| [Objective]Osteosarcoma is more common in children and adolescents,with high invasion and metastasis,and poor prognosis.Therefore,it is necessary to develop new drugs or treatment programs to improve the clinical cure rate.Aokefunan(C18H17NO6,6-acetyl-2-(1-aminoethyl)-7,9-dihydroxy-8,9b-dimethyl-9bh-diphenylpropylfuran-1)is a diphenylfuran compound extracted from Usnea.In this study,the effects of C18H17NO6 on the proliferation,apoptosis,invasion and migration of human osteosarcoma cells and the possible molecular mechanisms involved were studied through the culture of human osteosarcoma cells in vitro,which will provide an important theoretical basis for the in vivo experimental safety and efficacy observation of C18H17NO6 and even clinical experiments.[Methods](1)Conventional culture of human osteosarcoma cell MNNG/HOS C1#5[R-1059-D](KCB2016037YJ,Cell bank of Kunming Institute of zoology,Chinese Academy of Sciences),human osteosarcoma cell Saos-2(KCB200550YJ,Cell bank of Kunming Institute of zoology,Chinese Academy of Sciences).(2)CCK-8 was used to detect the viability of 0 μM,0.15625μM,0.3125μM,0.625 μM,1.25 μM,2.5 μM,5 μM,10 μM,100 μM C18H17NO6 on MNNG cells and Saos-2 cells after 24 h,48 h and 72 h.CCK-8 was used to detect the viability of 0 μM,0.15625 μM,0.3125 μM,0.625 μM,1.25 μM,2.5 μM,5 μM,10 μM,100 μM CDDP on MNNG cells for 48 h.The following experimental groups were:C18H17NO6 0μM;C18H17NO6 1.5 μM;C18H7NO6 3 μM;C18H7NO6 4 μM.(3)Flow cytometry and TUNEL staining were used to detect the apoptosis rate of MNNG cells treated with different concentrations of C18H17NO6 for 48 h.(4)Scratch test and Transwell chamber test were used to detect the effects of different concentrations of C18H17NO6 on the migration and invasion of MNNG cells for 48 h.(5)ELISA was used to detect the effects of different concentrations of Ci8H17NO6 on the secretion of MMP-2,MMP-9 and VEGF in MNNG cells for 48 h.(6)Western Blot was used to detect the effects of different concentrations of C18H17NO6 on the protein expression of PCNA、Ki67、Bcl-2、Bax、Cleaved Caspase-3、Cleaved Caspase-9、MMP-2、MMP-9、E-cadherin、N-cadherin,Vimentin、PI3K、AKT、p-PI3K、p-AKT in MNNG cells for 48 h.(7)qRT-PCR was used to detect the effects of different concentrations of C18H17NO6 on the mRNA expression levels of PCNA、Ki67、Bcl-2、Bax、Caspase-3、Caspase-9、MMP-2、MMP-9、VEGF in MNNG cells for 48 h.After introducing the PI3K/AKT signal pathway agonist 740Y-P and the PI3K/AKT signal pathway inhibitor LY294002,the experimental groups were:C18H17NO6 0 μM;740Y-P 20 μM;C18H17NO6 4 μM;C18H17NO6 4 μM+740Y-P 20μM;LY294002 20 μM;C18H17NO6 4μM+LY294002 20 μM.The experimental methods were the same as above.(8)Establishment of xenograft osteosarcoma model in nude mice0.2ml 5×106/ml MNNG cell suspension during logarithmic growth was injected into the right back skin of nude mice.SPF grade feeding.When the tumor volume reached 180-200 mm3,the nude mice were randomly divided into the control group and the experimental group,with 3 mice in each group.The experimental group was given 2 mg/kg C18H17NO6 intraperitoneal injection once a day.The control group was given 0.9%normal saline at the same dose and in the same way.(9)Tumor volume,body weight and liver and kidney function of nude mice were monitoredBody weight,tumor diameter and tumor length were recorded every 3 days.The nude mice will be killed by cervical dislocation on Day 20.Before killing the nude mice,collect as much retrobulbar venous blood as possible to test the levels of ALT,AST,CREA and UREA.(10)Biological detectionThe tumor was dissected into two parts.One part was preserved in 10%neutral formalin solution for HE staining,TUNEL staining and IHC staining.Some extracted proteins were used for western blot detection.The rest is stored in refrigerator at-80℃.