Research On The Biological Characteristics And Mechanism Of Breast Cancer Cells Regulated By KIF15

ObjectiveWith strong invasiveness and metastasis,breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death among females worldwide.Although significant advances in surgical treatments,chemotherapies,endocrinotherapies,targeted therapies and radiotherapies contribute to the eradication of primary tumors,the high recurrence rate and drug resistance seriously affect the prognosis of patients with breast cancer.Therefore,exploring the molecular mechanism of breast cancer progression and screening novel effective therapeutic targets and prognostic markers are of great practical significance for improving the therapeutic effects and survival rates of patients with breast cancer.Kinesin 15,also known as Hk1p2,is a member of the kinesin superfamily.It is mainly involved in cell mitosis,meiosis and macromolecular transport,and plays an important role in tumor drug resistance.Aberrant expression of KIF15 Leads to the development of tumors by causing abnormal distribution of intracellular genetic material.In preliminary study,over-expressed KIF15 is detedcted in BC tissues,being a compacted relationship with the prognosis of BC patients.Which reveals that KIF15 may be an important promoting factor in breast cancer and can be a potential target for treatment.As single-stranded,endogenous small non-coding RNA,miRNAs can lead to mRNA degradation or translation inhibition and regulate gene transcription by binding to the 3’untranslated region(3’-UTR)of the target gene.Studies have shown that multiple miRNAs participate in the occurrence,development and invasion of breast cancer.In this study,we detected the expression of KIF15 in breast cancer and adjacent tissues,and analyzed its correlation with clinicopathological characteristics of patients with breast cancer.Bioinformatics and experiments in vitro preliminarily explored the effect of upstream miRNA targeting KIF15 on breast cancer cell biology.The effect of KIF15 downregulation on proliferation and migration of breast cancer cells was studied by in vitro and in vivo experiments.To some extent,the molecular mechanism of KIF15 regulating the proliferation and migration of breast cancer cells was clarified,and its specific significance in the occurrence and development of breast cancer was clarified.Targeting KIF15 may be an effective strategy for breast cancer treatment.Methods1.Expression and clinical significance of KIF15 in breast cancer:The expression of KIF15 mRNA and protein in 55 cases of breast cancer and its corresponding adjacent tissues were detected by RT-qPCR and immunohistochemistry.The correlation between the expression of KIF15 and the clinicopathological characteristics of patients with breast cancer was analyzed.Kaplan-Meier overall survival analysis was used to explore the relationship between KIF15 expression and prognosis of breast cancer.2.Prediction and verification of upstream regulatory miRNA of KIF15 and its effect on the biological behavior of breast cancer cells:TargetScan and miRDB database were used to predict miRNAs targeting KIF15,and the dual luciferase reporter gene assay was used to preliminarily screen and verify the miRNAs and their binding sites.miR-578 had the most obvious inhibition effect on KIF15.CCK-8,colony formation and Transwell assay were used to the effects of miR-578 targeting KIF15 on the proliferation and migration of breast cancer cells.3.Effect of KIF15 downregulation on biological behavior of breast cancer cells:The expression level of KIF15 in siRNA transfected breast cancer cells was detected by RT-qPCR and Western blotting.CCK-8 assay,EdU cell proliferation assay,soft agar cell clone assay,plate clone assay and Transwell assay in vitro were used to detect the proliferation and migration of breast cancer cells after KIF15 downregulation.Western blotting was used to detect the expression of AKT,p-AKT,JNK,p-JNK,p53,PTEN,Cyclin D1,p21 and Cleaved Caspase3 after KIF15 down-regulation.Subcutaneous tumorigenesis model of nude mouse and lung tumor model of mouse were established to observe and analyze the subcutaneous tumorigenesis and lung tumor formation ability of breast cancer cells after KIF15 downregulation.Results1.Expression and clinical significance of KIF15 in breast cancer:With the results of RTqPCR and mmunohistochemistry,we found that comparing with paired adjacent tissues,there was significantly higher expression of KIF15 in breast cancer tissues,and the difference was statistically significant(P<0.01).Increased KIF15 expression was significantly correlated with tumor size(P=0.009),lymph node metastasis(P=0.046)and TNM stage(P=0.005),but not with age,Her-2 status,ER status and PR status(P>0.05).Moreover,Kaplan-Meier overall survival analysis indicated that higher expression of KIF 15 in breast cancer tissues is associated with worse prognosis of patients with breast cancer.2.Prediction and verification of upstream regulatory miRNA of KIF 15 and