| The incidence of nasopharyngeal carcinoma(NPC)in Guangxi Zhuang Autonomous Region is significantly higher than that in other regions,which is second only to Guangdong Province and ranking second in the world.According to statistics,the incidence rate of nasopharyngeal cancer in Guangxi was10.71/100,000,and the mortality rate was 5.15/100,000 in 2016,which was much higher than the national average(2.67/100,000 and 1.31/100,000).Nasopharyngeal carcinoma ranks 7th and 6th in the order of incidence and death of malignant tumors in Guangxi.Radiotherapy is the main treatment for NPC.Although radiation technology and equipment have improved,the prognosis of locally advanced NPC is still poor,with a 5-year survival rate of less than 60%.Abnormal tumor proliferation and apoptosis lead to resistance to radiation.Therefore,enhancing the radiosensitivity of tumor cells is the key to improving the effect of radiotherapy for NPC.Finding new functional genes which can increase the radiosensitivity of tumor cells has become one of the hot topics.In recent years,more and more evidence has shown that kinesin 15(kinesin family member 15,KIF15)participates in tumor organelles and vesicle transport,signal transduction,mitosis and other important functions,and promotes tumor development.However,the role of KIF15 in the radiosensitivity of NPC and its mechanism are not yet known.This study aims to explore the expression level of KIF15 in NPC,the influence of KIF15 on proliferation,apoptosis,radiosensitivity and its underlying mechanism of NPC through meta-analysis,bioinformatics analysis,and in vitro/vivo experiments.This research will provide new ideas for the development of new drugs to improve the radiosensitivity of NPC.Part 1: A systematic research on the expression,function and prognostic significance of KIF15 in malignant tumorsObjective: To clarify the expression and function of KIF15 in malignant tumors and its correlation with the poor prognosis of malignant tumors.Methods:(1)We firstly searched KIF15 related literature and datasets in Pub Med,Progno Scan,Chinese National Knowledge Infrastructure(CNKI),and Wanfang database.Based on the inclusion and exclusion criteria and established strategies,literature and microarray screening,literature quality evaluation,and data extraction were carried out.The Review Manager(Rev Man)5.3 software was applied to complete the pooled analysis of effect,heterogeneity detection,publication bias,etc.(2)In the TCGA,GEPIA2 and the KMplotter database,the expression of KIF15 and the impact of high KIF15 expression are analyzed in pan-cancer as well as head and neck squamous cell carcinoma(HNSCC).(3)Online systematic evaluations of the function of KIF15 are carried out in pancancer,including GO,KEGG,single cell functional status,protein-protein interaction network,tumor mutation burden,microsatellite instability,immune correlation,immune checkpoint,etc.Results:(1)The meta-analysis included 13 retrospective clinical studies and 49 GEO microarray datasets involving 12635 participants.Meta pooled effect analysis showed that high expression of KIF15 was significantly correlated with a high death risk of OS,RFS and DMFS in patients with pan-caner(OS,HR =1.25,95% CI = 1.14-1.37,P < 0.0001;RFS,HR = 1.31,95% CI = 1.13-1.53,P = 0.0003;DMFS,HR = 1.51,95% CI = 1.32-1.73,P < 0.0001).The detection of heterogeneity indicated that heterogeneity exited among the initial studies included in this meta-analysis,but removing the source of heterogeneity did not change the original trend and statistical significance of the pooled effect.(2)According to the TCGA and KMplotter database,it was observed that the expression of KIF15 was significantly increased in pan-cancer including HNSCC(P <0.05).(3)In GEPIA2 database,survival analysis showed that KIF15 can be used as a molecular marker for the increased risk of death in pan-cancer(OS)and disease-free survival(DFS)(OS: HR 1.6,P <0.001;DFS: HR 1.3,P <0.001).High expression of KIF15 was significantly associated with poor survival prognosis in HNSCC(OS: HR 1.8,P = 0.033;DFS: HR 2.2,P = 0.034).According to the KMplotter database,survival analysis also showed that high expression of KIF15 significantly reduced OS and recurrence-free survival(RFS)in locally advanced HNSCC patients(OS: HR 2.75,95% CI 1.11 – 6.83,P = 0.023;RFS: HR 9.02,95% CI 1.08-75.45,P = 0.017).