MiR-623 Inhibits Cell Proliferation And Epithelial-mesenchymal Transition By Repression Of TRIM44 In Glioma

Glioblastoma is the most common and destructive primary brain tumor in adults.Glioma is reported to be the most common primary brain tumor,originating from glial cells in the brain and spinal cord.At present,the standard clinical treatment of gliomas is mainly based on surgical resection combined with chemotherapy and radiotherapy.New treatments for gliomas have also emerged,such as immunotherapy.The latest treatments include targeted therapy,commonly used drugs are bevacizumab,gefitinib and CAR-T cell therapy.Glioblastoma multiforme(GBM)is the most invasive form of malignant glioma,which has a high risk of metastasis,leading to the poor prognosis.Neovascularization in brain tumors is directly related to their biological aggression,malignant degree and clinical recurrence,and is negatively correlated with prognosis.It is reported that GBM shows the highest degree of angiogenesis and endothelial cell proliferation,with the greatest effect of angiogenesis.Although the treatment methods have been continuously improved and the prognosis of patients have been significantly improved,the overall treatment strategy is still lack of breakthrough.Therefore,to find a new treatment of glioma is the focus of the research.miRNA is a single-stranded non-coding RNA,with a length of 21 to 23 bp,which may be involved in cell proliferation,apoptosis,invasion,migration and cycle and be related to plasma metabolism,cell differentiation,tumor occurrence and development.Many studies have confirmed that miRNAs have a very powerful biological function,they could regulate the function of 40%m RNA and play important role as the tumor suppressor genes or oncogenes in the occurrence and development of tumors.With the progress,more and more miRNA were reported to play important roles in the early diagnosis and treatment of tumors.Previous studies have shown that the main function of miR-623 are to protect the heart from hypoxia and anti-apoptosis.The target genes verified by miR-623 mainly included MMP1,CDK6,CCND1,XRCC5,GPRC5 A and so on.It can target the effect of cyclin on the cycle of tumor cells and can regulate the classical PI3 K / AKT signaling pathway.It had been reported that miR-623 played a role in the occurrence and development of breast cancer,lung cancer,liver cancer,pancreatic cancer and esophageal cancer.Most studies showed that miR-623 worked as tumor suppressor gene.It may regulate different signal pathways in different tumors and play different roles in the occurrence and development of different tumors.Therefore,the role and mechanism of miR-623 in malignant tumors need to be further studied.However,the expression,function and regulatory mechanism of miR-623 in gliomas are not clear.Epithelial-mesenchymal transformation((EMT))refers to the transformation process of epithelial-derived tumor cells,which are deprived of polarity and acquire stromal phenotypes.EMT is an effective way for epithelial cells to acquire the ability of migration,and plays an important role in the invasion and metastasis of malignant tumors.The study of EMT in glioblastoma has just begun,and miRNA has attracted wide attention in regulating the invasion and progression of EMT.In this study,through the analysis of the Chinese glioma genomic map database(CGGA),it was found that the expression of miR-623 was low-expression in gliomas and was related to the prognosis of the patients.The expression of miR-623 was detected in human astroglial normal cell line HEB and three glioma cell lines U87,U251,LN229 by reverse transcriptase real-time quantitative polymerase chain reaction(Quantitative real-time reverse transcription-polymerase chain reaction,qRT-PCR).The simulant of miR-623 was overexpressed by cell transfection technique.The effects of miR-623 on the proliferation,colony forming ability,invasion and migration of glioma cell lines LN229 and U251 were studied by MTS-way,transwell experiment and colony formation experiment.The relationship between miR-623 and epithelial mesenchymal transformation pathway was verified by Western blotting.Morever,three bioinformatics websites were used to analyze and predict the target sequence of downstream regulation of miR-623.Double luciferase reporter experiment was used to verify the targeting effect of miR-623 on downstream predictive target gene TRIM44.QRT-PCR and Western blotting were used to detect the changes of predicted target gene TRIM44 m RNA and protein expression in glioma cell lines LN229 and U251 after the high-expression of miR-623,so as to further clarify the regulatory effect of miR-623 on target genes.The biological function and effect on malignant phenotype of glioma cells of TRIM44 were verified in glioma cell lines LN229 and U251.The main contents and results are as followed:Part one The expression of miR-623 in glioma tissues and cells and its