| Background: Glioma is the most common primary intracranial neoplasm in adults.It is a heterogeneous,invasive neoplasm with poor prognosis.To study the genesis and development mechanism of glioma at the genetic and molecular level can provide a new theoretical basis for the diagnosis and treatment of glioma.Lnc RNAs can regulate gene expression from epigenetic level,transcriptional level and post-transcriptional level,and then widely participate in the pathophysiological process of the body,which is closely related to a variety of clinical diseases.Many kinds of Lnc RNAs can regulate the biological function of glioma cells through various signaling pathways,and play an important role in the diagnosis,prognosis and treatment of glioma.MIAT is a highly specific disease-related lnc RNA,which can affect the occurrence of many diseases and related cellular functions,such as proliferation,apoptosis and invasion.The regulation mechanism of MIAT is also very complex,involving a variety of signaling pathways or cytokines.In addition,MIAT is up-regulated in many cancer tissues or cells,such as gastric cancer,colorectal cancer,breast cancer and lung cancer.MIAT can also affect the proliferation,migration,invasion and apoptosis of cancer cells by regulating a variety of signaling pathways or micro RNAs.However,there are few studies on the relationship between MIAT and glioma.This study analyzed the role and mechanism of Lnc RNA MIAT in the development of glioma through cell experiments and clinical specimens detection,and provided theoretical basis for the diagnosis and treatment of glioma.Part Ⅰ Clinical Significance of Long Chain Noncoding RNA MIAT in GliomaObjective: To analyze the clinical significance of MIAT in glioma by detecting the relative expression of MIAT in tissues and blood of patients with glioma.Methods: From January 2016 to December 2017,200 patients with glioma were collected in a hospital.All patients were initially diagnosed with glioma and received no chemotherapy or surgery at the time of collection.In the HIS case inquiry system,we inquired about the size,location,staging and Ki-67 value of the tumors at the time of initial diagnosis.From the time of diagnosis,all the subjects were followed up regularly with an interval of 4 weeks.During the period of pathological collection,blood samples were collected from patients and healthy controls at the initial diagnosis of glioma in the laboratory of a hospital every day.Glioma tissues and adjacent tissues were collected from each patient by self-matching method.RT-PCR was used to detect the expression of Lnc RNA MIAT in blood and tissue samples.Results: 1.The relative expression of MIAT in cancer tissues and adjacent tissues was 0.76(0.68-0.83)and 0.32(0.21-0.45),respectively.The relative expression of MIAT in cancer tissues was significantly higher than that in adjacent tissues.2.The relative expression of MIAT in glioma tissues with tumors greater than or equal to 5 cm,Ki-67 greater than 10 and KPS score less than 70 was significantly higher than that in glioma tissues with tumors less than 5 cm,Ki-67 less than or equal to 10 and KPS score greater than or equal to 70.The relative expression level of MIAT in stage IV glioma tissues was higher than that in stage II and III glioma tissues.3.The median survival time of glioma patients in MIAT high-expression group and MIAT low-expression group was 21.4 and 30.5 months,respectively.There was significant difference in survival time between the two groups.4.Stage IV,tumor size greater than 5 cm,Ki-67 > 10,KPS score less than 70,multiple lobes involved and high MIAT expression are independent risk factors for the prognosis of glioma.5.The relative expression of MIAT in cancer tissues of recurrent glioma patients was significantly higher than that of non-recurrent glioma patients.6.The relative expression level of MIAT in blood of glioma patients was significantly lower than that of healthy controls.The area under curve,sensitivity and specificity of Lnc RNA MIAT in diagnosis of glioma were 0.891,0.875 and 0.804,respectively.Conclusion: MIAT is highly expressed in glioma tissues and blood,and is associated with the staging,size,Ki-67 value and KPS score of glioma patients.The high expression of MIAT is an independent risk factor for the prognosis of glioma and is associated with the recurrence of glioma.Blood MIAT has good early diagnostic value for glioma patients.Part 2 Effects of Long Chain Non-coding RNA MIAT on the Function of Glioma CellsObjective: To study the effects of MIAT knockout on the proliferation,invasion,migration and apoptosis of glioma cells.Methods: Human astrocyte HA1800,human glioma cell lines U251,U373 and SHG-44 and human polymorphic glioma cell T98 were purchased from the Institute of Basic Medicine,Chinese Academy of Medical Sciences.Grouping: U251 and T98 cells were divided into blank group,blank group and MIAT inhibition group.The cells in blank group were not treated.The blank plasmid and MIAT inhibitor plasmid were transfected into the blank plasmid and MIAT inhibitor plasmid respectively.The expression of Lnc RNA MIAT was detected by RT-PCR.CCK8,Transwell,flow cytometry and scratch assay were used to detect the proliferation,invasion,apoptosis and migration of glioma cells.Result: 1.The relative