(4)The top 100 genes related to KIF15 were screened out in the GEPIA2 database,and GO analysis using the DAVID database showed that KIF15 had important molecular functions such as protein binding,ATP binding,microtubule movement,chromosome separation,DNA replication,cell division,etc.;the results of KEGG analysis suggested that KIF15 was related to signal pathways such as p53,FOXO,mi RNA in cancer,DNA replication,and cell cycle.The results of protein-protein interaction network analysis showed the protein network associated with KIF15.Cancer SEA database analysis found that KIF15 was involved in multiple functional states such as apoptosis,cell cycle,DNA damage,DNA repair,tumor invasion,cell proliferation,and cell stemness in pan-cancer including HNSCC.In addition,KIF15 might also affect the immune cell infiltration and immune checkpoint status of many types of tumors,including HNSCC,or be related to tumor immunity.Conclusion:(1)The expression of KIF15 is significantly up-regulated in pancancer,including HNSCC,liver cancer,pancreatic cancer,and breast cancer.(2)In pan-cancer and HNSCC,the survival(OS,DFS,RFS,DMFS)of patients with high KIF15 expression is significantly worse than that of patients with low KIF15 expression.It is suggested that KIF15 may serve as a biomarker for prognosis of pan-cancer,including HNSCC.(3)KIF15 is involved in cell proliferation,cell cycle,apoptosis,DNA damage and repair,tumor immunity and other functions and biological processes,which has potential value for research.Part 2: The expression of KIF15 in NPC tissues and its relationship with the prognosis of NPC patientsObjective: To explore the expression level of KIF15 in NPC tissue and its influence on the prognosis of patients with NPC.Methods:(1)A bioinformatics analysis was performed on GSE12452,GSE61218 and GSE53819 in GEO database to determine the relative expression level of KIF15 in NPC tissues.The GSEA was applied to reveal the relevant pathways enriched by high expression of KIF15.(2)Immunohistochemistry(IHC)was applied to detect the expression of KIF15 protein in 98 patients with NPC and 18 controls,and a comparative analysis was performed.Kaplan-Meier survival curve analyzed the relationship between high expression of KIF15 and 5-year OS of NPC patients.(3)To evaluate the value of KIF15 as a diagnostic and prognostic marker for nasopharyngeal carcinoma.COX regression is used in statistical software to do single-factor/multi-factor analysis.The rms package of R software 3.6.3 is used for nomogram prediction model construction,calibration curve and consistency index calculation.The survival ROC and corrplot package is used for ROC analysis and Pearson correlation analysis,respectively.Results:(1)In the analysis of GSE12452,GSE61218 and GSE53819,the results showed that KIF15 was significantly up-regulated in NPC tissues compared to normal tissues(P <0.05),and it was verified in the 98 NPC tissues and 18 non-maliganant nasopharyngeal tissues by IHC(P <0.01).The GSEA results showed that the high expression of KIF15 was positively correlated with STAT3 signaling.Pearson correlation analysis suggests that in nasopharyngeal carcinoma tissues,KIF15 is positively correlated with STAT3 and its downstream related protein Bcl-2 and other proteins.(2)We divided 98 NPC patients into KIF15 high expression group(n = 49)and KIF15 low expression group(n = 49)based on the median value of expression.By statistical analysis,we found that the high expression of KIF15 was significantly related to higher T stage(P = 0.024).Kaplan-Meier survival curve showed that the 5-year OS of the KIF15 high expression group was significantly lower than that of the KIF15 low expression group(P = 0.043).(3)ROC analysis results in three nasopharyngeal carcinoma microarrays and tissue samples showed that KIF15 has a good ability to distinguish nasopharyngeal carcinoma from normal nasopharyngeal epithelium.COX regression univariate analysis showed that N stage and high expression of KIF15 were poor prognostic factors that predicted an increased risk of death in patients(P <0.05).The nomogram constructed based on clinicopathological characteristics and KIF15 has good classification ability,fit and prediction accuracy,suggesting that the model constructed based on KIF15 can predict the prognosis of nasopharyngeal carcinoma.Conclusion: Taken together,the expression of KIF15 is significantly upregulated in nasopharyngeal carcinoma tissues.High expression of KIF15 is closely related to the clinicopathological characteristics and poor prognosis of patients with nasopharyngeal carcinoma.Our findings suggest that KIF15 can act as diagnostic and prognostic biomarker.Part 3: KIF15 regulates the proliferation,apoptosis and radiosensitivity of nasopharyngeal carcinoma cells by activating the STAT3 signaling pathway in vitro and in vivoObjective: To investigate the mechanism of KIF15 regulating radiosensitivity of NPC cells in vivo and in vitro.Methods:(1)(1)In vitro,Western blot and q PCR were used to detect the expression of KIF15 in nasopharyngeal carcinoma cell lines C666-1,CNE1,CNE2,HONE-1 and normal nasopharyngeal epithelial cell line NP69.