Expression And Molecular Mechanism Of KIF20B To Promote Progression Of Tumor In Lung Adenocarcinoma

Background and objective:Lung cancer ranks first and second respectively in cancer-related deaths in men and women,with a dismal 5-year survival rate of only 10-15%.It was estimated that in China733.3 thousands new lung cancer cases and 610.2 thousands lung cancer deaths occurred in 2015,accounting for about 21.6%of all cancer deaths.The high incidence and mortality of lung cancer worldwide make this disease a notable health-care issue.In recent decades,with the development of molecular technology,and the intense research of tumor related genes,diagnosis and treatment of lung adenocarcinoma have undergone revolutionary changes.The introduction of targeted therapies against EGFR,such as erlotinib and afatinib,has revolutionized the treatment of a subset of lung adenocarcinomas that have EGFR-activating mutations;however,almost all patients acquired resistance after about 12 months.Meanwhile,even among Asian ethnicity female of non-smokers,the EGFR activating mutation rate in lung adenocarcinoma is only 51%.Therefore,new molecular target should be searched.Kinesin family member 20B(KIF20B)was identified through a subset of proteins which specifically phosphorylated at the G2/M transition.And KIF20B,which belongs to the kinesin superfamily,is a kind of plus-end-directed kinesin protein.Like all kinesin proteins,KIF20B also has a hyperconserved motor domain,and an ATPase activity dependent on microtubules.KIF20B can be combined with microtubules and use ATP enzyme hydrolysis to produce mechanical force,and then transport organelles,vesicles,intracellular substances to the proper position along microtube.Therefore,it plays an important role in maintaining cell morphology and function.In anaphase of mitosis,it surges in expression as into G2 phase,and then diffuse distribution in mitotic metaphase cells,Later,it is located in the central spindle,and end in the central body.The expression of KIF20B ensures that the cell centrosomes,spindles,chromosome sets and cytoplasmic divisions are performed precisely in mitosis.KIF20B can be detected in human brain,ovary,and kidney,especially higher in testis.Subsequent studies have found that the abnormal expression of KIF20B promotes the occurrence of multiple tumors such as bladder cancer,and drives the proliferation,invasion and self-renewal of tumor cells.The decrease of expression level can lead to mitotic disorder of tumor cells,cytokinesis anomaly,and then apoptosis,so as to control the tumor.KIF20B is likely to be a new target for tumor gene therapy.Currently,the expression of KIF20B in lung Adenocarcinoma is unknown and its role in the progression of lung Adenocarcinoma is unclear.This study,in which the correlation between KIF20B expression and prognosis of adenocarcinoma was predicted by bioinformatics,then the expression of KIF20B in lung adenocarcinoma was detected by Real Time PCR and immunohistochemistry,mainly deals with the correlation between KIF20B expression and patients'clinical parameters.TCGA database was used to validate the result.We knocked down the KIF20B by RNA interference technique in order to explore its function in the proliferation,invasion and motility of lung adenocarcinoma cells,and the fuction was further confirmed in-vivo xenograft lung adenocarcinoma model.The mechanism of KIF20B affecting tumor growth was also studied.Part 1 Expression of KIF20B in Lung Adenocarcinoma and its Relationship with the Clinical SignificanceMethods1)TCGA data sets were used to analyze the expression of KIF20B in adenocarcinoma.2)Real Time PCR was used to detect KIF20B mRNA expression in lung adenocarcinoma and adjacent normal lung epithelial tissues.3)The correlation between the expression level of KIF20B mRNA and the clinicopathological parameters was analyzed.4)Immunohistochemistry was performed to detect KIF20B protein expression in the lung adenocarcinoma and adjacent normal lung epithelial tissue.5)The relationship of KIF20B protein expression and clinicopathological parameters were analyzed in patients with lung adenocarcinoma.6)The relationship between KIF20B and prognostic of patients was analyzed.Results1)The analysis of TCGA data sets showed that the KIF20B mRNA expression in adenocarcinoma was significantly higher than that in adjacent normal lung epithelial tissue.2)The expression of KIF20B mRNA was significantly higher in 76 lung adenocarcinoma tissues than that in adjacent normal lung epithelial tissue correspondent(P<0.05).Unpaired analysis showed that the expression of KIF20B mRNA in 124 cases of lung adenocarcinoma was higher than that in 76 cases of adjacent normal lung epithelial tissue(P<0.05).3)The expression of KIF20B mRNA in lung adenocarcinoma patients was significantly correlated with tumor stage,lymphatic metastasis,and live state(P<0.05).4)The expression of KIF20B protein in lung cancer was significantly higher than that in adjacent normal lung epithelial tissue.KIF20B Protein was correlated with tumor stage,lymphatic metastasis and live state(P<0.05).5)Patients with higher expression of KIF20B had lower survival than those with lower expression(P<0.05).Summary1)The expression of KIF20B in lung adenocarcinoma tissues was higher than that in adjacent normal lung epithelial tissue.And it was associated with the tumor stage and lymphatic metastasis.The results show that KIF20B might play an important role in the development and progression of lung adenocarcinoma.2)The expression of KIF20B was significantly correlated with the survival of patients with lung adenocarcinoma,suggesting that KIF20B might be a prognostic factor in lung adenocarcinoma.Part 2 Effect of KIF20B on Biological Behavior of lung Adenocarcinoma cellsMethods1)KIF20B gene enrichment analysis was carried out through TCGA,and the correlation