its effect on the biological behavior of breast cancer cells:Bioinformatics analysis screened out the top 10 candidate miRNAs targeting KIF 15,RT-qPCR and WB showed that the expression of KIF 15 mRNA and protein was inhibited significantly after miRNA-578 mimics was transfected into MDA-MB-231 cell.The dual luciferase reporter gene confirmed that miR578 binds to specific sites of KIF15-3’UTR to inhibit KIF15 expression.The expression of KIF15 protein decreased with the increase of miR-578 transfection dose.CCK-8 cell proliferation assay showed that miR-578 mimics significantly reduced the proliferation ability of MDA-MB-435S,MCF7 and MDA-MB-231 cells.Plate cloning assay found that miR-578 mimics inhibited the colony-forming ability of MDA-MB-435S,MCF7 and MDAMB-231 cells.Transwell migration assay showed that miR-578 mimics inhibits the migration ability of MDA-MB-435S,MCF7 and MDA-MB-231 cells.3.Effect of KIF15 downregulation on biological behavior of breast cancer cells:The siKIF15 sequence was successfully designed,RT-qPCR and Western blotting analyses confirmed the expression of KIF 15 mRNA and protein in MCF7,MDA-MB-435S and MDA-MB-231 cells were significantly suppressed after transfection of siRNAs against KIF 15(P<0.05).The cell growth curves using CCK-8 assay showed that KIF 15 downregulation significantly reduced the proliferation rates of MCF7,MDA-MB-435S and MDA-MB-231 cells(P<0.05).The proliferation of EdU cells was remarkably reduced by decreasing the expression of KIF15 in MDA-MB-231 cells(P<0.05).Soft agar clone formation assay and plate clone formation assay showed the ability of growth and colony forming ability decreased significantly after KIF 15 was down-regulated in breast cancer cells(P<0.05).Transwell cell migration assay showed that KIF15 of MDA-MB-231,MDAMB-435S and 4T1 cells was down-regulated,which inhibited the migration of breast cancer cells(P<0.05).Results of Western blotting analyses reveled that KIF 15 down-regulated in MCF7 and MDA-MB-231 breast cancer cells strongly inhibited the phosphorylation of AKT and JNK,and Cyclin D1 expression was also suppressed.In contrast,both of p53 and p21 protein expression remarkably increased.In addition,Cleaved Caspase3 was not changed significantly.The model of subcutaneous tumorigenesis in nude mice was successfully established.After the lentivirus was transfected into MDA-MB-231 cells,green fluorescence was observed in the cells through a fluorescence microscope.Western blotting found that the expression level of KIF15 protein in the MDA-MB-231/LV-shKIF 15-1 group was significantly reduced.It was observed with naked eyes that all nude mice in the NC group formed subcutaneous tumors,and three nude mice formed subcutaneous tumors in the KIF15 low expression group.The subcutaneous tumors of the nude mice in the NC group were significantly larger than those in the KIF15 low expression group,and the weight of the subcutaneous tumors in the KIF15 low expression group was significantly lighter than that in the NC group.The mouse lung tumor model was established successfully,gross morphological observation,HE staining and immunohistochemistry confirmed the presence of tumor in mouse lung tissue.Weighing the mice after 3 weeks showed that the KIF15 low expression group mice were significantly heavier than the NC group mice.The number of lung tumors in the KIF15 low expression group was significantly less than that in the NC group,and the weight of lung tumors in the KIF15 low expression group was lighter than that in the NC group.Immunohistochemistry showed that after down-regulating KIF 15,the expressions of KIF15 and Vimentin in lung tumors in the KIF15 low expression group decreased,while the expressions of KLF4 and E-cadherin increased.Western blotting and immunohistochemistry were used to detect the expression of AKT,p-AKT,JNK,p-JNK,p53,PTEN,Cyclin D1,p21 and Cleaved Caspase3 in mouse lung tumors,and the results were consistent with in vitro experiments.Conclusionl.The results revealed that KIF15 was significantly overexpressed in breast cancer tissues,increased KIF15 expression in breast cancer tissues were positively related with tumor size,lymph node metastasis and TNM stage,but not with age,Her-2,ER,and PR status.Moreover,high KIF15 expression predicted a worse prognosis of patients with breast cancer.2.Bioinformatics and cell experiments showed that KIF15 was the downstream target gene of miR-578,and miR-578 mimics targeted KIF15 to inhibit the proliferation and migration of breast cancer cells.3.KIF15 down-regulated effectively inhibited the proliferation and migration of breast cancer cells,KIF15 down-regulated effectively inhibited the subcutaneous tumor-forming ability of MDA-MB-231 cells,and KIF15 down-regulated inhibited the lung growth ability of 4T1 breast cancer cells in mice.KIF15 down-regulation affected the expression of functional proteins related to cell cycle,proliferation and apoptosis.KIF15 may be involved in the activation of tumor-related signal pathways.