(2)A lentivirus vector of KIF15,sh RNA sequences targeting KIF15 m RNA and negative control were designed and constructed,respectively.The lentivirus vector for KIF15 overexpression was transfected to CNE2 cell(oe KIF15-CNE2),while the lentiviral PLKO.1/KIF15 sh RNA was transfected to CNE1 cell(sh KIF15-CNE1)for stable knock down of KIF15.We verified the efficiency of KIF15 knock-down and overexpression by q PCR and Western blot.(3)Cell viability,cell cycle and apoptosis of sh KIF15-CNE1,oe KIF15-CNE2 and control cells were detected by CCK8 assays and flow cytometry.Western blot was performed to determine the expression of KIF15,STAT3,p-STAT3,Bcl-2,Bax and PCNA.(4)Clone formation assays were performed after sh KIF15-CNE1,oe KIF15-CNE2 cells and control cells were irradiated with gradient doses(0,2,4,6,8,10 Gy).In addition,sh KIF15-CNE1,oe KIF15-CNE2 cells and control cells were treated with 4Gy radiation followed by CCK8 assays and flow cytometry to detect cell viability,cell cycle and apoptosis.Western blot was used to detect the expression of STAT3,p-STAT3,Bcl-2,Bax and PCNA between the groups.(5)In order to prove that KIF15 regulates the radiosensitivity of nasopharyngeal carcinoma through STAT3,oe KIF15-CNE2 cells were treated with 10 μmol/L STAT3 inhibitor AG490 with or without 4 Gy irradiation.Afterwards,cell viability,cell cycle and apoptosis experiment was detected by CCK8 assays and flow cytometry,Western blot was used to determine the expression of STAT3,p-STAT3,Bcl-2,Bax and PCNA.(6)The candidate proteins that directly interact with KIF15 were screened and identified through co-immunoprecipitation mass spectrometry(COIP-MS).(2)(1)In vivo,nude mice were subcutaneously transplanted with oe KIF15-CNE2 and oe NC-CNE2 cells to construct subcutaneous tumor xenograft models.The subcutaneous xenograft tumor growth in nude mice was recorded.The expression of KIF15 was detected by IHC.For evaluating the impact of KIF15 on NPC,The tumor growth and weight changes were recorded with or without 9Gy/f×1 fraction irradiation.The TUNEL assay was used to evaluate cell apoptosis.(2)With subcutaneous xenograft tumors in nude mice established by sh KIF15-CNE1 and sh NC-CNE1 cells,immunohistochemistry was applied to verify the expression of KIF15 in xenografts in nude mice,and tumor growth and weight changes were record.Results:(1)(1)In vitro,the m RNA and protein levels of KIF15 were significantly up-regulated in NPC cell lines(C666-1,CNE1,CNE2,HONE-1)compared with NP69 cells.(2)Results of western blot and q-PCR showed that,compared with the corresponding control group,the lentiviral p LVX-Puro/KIF15 transfected to CNE2 cells(oe KIF15-CNE2)significantly promoted the expression of KIF15.The transfection of lentiviral PLKO.1/KIF15 sh RNA into CNE1 cells(sh KIF15-CNE1)significantly inhibited the expression of KIF15,and the results showed that sh KIF15-1 and sh KIF15-2 had the highest knockdown efficiency.(3)CCK8 assay showed that the proliferation of sh KIF15-CNE1 cells was lower than that of control cells,while the proliferation of oe KIF15-CNE2 cells was higher than that of control cells.Flow cytometry analysis showed that knocking down KIF15 expression could induce cell apoptosis and cell cycle G1 arrest,while overexpression of KIF15 inhibited cell apoptosis and promoted the transition of cell cycle from G1 phase to G2 phase.