(11)The second generation high throughput sequencingAfter 48 h treatment with 3μM C18Hi7NO6,the miRNA differentially expressed in MNNG cells was detected by second generation high-throughput sequencing,and was verified by qRT-PCR.(12)Construction and transfection of plasmidAccording to the results of the second generation high-throughput sequencing and qRT-PCR validation,the over expression LV-miR-16-5p mimics and the inhibition expression LV-miR-16-5p inhibitor vectors were constructed with miR-16-5p sequence as template,and MNNG cells were transfected with LV-NC as control.(13)Biological behavior of MNNG cellsAfter 48 h treatment with 3μM C18H17NO6,the cell survival rate,apoptosis rate,invasion and migration were detected.[Results](1)C18H17NO6 can inhibit the proliferation of human osteosarcoma MNNG cells and Saos-2 cellsCCK-8 results showed that the inhibitory effect of C18H17NO6 on the proliferation of MNNG cells and Saos-2 cells was time-dose-dependent;C18H17NO6 had better inhibitory effect on MNNG cells than CDDP;C18H17NO6 significantly down regulated the expression of PCNA and Ki67 protein and mRNA in MNNG cells.MNNG cells were selected as the follow-up subjects,the experimental concentrations of C18H17NO6 were 1.5 μM,3μM,4 μM.(2)C18H17NO6 induced apoptosis of human osteosarcoma MNNG cells in a dose-dependent mannerFlow Cytometry and TUNEL staining showed that C18H17NO6 induced MNNG cells apoptosis in a dose-dependent manner;Western blot showed that C18H17NO6 significantly up-regulated the protein expression levels of Bax,Cleaved Caspase-3,Cleaved Caspase-9 and significantly down-regulated the protein expression levels of Bcl-2;qRT-PCR showed that C18H17NO6 significantly down-regulated the mRNA expression of Bcl-2 and Caspase-3.(3)C18H17NO6 inhibited human osteosarcoma MNNG cell migration and invasionScratch test and Transwell chamber test showed that C18H17NO6 significantly down-regulated the migration and invasion ability of MNNG cells;Western blot showed that C18H17NO6 significantly down-regulated the protein expression levels of MMP-2,MMP-9,N-cadherin and Vimentin,and significantly up-regulated the protein expression level of E-cadherin;ELISA showed that C18H17NO6 significantly down-regulated the secretion of MMP-2,MMP-9 and VEGF.(4)C18H17NO6 inhibits PI3K/AKT signal pathway activityWestern blot showed that C18H17NO6 significantly down-regulated the protein expression of p-PI3K and p-AKT,but did not affect the protein expression of total PI3K and total AKT.(5)C18H17NO6 plays an anti-osteosarcoma role by inhibiting the PI3K/AKT signaling pathwayCCK-8 showed that the cell survival rate of MNNG cells was significantly higher than that of C18H17NO6 group when C18H17NO6 and 740Y-P were co-incubated;Flow Cytometry showed that the apoptosis rate of MNNG cells was significantly lower than that of C18H17NO6 group when okofuran and 740Y-P were co-incubated;Western blot showed that the upregulation of 740Y-P on MMP-2,MMP-9,N-cadherin,Vimentin,p-PI3K and p-AKT protein was significantly weakened by the co-incubation of C18H17NO6 and 740Y-P on MNNG cells;In the detection of protein expression level of E-cadherin,we obtained the opposite experimental results;LY294002 significantly reduced the protein expression level of p-PI3K and p-AKT;when C18H17NO6 and LY294002 co-incubated MNNG cells,the down-regulation of p-PI3K and p-AKT protein was enhanced.(6)C18H17N06 inhibited the growth of xenograft tumor in nude miceAccording to the time-tumor volume curve,the tumor volume of the experimental group was significantly smaller than that of the control group;according to the time-nude mouse weight curve,there was no significant change in the body weight of the experimental group;the liver and kidney function test showed that the ALT(U/L),AST(U/L)and CREA(μmol/L)levels of the experimental group were significantly lower than that of the control group.