KIFC3 Promotes The Proliferation,migration And Invasion Of Non-small Cell Lung Cancer Through The PI3K/AKT Signaling Pathway

Objective: Lung cancer is the most deadly cancer worldwide.Lung cancer is mainly divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),of which non-small cell lung cancer accounts for the vast majority,accounting for about85%.Therefore,it is particularly important to explore the molecular mechanism of non-small cell lung cancer,and to explore potential biomarkers and new therapeutic targets.KIFC3 is a member of the Kinesin superfamily proteins(KIFs)and is located on chromosome 16q21.KIFs are a large molecular dynamics superfamily of 45 members that uses the energy of ATP hydrolysis to move along microtubules and support a variety of cellular functions,including mitosis,meiosis,and material transport.Family members share extensive homology within the globular head domain,the motor domain,that contains microtubules and ATP-binding sites.It has been shown that the balance of KIFC3 and EG5 tetrameric proteins controls the onset of mitotic spindle assembly,and at the onset of mitosis,KIFC3 becomes the main driver of centromeric cohesion to prevent premature spindle formation,counteracting EG5 segregation power.In mitosis,the timing of centrosome segregation is critical for spindle formation and the accuracy of chromosome segregation,and persistent centrosome cohesion can lead to chromosome misclassification.So,if the abnormal expression of the protein KIFC3,which controls the timing of centrosome separation,may promote the occurrence and development of tumors? We reviewed relevant literature and found that studies have shown that KIFC3 expression is positively correlated with biomarkers of hepatocellular carcinoma(HCC)cell migration and invasion,and KIFC3 overexpression is associated with shorter overall survival in HCC.Another study showed that a chimeric DAXX-KIFC3 fusion protein between death domain-associated protein(DAXX)and KIFC3 promoted osteosarcoma.However,the association of KIFC3 with non-small cell lung cancer has not been reported.Therefore,this study aimed to investigate the expression of KIFC3 in non-small cell lung cancer,its impact on biological functions and its mechanism of action.Methods:1.The pathological tissue samples of 109 patients with NSCLC in the pathology department of the First Affiliated Hospital of China Medical University were collected.The expression of KIFC3 in lung cancer tissues was detected by immunohistochemistry,and the correlation between KIFC3 expression and clinicopathological parameters of patients with non-small cell lung cancer was analyzed by statistics.2.In this study,A549 and H1299 cell lines were mainly used for in vitro experiments,and both cells were cultured in 1640 medium containing 10% fetal bovine serum.The cells were cultured in sterile culture flasks,which were incubated in a CO2 incubator at37°C.3.The localization of KIFC3 in each cell line was detected by immunofluorescence assay.4.The transfection reagents in this study were all Lipofectamine 3000.Si RNA and KIFC3 plasmid were transfected into the cells with Lipofectamine 3000,to down-regulate and up-regulate the protein expression level of KIFC3.LY294002,a specific inhibitor of PI3K/Akt signaling pathway,was used to inhibit the activity of PI3K/Akt pathway.Transient transfected A549 and H1299 cells were stably screened by G418,and stably transfected cell lines were successfully constructed.5.KIFC3 was interfered or overexpressed in A549 and H1299 cell lines,and the effect of KIFC3 on the proliferation ability of lung cancer cells was detected by MTT assay and colony formation assay,the effect of KIFC3 on the migration ability of lung cancer cells was detected by transwell migration assay,transwell invasion assay was used to detect the effect on the invasion ability of lung cancer cells.6.KIFC3 was interfered or overexpressed in A549 and H1299 cell lines,and the expression of key molecules of PI3K/Akt signaling pathway and their downstream proteins related to proliferation,migration and invasion were detected by Western blot.7.Co-immunoprecipitation assay was used to verify the interaction between KIFC3 and PI3 K.8.The effect of changed KIFC3 protein expression level on PI3 K m RNA expression level was detected by RT-PCR.9.To investigate the effect of KIFC3 on the proliferation ability and PI3K/Akt signaling pathway of NSCLC in vivo by subcutaneous tumorigenesis in nude mice.To explore the