analysis was used to analyze the related markers.2)Real Time PCR was performed to measure KIF20B mRNA expression in human lung adenocarcinoma cells lines and normal lung epithelial immortalized cells16HBE.Construct lung adenocarcinoma cell lines with stably KIF20B knockdown.Then Real Time PCR and Western Blot were used to detect the knockdown efficiency.3)CCK8 assay and Colony-forming assay were used to detect the effects of decreased KIF20B expression on the proliferation of lung adenocarcinoma A549 and Calu-3 cells.4)The flow cytometric analysis was performed to investigate the effect of KIF20B knockdown on apoptosis of lung adenocarcinoma cells.5)The Transwell migration assay and Wound Healing test were utilized to observe the effect of KIF20B knockdown on cell migration and invasion of lung adenocarcinoma cells.6)Ultra-low adhesion into the ball culture experiment was performed to examine the effect of KIF20B knockdown on self-renewal ability of lung adenocarcinoma.7)The in-vivo xenograft lung adenocarcinoma tumor model of nude mice was established to observe the effect of KIF20B knockdown on the tumor growth.The expression of KIF20B,Ki67,MMP9 on tumor site were detected.Results1)The gene enrichment analysis showed that KIF20B had a positive correlation with Ki-67 and CDK1,implying KIF20B was related with cell proliferation inadenocarcinoma.2)The expression of KIF20B mRNA was relatively higher in lung adenocarcinoma A549 and Calu-3 cells,thus those two cell lines were chosen to establish stable KIF20B knockdown cell lines.Transfection efficiency and knockout efficiency were achieving experimental standard.3)KIF20B knockdown could inhibit the proliferation,migration and invasion ability and promote apoptosis of lung adenocarcinoma cells.But it has little effects on self-renewal ability and cell cycle.4)In the in-vivo xenograft lung cancer model of nude mice,KIF20B knockdown could inhibit tumorigenicity.Summary1)KIF20B knockdown could inhibit migration and invasion and proliferation ability of lung adenocarcinoma cells,while promote apoptosis.2)KIF20B knockdown could inhibit the tumor formation in the in-vivo xenograft lung adenocarcinoma model of nude mice.Which indicates that KIF20B plays an important role in lung adenocarcinoma's development and malignant behavior.Part 3 KIF20B promotes the recruitment of monocyte/macrophages by regulating the activity of NF-?BMethods1)Cytometric Bead Array was performed to analyze the expression and changes of chemokines and cytokines in lung adenocarcinoma cells after KIF20B knockdown.The cytokine with the biggest fold changes was selected and then verified by ELISA.At the same time,Real Time PCR and Western Blot were used to detect the expression of CCL2 in A549 after KIF20B knockdown.2)The in-vitro Transwell assay was used to detect the effect of KIF20B knockdown on the recruitment of CD14~+cells isolated from healthy human peripheral blood.3)CD14~+monocytes isolated from healthy human peripheral blood were injected into the in-vivo xenograft lung Adenocarcinoma tumor model of nude mice to verify the effect of KIF20B suppression on the recruitment of CD14~+CCR2~+monocyte/macrophages.4)Real Time PCR was used to detect the expression of KIF20B,CCL2 and CCR2,CD163 in tumor xenografts of nude mice.and immunohistochemistry was used to detect the expression of KIF20B,CCL2,CCR2 and CD163.5)Western Blot was applied to analyze the effect of KIF20B knockdown on the NF-?B activity.The expression of CCL2 in A549 cells after the treatment of NF-?B inhibitor was examined by Real Time PCR and ELISA.6)Real Time PCR was performed to investigate the correlation between the mRNA expression of KIF20B and CCL2?CD14 and CD163 in lung Adenocarcinoma samples.7)The Kaplan–Meier survival curve was utilized to assess the association between the expression of CD163 mRNA and prognosis in patients with lung Adenocarcinoma.Results1)With the knockdown of KIF20B expression,the change of CCL2 in A549cells was the most obvious and significantly decreased.2)In vitro experiments,the decreased expression of KIF20B down-regulated CCL2 expression,resulted in a significant reduction in the number of monocytes recruited to tumor supernatant.3)The flow cytometry assay showed the significantly reduced number of CD14~+CCR2~+monocyte/macrophages recruitment to tumor sites upon KIF20B knockdown.4)KIF20B knockdown could reduce the in-vivo expression of CCL2,and CCR2mRNA and protein at tumor sites.5)KIF20B knockdown could inhibit the activity of NF-?B.After treatment of A549 cells with NF-?B inhibitor,the level of CCL2 decreased.6)The expression of KIF20B mRNA was positively correlated with the expression of CCL2 mRNA?CD14 mRNA and CD163 mRNA in lung adenocarcinoma tissues,P<0.05.7)The higher the expression of CD163 in lung Adenocarcinoma tissues was,the worse the prognosis of patients was.Summary1)The decreased production of CCL2 upon KIF20B knockdown in lung adenocarcinoma cell line A549 cells could reduce the number of monocyte/macrophages recruited to tumor sites,thus inhibiting the tumor.2)KIF20B regulates the expression of CCL2 by inhibiting the activity of NF-?B,then regulates the recruitment of monocyte/macrophages at the tumor site.Conclusion:1)KIF20B expression in lung adenocarcinoma was significantly increased,and the expression level was correlated with clinicopathological characteristics and prognosis.2)KIF20B knockdown inhibits the proliferation and migration and invasion capability of lung adenocarcinoma,promotes apoptosis and inhibits the tumorigenicity.It indicates that KIF20B plays an important role in the development of lung adenocarcinoma.KIF20B is associated with malignant progress of lung adenocarcinoma.3)KIF20B knockdown downregulates CCL2 expression and decreases the number of monocyte/macrophages recruited to tumor sites via inhibiting NF-?B activity.