Analysis Of The Inhibitory Effect Of 125I Radioactive Seeds On The Proliferation Of Human Chondrosarcoma Cells And Its Related Mechanism

Introduction:Chondrosarcoma is a common malignant bone tumor,accounting for about 9.2%of all primary malignant bone tumors,second only to osteosarcoma.The average age of onset is about 50 years old,slightly more in males than in females(55%vs 45%).Chondrosarcoma includes a variety of subtypes,of which traditional/classical chondrosarcoma accounts for about 85%.Other subtypes include dedifferentiated type,clear cell type,paracortical type,mucous type,mesenchymal chondrosarcoma and malignant chondroblastoma.The 5-year survival rate of traditional chondrosarcoma is about 70%.Extensive surgical resection is the main treatment at present.Because of rich tumor matrix and lack of blood supply,chemotherapy and radiotherapy are not sensitive to chondrosarcoma.Radiotherapy can be suggested when the tumor is located in the skull base,sacrum and other unresectable sites or when the tumor margin is positive after operation.¹²⁵I radioactive seed implantation is a kind of brachytherapy,which has been widely used in treatment of prostate cancer,pancreatic cancer,liver cancer,lung cancer,head and neck cancer,metastatic lymph nodes and other malignant tumors.however,there are few basic and clinical studies on primary malignant bone tumors,and the literature on the treatment of chondrosarcoma with ¹²⁵I seed implantation is even more rare.Up to January2021,we searched China knowledge Network,Wanfang Database and Pubmed with the subject words"¹²⁵I seed AND chondrosarcoma",only 3 literature reports were found,one of which was based on the results of this experimental study,and the other two were clinical reports.The author of one article reoperated on a case of postoperative recurrence of low-grade traditional chondrosarcoma of the vertebral body.During the operation,marginal resection of the tumor was performed,and ¹²⁵I radioactive seeds were implanted in the tumor bed for adjuvant treatment.There was no tumor recurrence during the two years follow-up.In the other article,the authors reported that 5 cases of recurrent spinal chondrosarcoma were treated with ¹²⁵I radioactive seed implantation.The patients were followed up for 15.2 months(2 to 41 months).The postoperative pain was significantly improved,the effective rate of analgesia was 100%,and the local control rate of tumor in1 year was 60%.These two clinical reports seem to provide an effective treatment for patients with chondrosarcoma who cannot be surgically removed widely.?-rays released by ¹²⁵I radioactive seeds can not only cause DNA breakage and cell necrosis in many kinds of malignant tumor cells,but also inhibit cell proliferation and promote cell apoptosis.however,there are no related reports on the effects on chondrosarcoma growth,cell proliferation and apoptosis.Therefore,we designed a new ¹²⁵I radioactive seed irradiation model in vitro to study the effects of ¹²⁵I radioactive seed irradiation on the proliferation,invasion,metastasis and apoptosis of chondrosarcoma cells,and then established a human chondrosarcoma SW1353 nude mouse tumor model to further clarify the effect of ¹²⁵I radioactive seeds on the tumor after implantation of ¹²⁵I radioactive seeds into chondrosarcoma.Finally,the miRNA expression profile was analyzed,and the miRNAs-mRNAs regulatory network was comprehensively analyzed and verified,which lays a foundation for revealing the mechanism of ¹²⁵I seed implantation in the treatment of chondrosarcoma and provides a theoretical basis for clinical treatment.Purpose:1.Evaluation of the effect of ¹²⁵I radioactive particles on SW1353chondrosarcoma cells.2.Evaluation of the effect of ¹²⁵I radioactive particles on transplanted tumor in nude mice.3.To explore the mechanism of ¹²⁵I radioactive particles on chondrosarcoma.4.To provide theoretical basis and reference for clinical application of ¹²⁵I seeds in the treatment of chondrosarcoma and its further study.Methods:1.Establishment of in vitro irradiation model of ¹²⁵I radioactive seeds.2.