relationship with clinical featuresObjective: in order to explore the potential predictive markers of glioma(glioblastomas,GBM)and whether they are related to survival and prognosis,we compared the changes of microRNA(miRNA)expression profile between low-grade glioma and high-grade glioma through CGGA(Chinese Glioma Genome Atlas)database,and looked for differentially expressed genes.The survival and prognosis were analyzed.Methods:1.Cluster analysis of differentially expressed miRNA of WHOII,WHOIII and WHOIV grade gliomas was carried out by integrating and analyzing CGGA database.2.Search for differentially expressed genes miR-623,to explore the expression of miR-623 in glioma tissues in the database.3.The expression of miR-623 in normal nerve cells and malignant nerve cells was detected by QRT-PCR technique.4.By integrating the clinical data of CGGA database,to analyze whether miR-623 is related to IDH mutation in glioma patients,and to analyze whether miR-623 is related to survival and prognosis of glioma patients.5.Through the analysis of CGG database,Cox multivariate survival analysis was carried out to analyze the clinical characteristics such as miR-623 expression,sex and age of patients.Results:1.CGGA database showed that there were 198 patients with glioma,including 60 patients with WHOII grade glioma,47 patients with WHOIII glioma and 91 patients with WHOIV grade glioma.2.There are many differences in the expression of miRNA among the three.A total of 123 differentially expressed genes were obtained(compared with WHOII,the expression ratio of WHOIV,WHOIII was more than 2 or < 0.5(P < 0.05).We carried out cluster analysis on these miRNA.2.GSE90603,the data set in GEO database,showed that compared with normal nerve tissue,the expression of miR-623 was low in glioma tissues(P = 0.0012).CGGA database showed that the expression of miR-623 in WHOIII and WHOIV gliomas was lower than that in WHOII gliomas(P < 0.001).Compared with patients with WHOIII grade glioma,the expression of miR-623 was low in patients with WHOIV grade glioma(P = 0.0387).3.The results of QRT-PCR showed that the expression of HEB,miR-623 in malignant glioma cells was significantly lower than that in normal nerve cells(U87 LN229 U251).4.CGGA database showed that miR-623 was highly expressed in glioma patients with IDH mutation(P=0.0799).Through the analysis of the expression of miR-623 and the survival prognosis of patients,it was found that the survival prognosis of patients with low expression of miR-623 was worse than that of patients with high expression of miR-623,and the difference was statistically significant(P<0.0001).For patients with primary glioma,the prognosis of patients with low expression of miR-623 was significantly lower than that of patients with high expression of miR-623(P<0.05).For patients with recurrent gliomas,patients with low expression of miR-623 had a poorer prognosis.5.The results of multivariate Cox survival analysis showed that the clinicopathological grade,chemotherapy were related to the survival prognosis of the patients.Conclusions: Compared with normal nerve tissue,the expression of miR-623 in glioma is lower.Compared with low-grade glioma tissues,the expression of miR-623 is lower in high-grade gliomas.Compared with normal glioma cells,qRT-PCR showed lower expression of miR-623 in malignant glioma cells.Compared with patients with high expression of miR-623,patients with lower expression of miR-623 had poorer survival and prognosis.Whether miR-623 has biological function or not needs further study.Part Two Screening of miR-623 in CGGA database and its expr-ession in gliomas.Objective: To explore the effect and mechanism of miR-623-transfected mimics on biological behavior and epithelial-mesenchymal transf-ormation pathway of glioma cells through the expression of miR-623-transfected mimics in glioma cells LN229 and U251.Methods: The mimics of miR-623 was transfected into glioma cells LN229 and U251,and the control group was used in the experiment.The experiment was divided into two groups: miR-623 mimics group and miR-623 NC group.QRT-PCR was used to detect the expression level of miR-623 after transfection.MTS-way,colony formation test and Transwell chamber test were used to detect the effect of miR-623 on the biological behavior of glioma cells LN229 and U251.Western blotting was used to detect the expression level of Epithelial-Mesenchymal Transition,EMT)-related protein E-cadherinN cadherin and MMP2,MMP9,and to analyze the effect of miR-623 on EMT of glioma cells.Results:1.In this study,the mimics of miR-623 was transfected into glioma cells LN229 and U251 by Lipofectamine 2000 transfection reagent.The results of qRT-PCR showed that the expression of miR-623 in miR-623 mimics group was significantly higher than that in miR-623 NC group(P<0.05).The results showed that the transfection was successful,which provided the basis for the followed experiments.2.The