Effect Of TNIP1 On Proliferation,invasion And Migration Of Glioma And Its Mechanism

Objectives:Glioma has the characteristics of infiltrative growth,poor differentiation,rapid proliferation and strong invasiveness,so the clinical treatment effect is not ideal.The purpose of this study is to explore the effect of TNFAIP3 Interacting Protein1(TNIP1)on the proliferation and motor ability of glioma cells in vivo and in vitro,and to determine whether TNIP1 affects the development of glioma through Mitogen Activated Protein Kinas(MAPK)signal pathway.Methods:The difference of TNIP1 gene expression between normal tissue and glioma tissue was detected by public tumor database The Cancer Genome Atlas(TCGA)and Chinese Glioma Genome Atlas(CGGA).Then we continue to explore the changes of TNIP1 expression in different grades of gliomas.Finally,we explore the relationship between the expression of TNIP and the survival and prognosis of glioma patients,and do Kaplan-Meier survival analysis in different subgroups to understand the effect of TNIP1 on the survival and prognosis of glioma patients.The samples of glioma tissue and normal brain tissue around the tumor were collected,and the expression of TNIP1 was analyzed by Western Blotting(WB)and immunohistochemistry.Human cerebellar astrocytes(Hac)and glioma cell lines(U87,U251)were selected to perform WB and Real-time Quantitative PCR experiments(RT-q PCR)to observe the expression of TNIP1 in glioma cell lines.Glioma cell lines with high expression of TNIP1(U251 and U87)were selected for stable transfection and transient transfection to knock down TNIP1 and TNIP1.The knock-down effect was verified by WB and RT-q PCR,and the one with the most obvious knock-down effect was selected for follow-up experiments.The proliferation ability of glioma was detected by CCK8,Ed U and clone test,and the migration and invasion ability of glioma was detected by Transwell test and scratch test.Finally,we carried out in vivo experiments to observe the effect of TNIP1 on the occurrence and development of gliomas.The nude mouse model was constructed,and the nude mouse model of subcutaneous tumor was established by selecting the U251 cell line stably transfected with knock-down TNIP1 and its negative control U251 cell line.The differences in tumor size,self-weight,mental state and survival time between the two groups were observed,recorded and analyzed.In order to clarify the potential effect of TNIP1 on the biological function of glioma cells.The U251 cell line stably transfected with TNIP1-sh RNA and its negative control group were selected for expanded culture.Then RNA-seq was used to detect differential genes and extract differential gene data to explore the potential function and possible regulatory signal pathways of TNIP1 in the occurrence and development of glioma by GO and KEGG enrichment analysis.Then we will verify the potential function and signal pathway of TNIP1 in the occurrence and development of glioma in vitro.WB was used to detect the changes of related pathway proteins after knocking down TNIP1.Results:1.High expression of TNIP1 was found in gliomas by TCGA database analysis.In CGGA database,it was found that the expression of TNIP1 increased with the increase of glioma grade;IDH wild type was higher than mutant type;recurrent type was higher than primary type;1p/19 q non-co-deletion was higher than co-deletion.In the TCGA database,it was found that the patients with high expression of TNIP1 had a shorter survival time and were reflected in different glioma subgroups.In clinical glioma samples,the results of WB and immunohistochemistry showed that the expression of TNIP1 in cancer tissues was significantly higher than that in paracancerous tissues.The protein and RNA levels of TNIP1 in glioma cell lines were higher than those in normal astrocytes,especially in U251 cell lines.2.From the phenotypic experiment in vitro,it was observed that the proliferation,invasion and migration ability of glioma cells decreased after knocking down TNIP1.3.The subcutaneous tumor model of nude mice was successfully established in vivo,and it was found that compared with the experimental group,the tumor in the control group grew rapidly,the volume was larger,the adhesion between the tumor and the surrounding tissue was heavier,and the blood vessels were abundant.However,there was no significant difference in weight,mental state and survival time of nude mice.4.RNA-seq analysis of sh-TNIP1 and its control group showed that there were a large number of differential genes,and GO enrichment analysis showed that the functions of different genes were enriched in mitosis,ATP enzyme activity,chromosome and so on.KEGG enrichment analysis showed that signal pathways were enriched in MAPK,Cell Cycle,PI3K-Akt and so on.The changes of proteins related to MAPK signal pathway were observed by WB experiment after knocking down TNIP1.It was found that the expression of phosphorylated C-RAF,phosphorylated MEK1/2 and phosphorylated ERK1/2 decreased,but the total protein content had no obvious downward trend.Conclusions:1.TNIP1 is highly expressed in gliomas,and the ability of proliferation,invasion and migration of glioma cells decreases after knocking down TNIP1.TNIP1 may be a new therapeutic target for glioma.2.TNIP1 may participate in the occurrence and development of glioma proliferation,invasion and migration through MAPK signal pathway.