| Background: Glioma,the most common primary intracranial malignancy,is highly heterogeneous and difficult to treat.According to the degree of malignancy,the WHO classifies gliomas into grade I-IV.Among them,the median survival time of patients with grade IV glioblastoma(GBM)receiving surgical treatment,concurrent chemoradiotherapy and TMZ maintenance therapy is less than 15 months,and the five-year survival rate is approximately 8-14%.Although the concept of diagnosis and treatment of glioma has been constantly updated in recent years,due to the high heterogeneity of glioma,the existing treatment methods have failed to have a significant impact on the survival rate and disease progress of glioma patients.Currently,TMZ is the most widely used drug for GBM,and its anti-glioma mechanism is that TMZ causes methylation of guanine at O6 position,which leads to the breakage of DNA double bond,thus inducing glioma cell apoptosis.However,due to various mutant molecules and complex molecular pathways in tumors,TMZ resistance has become increasingly prominent.Therefore,it is an urgent to develop new multi-target anti-glioma drugs in order to elevate the survival rate of glioma patients.TP53 is recognized as a gene that exert a function of repressing cancer,and p53 protein is the product of its transcription and translation.Meanwhile,in various human cancers,TP53 gene is also the gene with the highest mutation rate.wtp53 protein has a very short half-life and very low levels in normal cells under non-stress conditions.Nonetheless,under a variety of stress conditions such as DNA damage and oncogene activation,wtp53 will be activated by post-translational modifications, including phosphorylation,acetylation,and methylation.The activated wtp53 selectively binds to target genes and participates in the regulation of biological processes such as intracellular DNA repair,cell cycle,apoptosis,and oxidative stress to exert anticancer functions.However,more than half of cancers have mutations in TP53 gene.mutp53 usually loses its anti-cancer function and forms a tetramer in the presence of the remaining wtp53,thus blocking the anti-cancer function of the remaining wtp53.In addition,mutp53 tends to show carcinogenic activity in cells lacking wtp53.It is very difficult for mup53 to be degraded in cells and thus accumulate in cells,which is also the basis for mutp53 to obtain carcinogenic function.Therefore,promoting the degradation of mutp53 is an effective means to treat tumors with high expression of mutp53.Collagen is an essential component of the ECM and accounts for the highest proportion of protein in vivo.Collagen type XI ?1 chain(COL11A1)has been proved to be highly expressed in various cancers and promotes malignant progression of cancer.In addition,COL11A1 can be taken as an underlying tumor marker to calculate the prognosis of cancer patients,but the role of COL11A1 in glioma is still unclear.COL11A1 is involved in many intracellular signal transduction pathways.Many genes,including TGF-? 1,AKT,B-myb and Gli 1,can promote the transcription of COL11A1.High expression of COL11A1 promotes the expression of downstream related molecules such as Twist1 and MMP3,which are relevant to the drug resistance and invasion of tumors.Up to now,only a few drugs have been found to inhibit m RNA and promoter activity of COL11A1,which in turn reduced the expression of COL11A1.Berberine,a compound extracted from a variety of plants,has been used for many years as a non-prescription drug for the treatment of bacterial diarrhea in China.Berberine is also considered as a potential drug for the treatment of hyperlipidemia and diabetes.In recent years,berberine has also been proved to have anti-cancer effects.Studies have shown that berberine plays an anti-cancer role by regulating MAPK,ERK 1/2,JNK,AKT and Wnt/?-catenin signaling pathways.However,the effect of berberine on mutp53 and COL11A1 remains to be investigated.Objective: This research was aimed to illustrate the different influence of berberine on wtp53 and mutp53 in glioma cells,as well as the role of COL11A1 protein in glioma cells and to clarify the mechanism of berberine inhibiting COL11A1.Methods: In this study,the effect of berberine on the viability of U87,U251 and T98 G glioma cells was determined by CCK-8.Next,wtp53 cell line U87 cells and mutp53 cell line U251 cells(p53 R273H)are selected as research subjects,and the effect of berberine on the proliferation ability is detected through a cell cloning experiment.The influence of berberine on the cell cycle of wtp53 and mutap53 is analyzed by a flow cytometry,the influence of berberine on the content and phosphorylation levels of wtp53 and mutap53 proteins as well as on downstream proteins is detected by a Western blotting method,The inhibition of berberine on the proliferation of glioma cells in the presence of wtp53 and mutp53 was detected by lentivirus transduction,and the inhibition of berberine on the growth of different glioma cells in vivo was detected by a nude mouse subcutaneous xenograft model.The connection between COL11A1 and the glioma patients' survival rate was analyzed by the Chinese Glioma Genome Atlas(CGGA)database.Western blotting was used to analyze the expression of COL11A1 in different glioma cell lines.In