results of MTS-way assay showed that the cell proliferation ability of LN229 and U251 in miR-623 mimics group was significantly lower than that in miR-623 NC group at 24 h,48 h,72 h and 96 h after transfection(P < 0.05).3.The results of colony formation assay showed that the colony formation ability of LN229 and U251 in miR-623 mimics group was significantly lower than that in miR-623 NC group 24 hours after transfection and 14 days after fixation.4.The results of transwell experiment showed that 24 hours after transfection,the invasion and migration experiments were carried out to observe the number of cells passing through the bottom of the chamber.The ability of invasion and migration of LN229 and U251 cells in miR-623 mimics group was significantly lower than that in miR-623 NC group,and the number of cells passing through the bottom of the chamber was significantly lower than that in miR-623 NC group.Conclusions:1.miR-623 can inhibit the proliferation of glioma cells LN229 and U251.2.miR-623 can inhibit the colony formation,invasion and migration of glioma cells LN229 and U251.3.miR-623 can affect the epithelial-mesenchymal transformation pathway of glioma cells.Part Three miR-623 affects malignant phenotype and epithelialmesenc-hymal transformation of glioma cells by targeting TRI-M44.Objective: to study the role of miR-623 played in the regulation of target gene TRIM44,and to clarify the effect of miR-623 on biological behavior and epithelial mesenchymal transformation pathway of glioma cells by targeting TRIM44.Methods: prediction and screening were carried out on the bioinformatics websites of Target Scan,mi DIP and miRDB,and the possible target sequences were screened according to the literature and the effect of miR-623 on the proliferation of glioma cells.The targeting effect of miR-623 on selected candidate target genes was detected by double luciferase reporter gene analysis,and the expression of candidate target gene TRIM44 in glioma cell lines LN229 and U251 transfected with miR-623 was detected by qRT-PCR and Western blotting experiments to further verify the relationship between miR-623 and target gene TRIM44 expression.The Si RNA of TRIM44 was designed to transfect glioma cell lines LN229 and U251,and the m RNA expression level of TRIM44 was detected by QRT-PCR.The protein expression level of TRIM44 in glioma cells was detected by Western blotting to further verify the transfection efficiency.To verify the effect of TRIM44 silencing on cell proliferation,colony formation and other malignant phenotypes of glioma cells.(then the overexpressed TRIM44 plasmid or its control vector was transfected into glial cells,and further analyzed whether miR-623 affected the malignant phenotype of glioma by regulating the expression of TRIM44.miR-623 mimics and NC negative control cotransfected glioma cells with TRIM44 overexpression vector and control empty vector.The experiment was divided into three groups: a miR-623 NC,miR-623 mimics + p ENTER vector,miR-623 mimics + p ENTER-TRIM44.The proliferation of glioma cells was detected by MTS-way assay and the invasion and migration ability of cells was detected by transwell test,and the expressions of epithelial-mesenchymal transformation-related protein E-cadherin,Vimentin and N-cadherin were detected by Western blotting to verify the molecular mechanism of malignant phenotype.Results:1.The candidate target gene TRIM44 was predicted by Target Scan,mi DIP and miRDB bioinformatics websites.The protein expression of TRIM44 was detected to be decreased by Western blotting after transfection of miR-623 mimics,which proved that miR-623 could target TRIM44.2.Luciferase reporter experiment showed that compared with UTR+miR-623 mimics NC group,luciferase activity in UTR+ miR-623 mimics group was significantly lower.However,there was no significant difference in luciferase activity between UTR(mutant)+ miR-623 mimics group and UTR+ miR-623 mimics NC control group,which further proved that miR-623 could target TRIM44.3.In this study,the Si-RNA of TRIM44 was transfected into glioma cells LN229 and U251 by Lipofectamine 2000 transfection reagent.Western blotting showed that the expression of TRIM44 in LN229 and U251 glioma cells in TRIM44-Si RNA group was significantly lower than that in NC group.The results showed that the transfection was successful,and then the phenotypic experiment of glioma cells showed that the proliferation and colony formation ability of glioma cells decreased significantly.It was further verified that TRIM44 could also affect the malignant phenotype of glioma cells.4.After co-transfection of glioma cells with miR-623 mimics or control and TRIM44 over-expression vector or control vector,MTS-way results showed that after 24 hours,48 hours,72 hours and 96 hours,the proliferation activity of LN229 and U251 cells in miR-623NC+ p ENTER-TRIM44 group was significantly higher than that in miR-623NC+ vector group,while