(7)C18H17NO6 inhibited tumor cell proliferation and induced apoptosisHE staining showed that compared with the control group,the cell structure of tumor tissue in the experimental group was unclear,nuclear pyknosis,hyperchromatism,division and necrosis rate were significantly increased;TUNEL staining showed that the number of apoptotic cells in tumor tissue in the experimental group was significantly higher than that in the control group;IHC staining showed that the protein expression of PCNA and Ki67 in tumor tissue in the experimental group was significantly lower than that in the control group.(8)C18H17NO6 inhibited the expression of EMT and PI3K/AKT-related proteinsC18H17NO6 significantly down-regulated the protein expression levels of p-PI3K,p-AKT,N-cadherin and Vimentin in the tumor,and up-regulated the protein expression levels of E-cadherin.(9)Positive regulation of C18H17NO6 on miR-16-5p expression in MNNG cellsThe results of the second generation high-throughput sequencing showed that the 12 miRNAs of MNNG cells treated with C18H17NO6 were significantly different(p<0.05);the expression level of miR-16-5p was significantly up-regulated by qRT-PCR,which was consistent with the sequencing results.(10)miR-16-5p function acquisition up-regulate the inhibition of C18H17NO6 on the proliferation of MNNG cellsCompared with the control group and the negative control group,the survival rate of MNNG cells in the 3μM C18H17NO6+LV-miR-16-5p mimics group was significantly down-regulated;the survival rate of MNNG cells in the 3μM C18H17NO6+LV-miR-16-5p inhibitor group was significantly up-regulated;Western blot showed that the protein expression level of Ki67 and PCNA were up-regulated in 3 μM C18H17NO6+LV-miR-16-5p inhibitor group.(11)miR-16-5p function acquisition up-regulate the apoptosis of MNNG cells induced by C18H17NO6Flow Cytometry showed that the apoptosis rate of 3 μM C18H17NO6+LV-miR-16-5p mimics group was significantly up-regulated,the apoptosis rate of 3μM C18H17NO6+LV-miR-16-5p inhibitor group was significantly down-regulated;Western blot showed the protein expression levels of Bax,Cleaved Caspase-3 and Cleaved Caspase-9 were up-regulated in 3μM C18H17NO6+LV-miR-16-5p mimics group;the protein expression levels of Bax、Cleaved Caspase-3 and Cleaved Caspase-9 were significantly down-regulated and Bcl-2 protein expression was up-regulated in 3μM C18H17NO6+LV-miR-16-5p inhibitor group.(12)miR-16-5p function acquisition up-regulate the inhibition of C18H17NO6 on invasion and migration of MNNG cellsScratch test and Transwell chamber test showed that the migation and invasion ability of MNNG cell in 3μM C18H17NO6+LV-miR-16-5p inhibitor group were significantly up-regulated;Western blot showed the protein expression of E-cadherin in 3μM C18H17NO6+LV-miR-16-5p mimics group was up-regulated;the expression of E-cadherin in 3μM C18H17NO6+LV-miR-16-5p inhibitor group was down-regulated,while that of N-cadherin and Vimentin was up-regulated.(13)miR-16-5p function acquisition up-regulate the inhibition of C18H17NO6 on PI3K/AKT signaling pathwayWestern blot showed the protein expression of p-AKT and p-PI3K in 3μM C18H17NO6+LV-miR-16-5p mimics group were down-regulated.[Conclusions](1)C18H17NO6 can inhibit the proliferation,invasion and migration of MNNG cells,induce cell apoptosis;C18H17NO6 exerts its anti-osteosarcoma effect by inhibiting the PI3K/AKT signaling pathway.(2)C18H17NO6 significantly inhibited xenograft tumor growth,but did not affect body weight and liver and kidney function.(3)C18H17NO6 can up-regulate the expression of miR-16-5p in MNNG cells,and miR-16-5p function acquisition can up-regulate the inhibition of C18H17NO6 on the proliferation,migration and invasion of MNNG cells and the ability of C18H17NO6 to induce apoptosis of MNNG cells. |
PDF Full Text Request