| Glioma is the most common primary central nervous system tumor,accounting for about half of all intracranial primary tumors.Due to its strong invasiveness and the high recurrence rate,the five-year survival rate of glioma is only about 10%even after surgical resection combined with postoperative radiotherapy and chemotherapy,remaining it a serious threat to human race.MiRNAs have been found to play important roles in cell metabolism,apoptosis,growth and proliferation and other biological processes.Abnormal miRNA expression has been revealed in different types of tumors,which exert tumor suppressive or carcinogenic effects by directly regulating target genes.However,the mechanism of miRNA-424 influences the invasion,migration and epithelial to mesenchymal transition?EMT?in glioma cell is still unclear.We selected the miRNA-424 as the object to study the possible intrinsic mechanism of invasiveness,migration and EMT in glioma cells,to find and verify the miRNA-424downstream targets and to provide the basis for targeted therapy.KIF23 is a member of the kinesin family,located in the nucleus.It is closely related to tumor invasion ability.Previous studies have shown that KIF23 may activate Akt signaling pathway to promote proliferation and invasion of hepatocellular carcinoma and proliferation of glioma cells.However,the relationships between KIF23 and miR-424,as well as the prognosis of glioma remain unclear.We compared the expression differences of KIF23 gene between glioma tissue and normal brain tissue,glioma cell lines and human astrocyte lines through real-time quantitative PCR test.By real time quantitative PCR and Western blot test,we compared KIF23's mRNA and protein level changes in miR-424 mimcs group and miR-424 inhibitors group respectively with their corresponding NC group.Then through the dual luciferase reporter gene test,the downstream target of miR-424 is KIF23.So we guess KIF23 may be associated with development of glioma,miR-424 is likely to regulate the development of glioma by targeting KIF23.Kaplan-meier was used to analyze the relationship between KIF23 gene expression difference and patient survival time,and verify the relationship between KIF23 and patient's prognosis.In this study,the biological characteristics of miR-424 in glioma were discussed in the clinical cases,in vitro and in vivo,which was important in the invasion,migration and EMT regulation and also provided important clues for the further study of the function of miR-424.Part I miR-424 inhibits epithelial mesenchymal transformation and invasion and migration of glioma cellsObject:To explore the expression of miR-424 in glioma tissues and glioma cell lines and its correlation with the prognosis of patients,and to look for potential new markers of glioma.To investigate the relationship between miR-424 expression with the invasion,migration and EMT process of glioma.Materials And Methods:1.Tissue samples:those diagnosed with glioma in Rizhao People's Hospital from May2015 to April 2017 were selected.A total of 54 patients underwent intracranial tumor resection,and 54 glioma tissue samples were collected.Histopathological diagnosis of the patient is based on the revised international tumor grade.During the same period,12 cases of non-tumor and contusion brain tissue?normal brain tissue?were selected as the control group in appropriate traumatic brain injury patients?no tumor confirmed by imaging?.All patients did not receive radiotherapy or chemotherapy before surgery and provided written informed consent.This study was approved by the ethics committee of Rizhao People's Hospital.2.Cell lines:human glioma cell lines?A172,SHG-44,T98,LN18 and LN229?and Normal human astrocytes?NHA?were purchased from the USA ATCC.3.Cell culture:Cryostoraged cells after recovery and passage cultureed at 37?,5%CO2,saturated humidity cell incubator.4.RNA extraction and real-time quantitative RT-PCR:RNA was extracted by Trizol.The expression differences of miR-424 in glioma tissues and normal brain tissues,glioma cell lines and normal astrocytes were detected by real-time quantitative RT-PCR.5.LN229 and A172 cell lines were cultured for in-vitro cell function test.In the glioma cell lines used in this study,the expression level of miR-424 was the lowest in the LN229cell line and the highest in the A172 cell line.Mir-424 mimics and its negative control were transfected into LN229,and miR-424 inhibitor and its negative control were transfected into A172.6.After stable transfection,we investigated the effect of up-regulation and down-regulation of miR-424 expression on the invasion and migration abilities of glioma cells by Transwell