Expression Of Volume-regulated Anion Channel(LRRC8A)in Glioblastoma And Its Effect On Proliferation,Migration And Invasion Of Glioblastoma Cells

Objective: Glioblastoma(GBM)is the most invasive primary brain tumor in human beings.In recent years,although the comprehensive treatment program has been developed to a certain extent,the survival prognosis of GBM patients is still very poor,almost all patients will relapse,and many patients will endure multiple craniotomy in order to strive for more survival time.However,the effect of the current treatment is far from satisfactory.Ion channels participate in a number of physiological and pathological processes in the human body,which has been widely concerned by researchers.Six years ago,the LRRC8 family was identified as the molecular basis of volume-regulated anion channels,which consists of five subunits of LRRC8A-E,of which LRRC8 A is an essential subunit.studies have found that the expression of LRRC8 A is abnormal in many system malignant tumors,such as breast cancer,colon cancer,cervical cancer and so on.However,the biological role of LRRC8 A in the formation and rapid progression of glioblastoma is still unclear.The purpose of this study was to explore the expression of LRRC8 A in glioblastoma and its effect on the malignant biological characteristics of glioblastoma cells at the cellular level.Methods: In this experimental study,a total of 76 surgical specimens were collected(including 60 GBM tissue specimens and 16 normal controls).The patients were treated in the first affiliated Hospital of China Medical University and underwent craniotomy from June 2016 to June 2019.The specimens of patients included in the experiment must be patients who did not receive chemotherapy or radiotherapy before operation to avoid affecting the results of the experiment.In the end,the specimens that entered the experimental stage must be taken from patients with glioblastoma confirmed by pathological results after operation.in addition,the control group was 16 cases of non-tumor brain tissue from normal brain tissue removed during brain trauma or cerebral hemorrhage.This study was approved by the Ethics Committee of the first affiliated Hospital of China Medical University.In this study,human glioblastoma tissue was collected and glioma U87 cell line was used for experiment,the collected GBM tissue and normal brain tissue were tested respectively,the RT-PCR test was used to study the expression of LRRC8 A at RNA level between tumor tissue and normal brain tissue,and then Western blot related experiments were carried out to compare the expression of LRRC8 A between the experimental group and the control group at the protein level.Finally,we studied the localization of LRRC8 A in cells by immunohistochemical experiment,and verified the trend of protein expression.In the in vitro experiment,U87 glioma cells were cultured and LRRC8 A knockout group and normal control group were obtained by plasmid transfection.Plate cloning test,invasion test and cell scratch test were carried out respectively.The experimental results were compared with those of the control group to explore the changes of biological behavior of glioma cell lines after low expression of LRRC8 A.Results: 1.LRRC8 A was expressed in both normal brain tissue and glioblastoma tissue,and the expression level in glioblastoma tissue was significantly higher than that in normal brain tissue.2.In the clone formation experiment,the number of U87 cells in the experimental group(89 ±13)was significantly less than that in the control group(212 ±30).The results showed that the proliferation ability of glioma U87 cells after low expression of LRRC8 A was significantly lower than that of the control group,and there was significant difference between the two groups(P<0.01).3.In the experiment of invasiveness of tumor cells detected by Transwell method,the number of cells invading the subventricular layer in the experimental group with low expression of LRRC8 A was(68 ±10),which was significantly different from that in the control group(166 ±15).The experimental data are calculated,and the results show that the difference is statistically significant(P<0.01).4.In the cell scratch test,the scratch healing rate of U87 cells in the experimental group with low expression of LRRC8A(28±4)was significantly lower than that in the control group(54 ±3).The difference was statistically significant(P<0.01).Conclusion: Our results suggest that LRRC8 A may play an important role in the tumorigenesis and malignant progression of glioblastoma.The expression of LRRC8 A in glioblastoma tissue was significantly higher than that in normal brain tissue.Moreover,we also explored the effect of LRRC8 A on the biological behavior of glioma cell lines.After knocking down the expression level of LRRC8 A,it can significantly inhibit the proliferation,migration and invasion of glioma U87 cells in vitro.This study can provide new ideas for further research on the pathogenesis of glioblastoma and looking for new and more effective treatment in the future.