(4)The results of the colony formation after gradient dose irradiation suggested that compared with control group,overexpression of KIF15 significantly increased the survival rate of CNE2 cells,while knocking down KIF15 decreased the survival rate of CNE1 cells.The results of survival fraction curve proved that KIF15 could increase the radioresistance of NPC cells.Flow cytometry analysis showed that the apoptosis rate of CNE2 cells with KIF15 gene overexpression after 4Gy irradiation was significantly lower than that of the control group,while the apoptosis rate of CNE1 cells with KIF15 gene knockdown after 4Gy irradiation was significantly higher than that of the control group.CCK8 assay showed that the proliferation of oe KIF15-CNE2 cells after 4Gy irradiation was significantly higher than that of the control group,while the proliferation rate of sh KIF15-CNE1 cells after 4Gy irradiation was significantly lower than that of the control group.It was also found that the proportion of S phase cells of sh KIF15-CNE1 was lower than that of sh NC-CNE1 cells with or without 4 Gy irradiation,while the proportion of G1 phase cells in sh KIF15-CNE1 cells was increased compared with sh NC-CNE1 cells.In contrast,the proportion of S phase cells of oe KIF15-CNE2 was lower than that of oe NC-CNE2 cells with or without 4 Gy irradiation,while the proportion of G1 phase cells of oe KIF15-CNE2 cells was increased compared with oe NC-CNE2 cells.Western blot results showed that overexpression of KIF15 with or without 4Gy irradiation can lead to a significant increase in Bcl-2,PCNA and phosphorylated STAT3(p-STAT3)levels,accompanied by a decrease in Bax levels,while knocking down KIF15 lead to the opposite results.STAT3 inhibitor AG490 treatment of oe KIF15-CNE2 cells can reduce the level of p-STAT3 and eliminate the effect of KIF15 overexpression on CNE2 cell proliferation,apoptosis and cell cycle regulation.A total of 19 candidate proteins that inteact with KIF15 were identified by COIP-MS.The results of protein-protein interaction network suggested that the candidate proteins interact with STAT3,suggesting that KIF15 might regulate STAT3 by interacting with the candidate protein.Molecular correlation analysis indicated that KIF15 had expression correlation with STAT3,Bcl-2 and candidate proteins in head and neck squamous cell carcinoma and nasopharyngeal carcinoma.(2)(1)In vivo,the results of immunohistochemistry showed that the expression of KIF15 in oe KIF15-CNE2 transplanted tumors of nude mice was higher than that of oe NC-CNE2 transplanted tumors of nude mice,indicating that the overexpression of KIF15 was derived from inoculated oe KIF15-CNE2 cells.The results of oe KIF15-CNE2,oe NC-CNE2,oe KIF15-CNE2-9Gy and oe NC-CNE2-9Gy groups showed that the tumor volume and weight of the KIF15 overexpression group were higher than those of the control group(P <0.05).After receiving 9 Gy irradiation,the growthrate of the two transplanted tumors groups of nude mice slowed down,and the volume and mass were smaller than those of the non-irradiated group,but the oe KIF15-CNE2-9Gy group grew faster than the oe NC-CNE2-9Gy group(P <0.05).It was observed that KIF15 overexpression inhibited the apoptosis of nasopharyngeal carcinoma cells by TUNEL assay compared with the control group,and the difference was statistically significant(P <0.001).(2)The xenograft tumor with sh KIF15-CNE1 and sh NC-CNE1 cells showed that knocking down KIF15 significantly inhibited the growth of tumors.The volume and weight of tumors in the sh KIF15-CNE1 group were lower than those in the control group(P <0.05).The immunohistochemistry results showed that the expression of KIF15 in xenograft tumors in the sh KIF15 group was lower than in the control group.Conclusions:(1)KIF15 is up-regulated in nasopharyngeal carcinoma cells and is involved in the regulation of nasopharyngeal carcinoma radiosensitivity.Overexpression of KIF15 promotes the proliferation,inhibits apoptosis,increases the proportion of S-phase cells in vivo and in vitro,thereby reducing the radiosensitivity of nasopharyngeal carcinoma.Knockdown of KIF15 inhibits cell proliferation,promotes apoptosis,induces cell cycle G1 arrest,and increases radiosensitivity in vitro.(2)Up-regulation of KIF15 lead to up-regulation of pSTAT3,Bcl-2,PCNA expression and down-regulation of Bax expression,while down-regulation of KIF15 lead to down-regulation of p-STAT3,Bcl-2,PCNA expression and up-regulation of Bax expression.The STAT3 signaling pathway inhibitor AG490 can abolish the phenotype of KIF15 on nasopharyngeal carcinoma cells and the expression levels of Bcl-2,Bax and PCNA,indicating that KIF15 can affect the radiosensitivity of nasopharyngeal carcinoma through the STAT3 signaling pathway and its downstream related proteins.(3)The results of COIP-MS combined with bioinformatics analysis suggest that KIF15 may indirectly regulate STAT3 activation through its interaction with the candidate protein,but further research and verification are needed. |
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