test.7.After stable transfection,fluorescence real-time quantitative PCR and Western blot were used to detect the corresponding changes in mRNA and protein levels of EMT markers when miR-424 was high-expressed and low-expressed.So as to investigate the effect of miR-424 expression on the occurrence of EMT in glioma cells.8.The clinical significance of miR-424 in glioma patients was analyzed:the relationship between the expression level of miR-424 of glioma in different WHO grades,preoperative KPS score grades,gender,age and tumor volume were compared by t test.Kaplan-meier analysis was used to estimate the median survival time and overall survival rate,and log-rank test was used to test the relationship between the expression of miR-424 and the overall survival rate of glioma patients.Results1.Compared with normal brain tissue samples,miR-424 was significantly down-regulated in glioma tissue samples,and the difference was statistically different?P<0.01?.The expression of miR-424 in A172,SHG-44,T98,LN229 and LN18 cell lines was significantly lower than that in normal cell lines with statistic differences?P<0.05?2.After stable transfection of miR-424 mimics by Transwell assay,miR-424 was highly expressed in LN229 cells and its invasion and migration abilities were inhibited?P<0.05?.After stable transfection of miR-424 inhibitor,miR-424 was low expressed in A172cells and the cell invasion and migration abilities were improved?P<0.01?.3.After fluorescence quantitative PCR detection,the mRNA expression level of E-cadherin in LN229 cell line was increased compared with that of the negative control group,and the difference was statistically different?P<0.05?,while the mRNA expression levels of N-cadherin and Vimentin were decreased,and the difference was statistic?P<0.05?.After A172 cell line was transfected with miR-424 inhibitor,the mRNA expression level of E-cadherin in the miR-424 inhibitor group was decreased compared with the negative control group,and the difference was statistic?P<0.05?,while the mRNA expression levels of N-cadherin and Vimentin were increased,and the difference was statistic?P<0.05?.4.Western blot test showed that the expression of E-cadherin in LN229 cells with high expression of miR-424 was significantly increased,while the expressions of N-cadherin and Vimentin were significantly decreased.In addition,the low expression of miR-424 in A172cells significantly decreased the protein levels of E-cadherin and increased the protein levels of N-cadherin and Vimentin.5.According to the level of?=0.05,the expression levels of miR-424 were statistically different between the two groups with different WHO grades?P=0.0065?and preoperative KPS scores?P=0.0171?.There were no statistic differences in gender,age and tumor volume between glioma patients and the expression level of miR-424.Kaplan-Meier analysis was used to estimate the median survival time and overall survival rate.Log-rank univariate analysis showed that the overall survival rate of glioma patients with low expression of miR-424 was significantly reduced.Conclusion1.Compared with normal brain tissue samples,miR-424 was significantly down-regulated in glioma tissue samples.Compared with normal human astrocytes,the expression level of miR-424 in glioblastoma cell lines was significantly down-regulated,and the difference was statistic.It is speculated that it may have early diagnostic value in glioma.2.miR-424 negatively regulates the invasion and migration of glioma cells3.miR-424 is an inhibitor of the EMT process of glioma,and EMT may be a potential mechanism for miR-424 to regulate the invasion and migration of glioma cells4.The overall survival rate of glioma patients with low miR-424 expression was significantly reduced,which was correlated with high WHO grades and low preoperative KPS score,and the difference was statistic.The expression level of miR-424 may be one of the prognosis factors of glioma patients.Part?KIF23 is a direct target of miR-424,and its expression level is negatively correlated with the prognosis of patientsObject:To detect the expression of KIF23 in glioma tissues and cell lines,and its correlation with the prognosis of patients.To look for new potential markers of glioma.It was verified that KIF23 was the direct target of miR-424 in glioma cells.To verify the relationship between the high expression of miR-424 and the volume of glioma in vivo.Materials And Methods:1.Tissue samples:those diagnosed with glioma in Rizhao People's Hospital from May2015 to April 2017 were selected.A total of 54 patients underwent intracranial tumor resection,and 54 glioma tissue samples were collected.Histopathological