expression level of MIAT in U251,U373,SHG-44 and T98 cells was significantly higher than that in HA1800 cells,and the relative expression level of MIAT in U251,U373 and SHG-44 cells was significantly lower than that in T98 cells.2.In U251 and T98 cells,all kinds of intervention cells showed a trend of proliferation.The proliferation rate of control group and no-load group was faster,and that of MIAT inhibition group was slower.In U251 cells,the cell proliferation multiples in blank group,blank group and MIAT inhibitory group were 5.12±1.14,4.78±1.25 and 2.03±1.02,respectively.In T98 cells,the cell proliferation multiples in blank group,blank group and MIAT inhibitory group were 7.25±1.59,6.51±2.08 and 3.15±1.14,respectively.In U251 and T98 cells,the proliferation of MIAT-inhibited cells was significantly lower than that of blank and empty groups.3.In U251 cells,the number of penetrating cells in blank group,blank group and MIAT inhibitory group was 153±21.0,176±26.2 and 52.1±10.5,respectively.In T98 cells,the number of perforating cells in blank group,no-load group and MIAT inhibitor group was 225±18.6,253±31.2 and 92.3±15.6,respectively.In U251 and T98 cells,the number of penetrating cells in MIAT inhibitory group was significantly lower than that in blank group and empty groups.4.In U251 cells,the healing rates of blank group,blank group and MIAT inhibitory group were 16.3±2.51,14.5±3.26 and 5.26±2.17,respectively.In T98 cells,the healing rates of blank group,blank group and MIAT inhibitory group were 24.7±5.16,25.1±5.47 and 8.73±3.15,respectively.In U251 and T98 cells,the cell healing rate in MIAT inhibition group was significantly lower than that in blank group and empty groups.5.In U251 cells,the apoptotic rates of blank group,blank group and MIAT inhibitory group were 25.6±5.19,26.1±5.96 and 39.5±8.15,respectively.In T98 cells,the apoptotic rates of blank group,no-load group and MIAT inhibitory group were 15.6±3.82,12.8±4.18 and 31.8±5.79,respectively.In U251 and T98 cells,the apoptotic rate of MIAT inhibitory group was significantly higher than that of blank group and empty groups.Conclusion: MIAT is highly expressed in glioma cells.Knocking out MIAT gene can significantly reduce the proliferation,invasion and migration of U251 and T98 cells,and significantly increase the apoptotic level.Part 3 Long-chain non-coding RNA MIAT regulates chemosensitivity of glioma through micro RNA-29aObjective: To analyze the effects of MIAT and micro RNA-29 a on the treatment of glioma.In cell experiments,the effects of MIAT and micro RNA-29 a on the sensitivity of glioma cells to temozolomide and their relationship were analyzed.Methods: According to the criteria for evaluating the curative effect of solid tumors,the short-term therapeutic effect of the patients was evaluated.Human glioma cell U251 was purchased from Nanjing Kebai Biotechnology Co.,Ltd.Human glioma drug-resistant cell line U251/TMZ was cultured by distribution induction method.U251/TMZ cells were divided into blank group,no-load group,MIAT inhibitory group,micro RNA-29 a group and MIAT+micro NA-29 a inhibitory group.The blank group was not treated,no-load group,MIAT inhibition group,and micro RNA-29 a group were transfected with no-load plasmid,MIAT inhibitor plasmid and micro RNA-29 promoter plasmid respectively,while the MIAT + micro RNA-29 a inhibition group was transfected with MIAT and micro NA-29 a inhibitor plasmid.TMZ solutions with concentrations of 5,10,20,50,100,200,400 and 800 M were used to intervene the cells in each group.After 48 hours of culture,CCK-8 assay was used to detect the cell survival rate.Using TMZ concentration as abscissa and cell viability as ordinate,dose response curves were drawn and drug resistance index(drug resistance index = IC50U251 / TMZ / IC50U251)was calculated according to the formula.The expression of micro RNA29 a in tissues and cells was detected by q RT-PCR.Result: 1.The relative expression level of MIAT disease control group(CR + PR + SD)glioma patients was significantly lower than that of uncontrolled group(PD).The relative expression level of micro RNA29 a in glioma tissues in disease control group was significantly higher than that in uncontrolled group.2.The relative expression level of MIAT in U251/TMZ cells was significantly higher than that in U251 cells(t=6.12,P=0.002).The relative expression level of micro RNA-29 a in U251/TMZ cells was significantly lower than that in U251 cells(t=5.33,P=0.009).3.Compared with U251 cells,the dose-response curve of U251/TMZ cells shifted to the right,indicating an increase in drug resistance.The IC50 of U251 and U251/TMZ cells was 21.6 and 141.8 Mm respectively,and the drug resistance index of U251/TMZ cells was 6.56.4.Compared with the blank group and the empty groups,the dose-response curve of MIAT inhibitory group shifted to the left obviously,which indicated that the drug resistance of MIAT inhibitory group was weakened.When the concentration of TMZ was 100 m M,the cell survival rate of MIAT inhibitory group was significantly lower than that of blank group and empty groups.5.The relative expression levels of micro RNA29 a in blank group,no-load group and MIAT suppression group were 0.960.13,0.860.28 and 2.080.34,respectively.The relative expression level of micro RNA29 