effect of KIFC3 on the migration and invasion of NSCLC in vivo through a nude mouse model of lung metastasis.10.SPSS 21.0 and Graph Pad-Prism 8.0 software were used for statistical analysis.Chi-square test was used to analyze the relationship between KIFC3 expression and clinicopathological factors,and paired t-test was used to analyze the differences between groups.Results: 1.The expression of KIFC3 in the pathological tissues of 109 patients with NSCLC was analyzed by immunohistochemistry,and it was found that KIFC3 was mainly expressed in cytoplasm and nucleus.KIFC3 was weakly expressed in normal alveolar and bronchial tissues,but increased in lung cancer tissues.We statistically analyzed the histochemical scoring results and clinicopathological factors of 109 tissue samples,and found that the expression of KIFC3 was associated with the degree of differentiation of non-small cell lung cancer(P=0.037),tumor size(P=0.033),lymph node metastasis(P=0.003)and TNM stage(P=0.025).There was no significant correlation with age(P=0.949),gender(P=0.284)and histological type(P=0.158).The Kaplan-Meier database was used for prognostic analysis.The results showed that high expression of KIFC3 protein was associated with short overall survival and poor prognosis in patients with non-small cell lung cancer(P=0.029).2.In A549 and H1299 cell lines,we transfected si RNA and c DNA plasmids to down-regulate or over-express the protein expression level of KIFC3 to evaluate the biological function of KIFC3 in NSCLC.The proliferation ability of tumor cells was verified by MTT assay and colony formation assay,the migration ability of tumor cells was detected by transwell migration assay,and the invasion ability of tumor cells was detected by transwell invasion assay.The results showed that the overexpression of KIFC3 protein promoted the proliferation,migration and invasion ability of NSCLC cells.On the contrary,down-regulation of KIFC3 protein level decreased the proliferation,migration and invasion ability of tumor cells.3.The screening of tumor related pathways showed that in A549 and H1299 cell lines,when the protein expression level of KIFC3 was up-regulated,the protein expression level of PI3Kp85α and p-Akt was stably and significantly increased,meanwhile,the protein expression levels of proliferation,migration and invasion related proteins such as cyclin D1,CDK4,CDK6,Rho A,Rho C and MMP2 which are downstream of PI3K/Akt signaling pathway were increased.Down-regulated the protein expression level of KIFC3 showed the opposite results.4.The overexpression of KIFC3 protein can activate the PI3K/Akt signaling pathway,and the expression levels of proliferation,migration and invasion related proteins downstream of the pathway are increased,while this promotion effect can be inhibited by the specific inhibitor of PI3K/Akt pathway,LY294002.5.Co-immunoprecipitation assay confirmed the interaction between KIFC3 and PI3Kp85α.6.The results of subcutaneous tumor-forming experiments in nude mice showed that,compared with the control group,the volume,weight and growth rate of subcutaneous tumorigenesis in nude mice were significantly increased after the injection of A549 and H1299 cells stably transfected with KIFC3 overexpression plasmid.Western blot analysis of tumor tissue proteins showed that the protein expressions level of PI3Kp85α and p-Akt were significantly increased after KIFC3 overexpression.The results of lung metastasis experiment in nude mice showed that the number of lung metastatic nodules in nude mice injected with KIFC3 overexpression lung cancer cells was significantly higher than that in the control group.The results of HE staining showed that the tumor nests in the lung tissues of nude mice injected with KIFC3 overexpressing lung cancer cells were significantly larger than those of the control group.In vivo experiments confirmed that KIFC3 overexpression can promote the proliferation,migration and invasion of NSCLC cells through the activation of PI3K/Akt signaling pathway.Conclusion: 1.The high expression of KIFC3 was correlated with the degree of differentiation,tumor size,lymph node metastasis,TNM stage and prognosis of NSCLC.2.In vitro experiments showed that KIFC3 promoted the proliferation,migration and invasion of NSCLC cells.3.KIFC3 regulated the malignant behavior of NSCLC cells through PI3K/Akt signaling pathway in vitro.4.KIFC3 promoted the proliferation,migration and invasion of NSCLC through PI3K/Akt signaling pathway in vivo.