KIF15 Promotes Pancreatic Ductal Adenocarcinoma Proliferation And Invasion Via The MEK-ERK Signaling Pathway

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The expression and clinical significance of KIF15 in pancreatic cancer tissueObjective: Screen potential gene therapy targets for pancreatic cancer and validate the results.Detect KIF15 expression in pancreatic cancer tissues.Investigate the expression and clinical significance of KIF15.Methods: Agilent m RNA microarray analysis of m RNAs from seven pancreatic cancer tissue and seven healthy control tissue was performed.22 genes were selected for high-content si RNA screening to validate the microarray results and verify the function.Immunohistochemical(IHC)staining of 90 pairs of matched PC and normal pancreatic tissue was performed to detect the expression of KIF15 and the relationship with clinical stage and prognosis.Detect the expression of KIF15 in 27 pair of pancreatic cancer and normal tissues by q RT-PCR.Results: 1.The results indicated that 892 m RNAs were upregulated and 568 m RNAs were downregulated.2.Data analysis identified four candidate genes: FAM54 A,GMFB,KIF15,and ZWINT.Knockdown of each gene reduced the cell proliferation and KIF15 knockdown resulted in the lowest proliferation rate in PANC-1 cells.3.Immunohistochemical(IHC)staining of tissue microarrays shows that KIF15 is mainly localized to the cytoplasm of human PC cells,whereas no KIF15 staining was present on adjacent normal pancreatic tissue.Higher KIF15 expression is associated with poorer tumor differentiation(according to NCCN staging)and shorter overall patient survival time.The expression of KIF15 was significantly higher in tumors from patients with lymph node involvement and/or distant metastasis than in those without that.KIF15 m RNA expression was upregulated in PC tissues detected by RT-PCR.Conclusion: KIF15 is up-regulated in pancreatic cancer tissue and correlate with a poor prognosis.KIF15 may play a crucial role in the development of pancreatic cancer,which can be used as a further study.Part II The effect of KIF15 expression on proliferation of pancreatic cancer cellsObjective: To investigate the effect of KIF15 expression on proliferation of pancreatic cancer cells in vivo and vitro.Methods: si RNA sequences for knockdown KIF15 and lentiviral vectors of KIF15 including overexpression,knockdown and negative control were designed and constructed,and were infected into pancreatic cancer cell lines PANC-1 and MIA Pa Ca-2 respectively.RT-PCR was used to detect the expression of KIF15.CCK-8 was employed to detect cell proliferation.Flow cytometry was used to detect cell cycle and apoptosis.The subcutaneous tumor model of nude mice was established to detect the effect of KIF15 on proliferation in vivo.The expression of Ki67 for detecting proliferation in mice tumor by IHC.Western bolt was used to detect the expression of cyclin protein.Results: CCK-8 and subcutaneous tumor model showed that KIF15 could promote cell proliferation in vivo and vitro.Meanwhile,inhibition of KIF15 expression made cell cycle of pancreatic cancer arrest in G1 phase.Nevertheless,there was no significant in cell apoptosis.The expression of immunohistochemistry index Ki67 was up-regulated in mice tumor.Western bolt result showed KIF15 up-regulated the expression of cyclin D1,CKD2,and p-RB.Conclusion: Our results suggested that KIF15 could promote proliferation of pancreatic cancer cell,and inhibition of KIF15 induced the cell cycle arrest in G1 phase.Part ? The effect of KIF15 expression on migration and invasion of pancreatic cancer cellsObjective: To investigate the effect of KIF15 expression on invasion of pancreatic cancer cells in vivo and vitro.Methods: Transwell assay and the model of liver metastasis were employed to detect cell invasion ability.The influence of KIF15 on cytoskeleton was detected by immunofluorescence technique.Western bolt was used to detect the expression of Matrix metalloproteinases.Results: Transwell assay result showed up-regulated KIF15 increased the invasive cell than negative control,and overexpression of KIF15 induced more liver metastasis and poor prognosis in nude mice.Immunofluorescence test showed that up-regulated KIF15 in pancreatic cancer cell PANC-1 and MIA Pa Ca-2,F-actin was depolymerization and down-regulated.As the result,cells lost movement structure and polarity and their migration ability was weakened.Meanwhile,up-regulated KIF15 increase the expression of MMP2 and MMP14.Conclusion: Our results suggested that KIF15 could promote invasion and migration of pancreatic cancer cell,and inhibition of KIF15 induced less liver metastasis and improved prognosis of nude mice.The underline mechanism might was that overexpression of KIF15 induce up-regulated MMP2 and MMP14,which contributed to the degradation of extracelluar matrix.Part ? KIF15 promotes pancreatic ductal adenocarcinoma proliferation and invasion via the MEK-ERK signaling pathwayObjective: To investigate the underline mechanisms that KIF15 promotes pancreatic cancer proliferation and invasion.Methods: Agilent m RNA microarray analysis of m RNAs from 3 pair KIF15 knockdown PANC-1 and negative control PANC-1 was performed.Surprisingly,many key genes in the MEK–ERK signaling pathway were downregulated in the KIF15 knockdown group.Pathway analysis confirmed that the MEK–ERK pathway was most closely connected with KIF15,so we preliminary analysis that KIF15 might activated MEK-ERK pathway.After that,we use western bolt to detect the expression of MEK-ERK pathway related proteins,and utilized co-immunoprecipitation and immunofluorescence to detect interaction in MEK-ERK pathway related proteins.Then we use inhibitor of MEK-ERK pathway to detect the change of biological effect conducted by KIF15.Meanwhile,the liver metastasis model was established to detect the liver metastasis with injection of PD98059 induced by KIF15.Western bolt was used to detect the expression of cycle protein and MMPs after adding inhibitors of MEK-ERK pathway.Results: we find out that overexpression of KIF15 increased the expression of p-c-Raf,p-MEK and p-ERK,which were the key proteins in MEK-ERK pathway,and KIF15 could interact directly with p-c-Raf and p-MEK.KIF15 knockdown in PANC-1 and MIA-Pa Ca-2 cells suppressed p-ERK translocation from the plasma membrane to the nucleus.What's more,inhibitor of MEK-ERK pathway could block the activation,which reduced the expression of MMPs and cycle proteins.Meanwhile,the role of KIF15 in promoting proliferation and invasion was suppression with the inhibitor of MEK-ERK pathway.The model of liver metastasis revealed that PD98059 could reverse KIF15 in promoting liver metastasis,decreased the metastasis and improved the condition of nude mice.Conclusion: Our major research suggested that KIF15 could interact directly with p-c-Raf and p-MEK,which activated the downstream p-ERK.And p-ERK translocated from the plasma membrane to the nucleus,increased the transcription and induced up-regulated cyclin D1 and MMPs.Finally,the increased protein promoted proliferation and invasion.