diagnosis of the patient is based on the revised international tumor grade.During the same period,12 cases of non-tumor and contusion brain tissue?normal brain tissue?were selected as the control group in appropriate traumatic brain injury patients?no tumor confirmed by imaging?.All patients did not receive radiotherapy or chemotherapy before surgery and provided written informed consent.This study was approved by the ethics committee of Rizhao People's Hospital.2.Cell lines:human glioma cell lines?A172,SHG-44,T98,LN18 and LN229?and Normal human astrocytes?NHA?were purchased from the USA ATCC.3.Cell culture:Cryostoraged cells after recovery and passage cultureed at 37?,5%CO2,saturated humidity cell incubator.4.RNA extraction and real-time quantitative RT-PCR:RNA was extracted by Trizol.The expression differences of KIF23 in glioma tissues and normal brain tissues,glioma cell lines and normal astrocytes were detected by real-time quantitative RT-PCR.5.LN229 and A172 cell lines were cultured for in-vitro cell function test.In the glioma cell lines used in this study,the expression level of miR-424 was the lowest in the LN229cell line and the highest in the A172 cell line.miR-424 mimics and its negative control were transfected into LN229,and miR-424 inhibitor and its negative control were transfected into A172.6.After stable transfection,the corresponding changes in mRNA and protein levels of KIF23 gene were detected by fluorescence real-time quantitative PCR and Western blot,to study the effect of miR-424 expression on KIF23.7.Bioinformatics methods were used to predict the possible downstream targets of miR-424,to study the expression difference of KIF23 between glioma and normal brain tissue,and to predict its clinical significance.8.Dual luciferase reporter gene test verified the direct target gene of miR4249.Kaplan-Meier was used to analyze the relationship between KIF23 expression and the overall survival rate of glioma patients10.Xenoblastoma test in nude mice verified the relationship between the high expression of miR-424 and the growth rate of glioma.Results1.Bioinformatics study:possible target genes of miR-424 were retrieved from Targetscan database and microRNA.org,and KIF23 was the target gene with a high possibility among all candidate genes.A search of the Oncomine database showed that KIF23 expression was higher in gliomas than in normal brain tissue.Oncolnc database was searched to predict significant differences in the total survival time of glioma patients with different levels of KIF23 expression.After univariate analysis of log-rank between the two groups,the total survival time of glioma patients in the KIF23 high-expression group was significantly lower than that in the low-expression group.2.Real-time PCR:compared with normal brain tissue samples,KIF23 was significantly up-regulated in glioma tissue samples,and the difference was statistic?P<0.01?.Compared with normal astrocytes,the expression of KIF23 in A172,SHG-44,T98,LN229 and LN18cell lines was significantly higher than that in normal astrocytes?P<0.05?.3.The fluorescence Real-time quantitative PCR analysis showed that the mRNA expressions of KIF23 gene in the two transfected cell lines-LN229 and A172-were decreased and increased respectively.And the differences were statistic?P<0.05?.Western blot analysis confirmed that KIF23 protein levels in glioma cell lines LN229 and A172 were decreased and increased respectively after transfection.4.Dual luciferase reporter assay confirmed that KIF23 was a direct target gene of miR-424,and miR-424 could affect the expression of KIF23 by directly binding KIF23-3'-UTR5.Kaplan-Meier analysis showed that the expression of KIF23 was correlated with the prognosis of glioma patients,and the overall survival rate of glioma patients with high expression of KIF23 was lower than that of glioma patients with low expression of KIF23.6.Xenoblastoma test in nude mice:the high expression of miR-424 in glioma can significantly inhibit tumor growth.Conclusion1.KIF23 is a direct downstream target of miR-424.miR-424 may inhibit the invasion,migration and EMT of glioma cells by directly targeting KIF23.2.The expression level of KIF23 is related to the prognosis of glioma patients,and the overall survival rate of glioma patients with high expression of KIF23 is lower than that of glioma patients with low expression of KIF23.3.In vivo experiments,the high expression of miR-424 in glioma can significantly inhibit tumor growth.Molecular therapy targeting miR-424 to inhibit EMT may become feasible for the treatment of glioma in the future. |
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