the proliferation ability of glioma cells in miR-623 mimics+ vector group was significantly lower than that in miR-623 mimics+vector group.Compared with miR-623+vector group,the proliferation activity of LN229 and U251 cells in miR-623mimics+TRIM44 group was significantly higher than that in miR-623mimics+TRIM44 group.And the proliferation activity of miR-623 mimics+p ENTER-TRIM44 group was higher than that in other two groups,and the salvage experiment further proved that miR-623 could target TRIM44.5.After co-transfection of glioma cells with miR-623 mimics or control and TRIM44 over-expression vector or control vector,Transwell chamber assay showed that the number of glioma cells passing through the chamber bottom in miR-623NC+ p ENTER-TRIM44 group was significantly more than that in miR-623NC+ vector group,while that of glioma cells in miR-623 mimics+vector group was significantly less than that in miR-623 mimics+ vector group.Compared with the miR-623+vector group,the number of LN229 and U251 cells passing through the bottom of the chamber in the miR-623mimics+ TRIM44 group was significantly more than that in the miR-623mimics+ TRIM44 group(P<0.05).And the proliferation activity of miR-623 mimics+p ENTER-TRIM44 group was higher than that in other two groups,and the salvage experiment further proved that miR-623 could target TRIM44.6.Western blotting showed that the expression of E-cadherin in miR-623 NC group was significantly higher than that in miR-623 mimics + p ENTER vector group,while the expression level of Vimentin and N-cadherin decreased significantly.Next,compared with miR-623 mimics + p ENTERTRIM44 group,the expression level of E-cadherin in miR-623 mimics + p ENTER vector group decreased significantly,while the expression level of Vimentin and N-cadherin increased significantly.The addition of p ENTERTRIM44 plasmid can save the effect of miR-623 mimics on epithelialmesenchymal transformation-related proteins.Conclusions:1.TRIM44 could be regulated by miR-623.2.TRIM44 can affect the malignant phenotype of glioma cells.3.miR-623 can inhibit the expression of TRIM44 and regulate the proliferation of glioma cells and the pathway of epithelial-mesenchymal transformation.Part Four The effect of miR-623 on the growth of glioma cells in nude mice and its mechanism.Objective: To detect the effect of miR-623 on the tumorigenesis of glioma cells in nude mice.The tumor-bearing experiment in nude mice was used to explore its mechanism in the occurrence and development of glioma.Methods: The stable transduced cell lines of miR-623 overexpression plasmid and control vector were constructed,and the stable transduced cell lines were screened.The stable cell lines were screened for the expression of miR-623 m RNA in qRT-PCR assay,and the phenotype of stable cell lines was verified by MTS-way and colony formation experiments.The stable cells were cultured,subcultured and collected,and the cells were injected subcutaneously into nude mice to establish the tumor-bearing model of nude mice.The tumor volume of tumor-bearing nude mice was measured.Four weeks later,the nude mice died suddenly by necking method.Each animal was dissected,the organs were separated,the size of the tumor was observed and photographed,each tumor was removed,and the data were recorded.The expression of miR-623 m RNA in nude mice tumors was detected by qRT-PCR,and the expression levels of TRIM44 and epithelial-mesenchymal transformationrelated proteins in nude mice tumors were detected by Western blotting,and the effect of miR-623 on the proliferation of glioma cells in vivo was further analyzed.Results:1.The proliferation activity and colony formation ability of stable glioma cells were similar as those of transient transfection,which further proved the effectiveness of stable transfection cells.2.The number,volume and weight of tumors in miR-623 group were significantly lower than those in control group(P < 0.05).3.The results of QRT-PCR experiment showed that m RNA in miR-623 over-expression group was higher than that in control group.The level of TRIM44 m RNA in miR-623 group was significantly lower than that of the control group.4.Western blotting showed that the expression of TRIM44 in the tumor tissue of nude mice in the miR-623 group decreased significantly,while the expression of epithelial mesenchymal transformation-related protein E-cadherin increased significantly and the expression of Vimentin and N-cadherin decreased significantly compared with the control group.Conclusions: The vivo experiments showed that miR-623 could inhibit the growth of transplanted tumor in nude mice.In a word,miR-623 was low-expression in glioma patients and was related to survival.miR-623 inhibits the proliferation of glioma cells in vivo and in vitro by targeting TRIM44 3’UTR,and inhibits the occurrence of epithelial-stromal transformation pathway.miR-623 is expected to become a new target for glioma therapy.