the result,the cells with high expression of COL11A1,U87 and T98 G cells,were selected as the research subjects.COL11A1 gene in glioma cells was knocked out by si RNA,and the effect of COL11A1 on the invasion and drug resistance of glioma cells was investigated.The influence of berberine on the migration ability of glioma cells is detected by a wound healing experiment,the influence of berberine on the invasion ability of U87 and T98 G cells is detected by a transwell test,the influence of berberine on the cytoskeleton of glioma cells is detected by an F-actin probe,the concentration of TGF-?1 in U87 and T98 G cells culture supernatant is detected by an enzyme-linked immunosorbent assay(ELISA),Western blotting was served to survey the impact of berberine on TGF-?1/COL11A1 pathway and its downstream proteins,and HE staining and immunofluorescence were used to analyze the effect of berberine on collagen and COL11A1 in glioma xenografts in vivo.Results: 1.CCK-8 results showed that berberine inhibited glioma cell viability in a time-dependent and concentration-dependent manner.Cell cloning experiments showed that berberine inhibited the proliferation of U87 and U251 cells.The results of flow cytometry showed that berberine caused U87 in G1 phase arrest and U251 in G2 phase arrest.2.Western blotting results showed that berberine increased the phosphorylation level of wtp53 protein,promoted the expression of p21 protein and inhibited the expression of cyclin D1 protein in U87 cells.In U251 cells,berberine repressed both the expression of mutp53 protein and the phosphorylation of mutp53 protein,as well as the expression of p21 protein and cyclin D1 protein.3.After the lentivirus was used to transfer wtp53 into U251 cells,berberine had a stronger cycle arrest effect.HA-tag showed that berberine had no significant effect on the content of exogenous wtp53.4.The subcutaneous transplanted tumor model in nude mice showed that berberine significantly inhibited the growth of U87 and U251 cells in vivo.Immunohistochemical results showed that berberine promoted the phosphorylation of wtp53 and reduced the content and phosphorylation level of mutp53 in vivo.HE staining of the major organs in nude mice proved that berberine had no obvious damage to the organs of nude mice,which confirmed the safety of berberine in vivo.5.CGGA database analysis shows that compared with patients with low expression of COL11A1 glioma,patients with high expression of COL11A1 glioma have significantly shorter survival time,which is statistically significant.The level of COL11A1 in seven glioma cell lines was detected by Western blotting.The results showed that,except A172 cells,the other six glioma cell lines all highly expressed COL11A1.U87(MGMT-deficient)and T98G(MGMT-expressed)cells were selected as research objects.After knocking out COL11A1 gene in glioma cells by si RNA technology,it was found that the migration and invasion ability of U87 and T98 G cells were significantly decreased,and the glioma cells were more sensitive to TMZ. Western blotting results showed that after COL11A1 knockout,the expression of MMP2,MMP3 and MMP9 in cells decreased significantly,and the contents of MGMT and snail protein also decreased significantly.6.Wound healing experiment and transwell test showed that berberine inhibited the migration and invasion of U87 and T98 G cells in a dose-dependent manner.After observing the morphology of glioma cells under high magnification microscope,it was found that berberine reduced the number of plasma membrane ruffles of glioma cells,and F-actin probe showed that berberine changed the arrangement of actin cytoskeleton in U87 and T98 G cells.The Western blotting results showed that berberine reduced the contents of TGF-?1,COL11A1,MMP2,MMP3,MMP9 protein in glioma cells.ELISA results showed that berberine reduced the level of TGF-?1 in T98 G cell culture supernatant,but had no significant effect on the level of TGF-?1 in U87 cell culture supernatant.Adding TGF-?1 into the cell culture medium could partially reverse the inhibitory effect of berberine on the migration and invasion ability of glioma cells.7.CCK-8 experiment and DAPI staining showed that berberine could increase the sensitivity of glioma cells to TMZ.Western blotting results showed that berberine inhibited the expression of snail protein in U87 cells,and inhibited the expression of MGMT and snail protein in T98 G cells.The results showed that berberine changed the arrangement of glioma cells and increased the intercellular space in vivo.Immunofluorescence results showed that berberine inhibited the expression of COL11A1 protein in vivo.Conclusion: 1.Berberine inhibits the proliferation of glioma cells in vitro and in vivo.2.Berberine promotes the phosphorylation of wtp53,thus promoting its anti-cancer function;Berberine inhibits the expression and phosphorylation of mutp53 and inhibits its carcinogenic effect.3.The high expression of COL11A1 is related to the poor prognosis of glioma patients.4.COL11A1 is related to the migration and invasion ability of glioma cells as well as TMZ resistance.5.Berberine inhibits the migration,invasion and TMZ resistance of glioma cells by inhibiting the TGF-?1/COL11A1 pathway. |
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