Effect Of Connexin43 On Biological Behavior And Temozolomide Sensitivity In Human Glioma U87 Cells

Glioblastoma(GB)is the most common malignant tumor of the central nervous system.Patients with GB have very low 3-year survival after aggressive treatment.In addition,the chemotherapeutic resistance of GB also brings great obstacles to clinical treatment.Gap junctions exist between two adjacent cells,which are recognized as cell-cell direct connections except between blood cells and skeletal muscle cells.The formed gap junction closely connects the cytoplasm of neighboring cells,thereby regulating the exchange of small molecular metabolites and ions.Therefore,abnormal gap junction expression can alter cell metabolism and contribute to the development and progression of cancer.Highly expressed in astrocytes and neural precursor cells,Connexin 43(Connexin43,Cx43),is often reduced in most astrocytomas.The role of Cx43 in tumor cell growth,migration,and invasion has been shown to be complex and controversial.At the same time,its effect on tumor cell chemotherapeutic drug sensitivity is also worth exploring.Purpose:To investigate the effects of Cx43 on the growth,migration,invasion and chemotherapeutic drug sensitivity of glioma U87 and the related molecular mechanisms.It provides a new idea for the clinical treatment of glioma and the study of TMZ sensitivity.Methods and results:Based on the construction of U87 stable transfected cell lines with overexpression and silencing of Cx43,the following studies were conducted in this study:1.Construction and identification of glioma U87 cell line that stably overexpresses and silences Cx43Lentiviral vector was used to construct glioma U87 cells with stable overexpression and silencing of Cx43.U87 cells infected with lentivirus carrying Cx43 were labeled as U87-O-Cx43,and the no-load group was labeled as U87-NG-O-Cx43.U87 cells infected with lentivirus carrying the silenced Cx43 sequence were labeled as U87-Sh-Cx43,and the no-load group was labeled as U87-NG-Sh Cx43.The expression of Cx43 in U87-O-Cx43 cells was significantly higher than that in U87 and U87-NG-O-Cx43 groups,and the expression of Cx43 in U87-Sh-Cx43 cells was significantly lower than that in U87 and U87-NG-Sh Cx43 groups.The stable overexpression and silenced Cx43 cells were obtained successfully.2.Effects of Cx43 on U87 cell growthOur research use U87-O-Cx43,U87-NG-O-Cx43,U87,U87-NG-Sh Cx43,U87-Sh-Cx43 cell,counted and planted them in 96 well plates for 10% FBS DMEM medium,respecitivily.The culture lasted for 6 days,and the medium was replaced on the 3rd day.The absorbance of the corresponding groups was measured with CCK-8every 24 h to get the cell proliferation curve.Experimental results showed that the absorbance of the five groups of cells was very similar from 1 to 6 days,and there was no significant difference through statistical analysis.These results indicated that Cx43 inhibited the invasion of U87 cells.These results indicated that Cx43 had no significant effect on the proliferation of U87 cells.3.Effects of Cx43 on U87 cell migrationIn this part,Transwell migration assay was used to detect the effect of Cx43 on glioma U87 cell migration.This migration assay showed that under the same treatments,the number of U87-O-Cx43 cells migrated to the bottom of transwell was much lower than that of U87-NG-O-Cx43 cells,U87 cell group;while U87-Sh-Cx43 cells migrated to the lower chamber of transwell is far more than U87 cells,U87-NG-Sh Cx43 cells.These results indicated that Cx43 inhibited the migration of U87 cells.4.Effects of Cx43 on U87 cell invasionFor previous part,we have tested the effect of Cx43 for cell migration by transwell migration assay.In this part,we want to detect the effect of Cx43 for cell invasion,with matrigel to simulate ECM in the chamber of transwell.Consistently,the number of U87-O-Cx43 cells crossed to the bottom of transwell was much lower than that of U87-NG-O-Cx43 cells,U87 cell group;while U87-Sh-Cx43 cells arrived to the lower chamber of transwell is far more than U87 cells,U87-NG-Sh Cx43 cells.These results indicated that Cx43 inhibited the invasion of U87 cells.5.Effects of Cx43 on the sensitivity of U87 cells to TMZAt present,most of the clinics use post-surgical adjuvant radiotherapy and chemotherapy to treat GBM patients.As an alkylating agent,TMZ,is currently the most effective chemotherapeutic showing excellent bioavailability.It has been proved that TMZ of 1000 u M can induce apoptosis of glioma.To further explore whether Cx43 affects the sensitivity of U87 cells to TMZ,we used the above five groups of cells,measured their drug response dose curves,and calculated the IC50 value,respectively.The results indicated that the IC50 value of U87-O-Cx43 cells was lower than that U87-NG-O-Cx43 and U87 cell groups;while the U87-Sh-Cx43 cell group and U87 cells and U87-NG-Sh Cx43 cells were basically the same.Conclusion:1.Cx43 did not affect the proliferation ability of U87 cells.2.Cx43 inhibited the migration and invasion of U87 cells.3.Overexpression of Cx43 promoted the sensitivity of U87 cells to temozolomide,while silencing of Cx43 had no effect.