Effect Of CN-3,the Active Ingredient Of Culcita Novaeguineae,Inhibiting The Migration And Invasion Of Glioma

【Objective】The infiltrative growth of glioma cells,which leads to recurrence,and the mediocre efficacy of current chemotherapeutic agents have both contributed to the poor prognosis of glioma,therefore,it is crucial to develop new chemotherapeutic drugs that really can inhibit the infiltrative growth of glioma.As a traditional folk Chinese herbs,starfish was first published in The Compendium of Materia Medica,and has been recorded in The Annals of Chinese Medicinal Animals and The Chinese Materia Medica,etc.It has received attention in anti-tumor research for its"softening and dispersing"effect.Saponins are an important structural type of active ingredients in Traditional Chinese Medicine.Asterosaponin,a broad steroid saponins,are the most representative active ingredients of starfish and have potential prospects for antitumor drug development.Aiming at the characteristics of glioma which are prone to invasion and high recurrence,the study explored the effect of Culcita novaeguineae-3（CN-3）,an active ingredient of starfish,on the invasive growth of glioma cells,and then explored the key efficacy target of CN-3 to play the role of drug efficacy.The study is anticipated to provide certain experimental foundation for clarifying the pharmacodynamic substance basis of starfish against glioma,to promote the development and application of starfish,to present research ideas for understanding the mechanism of action of asteroidean active component CN-3,and to give theoretical backing for clinical medical therapy of saponin compounds against glioblastoma.【Methods】（1）The active component CN-3 in Culcita novaeguineae was separated and prepared by using silica gel column chromatography,Sephadex LH-20 gel column chromatography,reversed-phase C-18 column chromatography and high performance liquid chromatography.（2）The cell counting kit-8（CCK-8）method was used to identify the inhibitory doses of CN-3 on the glioma cells LN229 and U373-MG.The half maximal inhibitory concentration(IC₅₀)values were then determined to serve as a guide for the dosing concentrations in further investigations.（3）The proliferation viability curves of cells were recorded using the real time cellular analysis（RTCA）system.（4）The effect of CN-3 on the migration and invasion ability of glioma cells was examined by Transwell migration assay and Transwell invasion assay（using Matrigel-coated subcompartment membrane）.（5）The Cancer Genome Atlas（TCGA）was used to study the expression profile and clinical information of the high mobility group protein A1（HMGA1）in 1116 samples related to glioma.（6）Overexpression and interference with HMGA1 expression was achieved by lentiviral infection of two kinds of glioma cells to construct stable transgenic strains.The effects of HMGA1 on the proliferation viability,migration and invasion of glioma cells as well as the efficacy of CN-3 combination were examined.（7）The conserved binding relationship between Hsa-miRNA-4465 and HMGA1 was predicted by Targetscan.Based on this prediction,a luciferase reporter gene plasmid was constructed,transfected and validated to determine whether miRNA-4465 targets HMGA1in this manner.（8）The effects of miRNA-4465 on proliferation viability,migration and invasion of glioma cells and on the efficacy of CN-3 were examined by transfecting miRNA-4465mimics and inhibitor.（9）RT-PCR was used to detect the HMGA1 and miRNA expression levels in glioma cells.（10）By using Western blot,the expression of proteins involved in migration and invasion in glioma cells was examined before and after CN-3 administration,as well as after HMGA1 or miRNA4465 interference.（11）Inhibition of glioma by CN-3 in vivo studied by a glioma subcutaneous tumor model in nude mice.（12）The physiological function of HMGA1 in vivo was studied by glioma in situ model in nude mice.【Results】（1）By comparing with the previous reference substance,CN-3 reagent 212.6 mg was successfully extracted and isolated from the bread starfish with a wet weight of 30 kg.（2）Compared with the control group,low doses of CN-3 induced the development of glioma cell inhibited the migration,invasion and angiogenesis of glioma LN229 and U373-MG cells.Western blot demonstrated that CN-3 inhibited the expression of neuronal cadherin（N-cad）and upregulated the expression of epithelial cadherin（E-cad）,thus inhibiting the degree of reduced adhesion between glioma cells;and decreased the expression of the matrix metalloproteinase 9（MMP9）and the matrix metalloproteinase 2（MMP2）to inhibit the degradation of extracellular matrix;reduce the expression of vascular endothelial growth factor receptor-2（VEGFR2）to inhibit angiogenesis;and inhibit the expression of nuclear factor kappa-B（NF-κB）to stop the process of epithelial mesenchymal transition.（3）The expression of HMGA1 in glioma cells is inhibited by CN-3,and patient prognosis is negatively correlated with HMGA1 expression.HMGA1 is a crucial component of CN-3’s anti-glioma migration and invasion impact.Compared with the control group,interference with the expression of HMGA1 significantly inhibited the proliferative activity,migration and invasion ability of glioma cells,while the expression of infiltrative growth-related proteins was also down-regulated;reversionary experiments showed that overexpression of HMGA1 significantly enhanced the proliferative activity and migratory invasion ability of glioma cells.The reversion assay showed that overexpression of HMGA1 significantly enhanced the proliferative activity,migration and invasion ability of glioma cells,and overexpression of HMGA1 antagonized the inhibitory effect of CN-3 on the invasion of glioma cells.（4）The presence of conserved binding of miRNA-4465 to HMGA1 was confirmed by transfection with a luciferase reporter plasmid.miRNA-4465 exhibited an inhibitory effect on infiltrative growth in glioma cells.（5）CN-3 can inhibit HMGA1 by upregulating the expression of miRNA-4465,which in turn exerts an inhibitory effect on the migration and invasion of glioma cells.【Conclusions】CN-3 has the ability to inhibit the migration and invasion of glioma LN229 and U373-MG cells,and has the potential to be developed as a chemotherapeutic agent to inhibit the infiltrative growth of glioma;CN-3 can inhibit HMGA1 by up-regulating the expression of miRNA-4465,and then inhibit the transcription of NF-κB,thus producing the effect of inhibiting the infiltrative growth of glioma cells.