer progression and multidrug resistance genes in breast cancer cell lines treated with 5-Fu were detected by fluorescence quantitative PCR.The results showed that ECM of breast cancer tissue significantly promoted the proliferation of breast cancer cell lines,the expression of Kif11 gene and protein,ECM remodeling process,EMT process and the expression levels of hub genes of breast cancer progression compared with ECM of adjacent tissues.Moreover,ECM of breast cancer tissue could significantly promote the expression of multidrug resistance genes in breast cancer cell lines,which further enhanced the cell resistance abilities to 5-Fu.In the last part of this manuscript,the 3D culture system was treated by Kif11inhibitor-SB743921 to further analysis the effects of ECM on the correlations between Kif11 gene expression level and breast cancer progression in 3D culture system.After the down-regulation of Kif11 gene and protein in 3D culture systemconfirmed by fluorescence quantitative PCR and western blot,the expression of PCNA,genes related with ECM remodeling process,EMT transcription factors and hub genes of breast cancer progression were detected by fluorescence quantitative PCR.The results showed that after down-regulation of Kif11 gene expression,ECM of adjacent tissues could significantly inhibit breast cancer progression in 3D culture system.Meanhwhile,ECM of breast cancer tissue could maintain the proliferation of breast cancer cells and breast cancer progression.The above results indicated that the correlations between Kif11 gene expression levels and breast cancer progression in3 D culture system were positively related to ECM and the 3D culture system based on ECM could provide an ideal 3D culture model for the further correlation studies between Kif11 gene expression levels and breast cancer progression. |
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