a in MIAT inhibitory group was significantly higher than that in blank group and empty groups.6.Compared with the blank group and the blank group,the dose-response curve of the micro RNA-29 a group shifted significantly to the left,indicating that the drug resistance of the micro RNA-29 a group was weakened.When the TMZ concentration was 100 m M,the cell survival rate of the micro RNA-29 a group was significantly lower than that of the blank group and the empty groups.7.Compared with MIAT inhibitor group and micro RNA-29 a group,the dose-response curve of MIAT+micro RNA-29 a inhibitor group shifted significantly to the right,indicating that the drug resistance increased.When the TMZ concentration was 100 m M,the cell survival rate of MIAT +micro RNA-29 a inhibitor group was significantly higher than that of MIAT inhibitor group and micro RNA-29 a group.Conclusion: The expression of micro RNA29 a is positively correlated with the therapeutic effect of glioma and is low in U251/TMZ cells.Mi RNA-29 a can reduce the resistance of U251/TMZ cells to TMZ.MIAT was negatively correlated with the therapeutic effect of glioma and was highly expressed in U251/TMZ cells.MIAT inhibition can increase the expression of micro RNA-29 a in U251/TMZ cells,and reduce the cell resistance to TMZ.Part Ⅳ Long Chain Non-coding RNA MIAT Regulates Wnt/beta-catenin Signaling Pathway in Glioma by Micro RNA-200aObjective: To further analyze the relationship between MIAT and micro RNA-200 a,Wnt/beta-catenin signaling pathways in glioma patients and glioma cells,and their effects on glioma cells,so as to provide evidence for elucidating the mechanism of MIAT in glioma.Methods: U251 was divided into blank group,no-load group,MIAT inhibition group and MIAT inhibition + micro RNA-200 a group.The blank group was not treated,the blank group and the MIAT inhibitory group were transfected with the blank plasmid and the MIAT inhibitory plasmid respectively.MIAT inhibitory + micro RNA-200 a group was transfected with MIAT inhibitory plasmid and micro RNA-200 a overexpression plasmid.The expression levels of beta-catenin,p-GSK-3beta,APC and Axin in tissues and cells were detected by Western blotting.Result: 1.The relative expression levels of micro RNA-200 a in adjacent tissues and glioma tissues were 0.86±0.23 and 0.35±0.14,respectively.The relative expression levels of micro RNA-200 a in glioma tissues were significantly lower than those in adjacent tissues.The relative expression level of beta-catenin in glioma tissues was significantly higher than that in adjacent tissues.The relative expression levels of p-GSK-3beta,APC and Axin protein in glioma tissues were significantly lower than those in adjacent tissues.2.There was a significant positive correlation between MIAT level and the relative expression of beta-catenin in glioma tissues,and a significant negative correlation between MIAT level and the relative expression levels of micro RNA-200 a,p-GSK-3beta,APC and Axin.The level of micro RNA200 a was negatively correlated with the relative expression level of beta-catenin,and positively correlated with the relative expression levels of p-GSK-3beta,APC and Axin.3.The relative expression levels of micro RNA-200 a in glioma cells of blank group,empty group and MIAT-inhibited group were 1.09±0.26,1.24±0.21 and 2.31±0.34),respectively.The relative expression levels of micro RNA-200 a in glioma cells of MIAT-inhibited group were significantly higher than those of blank group and empty groups.4.The levels of beta-catenin protein in MIAT-inhibited group were significantly lower than those in blank group and empty groups.The levels of p-GSK-3beta,APC and Axin protein in MIAT-inhibited group were significantly higher than those in blank group and empty group.5.The levels of beta-catenin protein in the micro RNA-200 a group were significantly lower than those in the blank group and the empty group.The levels of p-GSK-3beta,APC and Axin protein in the micro RNA-200 a group were significantly higher than those in the blank group and the empty group.6.The levels of beta-catenin protein in MIAT+mi RNA-200 a inhibitory group were significantly higher than those in MIAT inhibitory group and MIAT-200 a inhibitory group.The levels of p-GSK-3beta,APC and Axin protein in MIAT+mi RNA-200 a inhibitory group were significantly lower than those in MIAT inhibitory group and MIAT-200 a inhibitory group.7.Proliferation multiple,number of transmembrane cells and healing rate in MIAT inhibitor group and micro RNA-200 a group were significantly lower than those in blank group and empty group.The proliferation multiple,number of transmembrane cells and healing rate in MIAT+micro RNA-200 a inhibitor group were significantly higher than those in MIAT inhibitor group and micro RNA-200 a group.The apoptotic rate of MIAT inhibitor group and mi RNA-200 a group was significantly higher than that of blank group and empty group,and that of MIAT + mi RNA-200 a inhibitor group was significantly lower than that of MIAT inhibitor group and mi RNA-200 a group.Conclusion: MIAT knockout can promote the expression of micro RNA-200 a,inhibit Wnt/beta-catenin signaling pathway,glioma cell proliferation,invasion and migration,and promote cell apoptosis. |
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