LRIG3 Regulates VEGFA-Mediated Glioma Angiogenesis Via PI3K/AKT Pathway

PART ?The effects of LRIG3 overexpression in glioma cells on the biological function of vascular endothelial cells Objective: To investigate the effects of glioma cells overexpressing LRIG3 on the migration,proliferation,and tube formation of vascular endothelial cells.Methods: We performed in vitro gain-of-function analysis by overexpressing LRIG3 with a lentiviral vector in U87 and U251 cells.Altered expression of LRIG3 in glioma cells was confirmed by western blot analysis.The supernatant was collected from each group as conditioned medium(CM),which was used to treat human umbilical vein endothelial cells(HUVECs).The wound healing assays and transwell migration assays were carried out to demonstrate the function of LRIG3 upregulation on HUVECs migration.We performed the tube formation assays to explore the effect of each CM on the ability of HUVECs to form vascular lumen like structures.We also performed the MTT proliferation assays to determine the cell viability of HUVECs treated with different CM.Results: Compared with the control group,the CM derived from glioma cells overexpressing LRIG3 significantly inhibited the migration,proliferation,and tube formation of HUVECs.Conclusion: The overexpression of LRIG3 in glioma cells could inhibit the glioma angiogenesis in vitro.PART ?The effects of LRIG3 downregulation in glioma cells on the biological function of vascular endothelial cells Objective: To investigate the effects of LRIG3-silenced glioma cells on the migration,proliferation,and tube formation of vascular endothelial cells.Methods: We performed in vitro loss-of-function analysis by silencing LRIG3 expression in U87 and U251 with siRNAs.Altered expression of LRIG3 in glioma cells was confirmed by western blot analysis.The supernatant was collected from each group as conditioned medium(CM),which was used to treated human umbilical vein endothelial cells(HUVECs).The wound healing assays and transwell migration assays were carried out to demonstrate the effects of LRIG3 downregulation on HUVECs migration.We performed the tube formation assays to explore the effect of each CM on the ability of HUVECs to form vascular lumen like structures.We also used the MTT proliferation assays to determine the cell viability of HUVECs treated with different CM.Results: Compared with the control group,the CM derived from LRIG3-silenced glioma cells significantly promoted the migration,proliferation,and tube formation of HUVECs.Conclusion: The downregulation of LRIG3 in glioma cells could promote the glioma angiogenesis in vitro.PART ?The effects of LRIG3 on glioma angiogenesis in vivo Objective: To investigate the effects of LRIG3 on glioma angiogenesis in vivo.Methods: We first investigated the role of LRIG3 in regulating the angiogenesis of glioma in vivo using intradermal angiogenesis assays.The number of intradermal tumor-directed capillaries in different groups was used to evaluate the effect of LRIG3 on glioma angiogenesis.Next,we established the intracranial xenograft mice by using LRIG3-overexpressed U87 cells and the control cells.MRI was used to monitor the tumor progression.A (13)^NNH₃·H₂O PET scan was performed to evaluate the effect of LRIG3 on the blood flow of intracranial xenografts.Parallel histological analysis of intracranial xenograft tumors was performed to evaluate the microvessel density(MVD)in the two groups.Results: In intradermal angiogenesis assays,we found that glioma cells stably overexpressing LRIG3 exhibited a significantly lower number of tumor-directed capillaries relative to cells transfected with the control vector.Conversely,LRIG3-silenced glioma cells attracted significantly more blood vessels compared to cells transfected with control siRNAs.A (13)^NNH₃·H₂O PET-CT scan revealed that xenografts carrying LRIG3-overexpressing U87 cells exhibited a significantly lower (13)^NNH₃·H₂O uptake tumor/reference(T/R)ratio relative to the xenografts carrying control cells.Parallel histological analysis of orthotopic xenograft tumors revealed significantly lower MVD in tumors formed by LRIG3-overexpressed U87 cells.Conclusion: LRIG3 could suppress glioma angiogenesis in vivo.PART ?The mechanism of LRIG3 regulating angiogenesis in glioma Objective: To explore the specific mechanism of LRIG3 regulating angiogenesis in glioma.Methods: We performed qRT-PCR to quantify the levels of mRNAs for angiogenesis-related factors in the glioma cells following LRIG3 knockdown or overexpression.Western blots and ELISA assays were used to confirm the changes of protein expression of the corresponding factor.Results: The results showed that LRIG3 knockdown significantly elevated levels of VEGFA mRNA in U87 and U251 cells,whereas LRIG3 upregulation significantly repressed the VEGFA mRNA expression in glioma cells.Western blots and ELISA results indicated that levels of VEGFA protein were consistently upregulated or downregulated in LRIG3-knockdown or LRIG3-overexpressing glioma cells respectively,compared to control cells.Conclusion: LRIG3 suppressed glioma angiogenesis by inhibiting VEGFA expression in glioma cells.PART ?The mechanism of LRIG3 regulating VEGFA expression in glioma Objective: Previous studies have shown that LRIG3 can inhibit the activation of PI3K/AKT pathway and ERK pathway.We intend to clarify the role of PI3K/AKT pathway and ERK pathway in the LRIG3-regulated VEGFA expression.Methods: To ascertain the pathway responsible for LRIG3-mediated VEGFA expression,we used the specific inhibitors of the ERK(PD98059)and AKT(LY292002)pathways to block their activation in glioma cells following LRIG3 knockdown.Then,we performed western blots to quantify the levels of VEGFA in each group.Parallel changes of VEGFA in conditioned medium(CM)were detected by ELISA assay.The effects of conditioned medium on HUVECs were detected by functional assays.Finally,we validated the clinical association between LRIG3 with p-AKT,and VEGFA in patients,using IHC staining and western blots of these proteins in glioblastoma samples.Results: The results indicated that inhibition of the AKT,not the ERK pathway,caused changes of VEGFA expression in LRIG3-silenced glioma cells.In the functional assays,we treated HUVECs with the indicated CM to detect the proliferation,migration,and tube formation of endothelial cells,and the results consistently showed that inhibition of the AKT pathway,not the ERK pathway,repressed the ability of LRIG3-silenced glioma cells to induce angiogenesis.In glioma samples,tumors with high LRIG3 levels tended to express low levels of p-AKT and VEGFA,and the reverse was true for tumors with low LRIG3 expression.Conclusion: LRIG3 suppressed VEGFA expression by inhibiting the activation of PI3K/Akt pathway in glioma cells.