¹²⁵I radioactive seeds model was used to irradiate human chondrosarcoma cells in vitro.3.The cell viability was determined by MTT method and the cell growth curve was drawn.4.The proliferation,migration,invasion and apoptosis of human chondrosarcoma cells were analyzed by clone formation test,cell scratch test,transwell test and TUNEL test.5.The transplanted tumor model of SW1353 human chondrosarcoma in nude mice was established.6.After the successful construction of the model,seeds were implanted into the transplanted tumor to evaluate the effect of ¹²⁵I radioactive seeds on the growth of transplanted tumors in nude mice,and the effects of ¹²⁵I radioactive seeds on tumor tissue necrosis,proliferation and apoptosis were evaluated by h E staining and immunohistochemistry.7.TheRNA of SW1353 cells irradiated by 125I radioactive seeds was extracted and and the differentially expressed miRNAs was analyzed by bioinformatics analysis technology,and the mRNA,regulated by it was predicted to find out the miRNAs and mRNA related to the experimental results and verified by experiments.8.To determine the key miRNA,for the increase of apoptosis of human chondrosarcoma cells irradiated by ¹²⁵I radioactive seeds,the cells transfected with miRNA-specific mimic NC,mimic,inbibitor NC and inbibitor were studied on proliferation,invasion,migration and apoptosis,and the effect of key miRNA on human chondrosarcoma was further determined.9.TheRNA of transfected SW1353 cells was extracted and the transcriptional group was sequenced,bioinformatics analysis and experimental verification to find out the differentially expressed target genes.10.Using real-time PCR,Western Blot and other experimental methods to further verify the selected differentially expressed target genes,and to clarify some of the mechanisms of proliferation,invasion,migration and apoptosis of human chondrosarcoma cells irradiated by ¹²⁵I radioactive seeds.Results:1.The effects of ¹²⁵I radioactive seed irradiation on the proliferation,invasion,migration and apoptosis of human chondrosarcoma cells.The main results were as follows:(1)The growth of SW1353 and CAL78 cells became unstable within 2 days after exposure to 2 Gy total radiation,and the percentage of cell growth inhibition increased gradually from day 2 to day 7.When SW1353 and CAL78 cells were exposed to a total dose of 4 Gy radiation,the inhibition of cell growth increased gradually from day 1 to day 7 after exposure.however,the growth inhibition of SW1353 cells in 6 Gy and 8 Gy groups occurred almost instantly after irradiation,and the growth of SW1353 cells was significantly inhibited from the first day after irradiation.The percentage of cell growth inhibition increased with the increase of radiation dose from 6 Gy to 8 Gy,and reached the highest value on the 7th day after exposure.On the7th day after irradiation,the growth inhibition rates of SW1353 cells exposed to the total dose of 2,4,6 and 8 Gy radiation were 14.64%,17.25%,22.41%and 26.39%,respectively,and the growth inhibition rates of 12.30%,15.25%,24.08%and 28.39%of CAL78 cells were 12.30%,15.25%,24.08%and 28.39%,respectively(P<0.05).(2)Compared with the normal control group,the 24-hour cell migration rate of SW1353 and CAL78 cells in the experimental group exposed to 6 Gy total radiation dose was significantly lower than that in the control group(P<0.05).The migration rate of SW1353 cells exposed to a total dose of 6 Gy radiation was 44.67%.The migration rate of trained CAL78 cells was 31.0%.(3)Compared with the normal control group,the number of SW1353 cells and CAL78 cells passing through the basement membrane of Transwell chamber covered with Matrigel matrix glue in the experimental group exposed to 6 Gy total radiation dose was significantly decreased,and the cell invasiveness was significantly decreased(P<0.05).(4)The results of TUNEL experiment showed that apoptosis occurred in some cells in the experimental group,showing abnormal manifestations such as nuclear fragmentation,cavity,DNA fragmentation and so on.The apoptosis rate of SW1353 and CAL78 cells in the control group was 0.33%,while that of SW1353 cells in the experimental group was 25.21%,and that of CAL78 cells in the experimental group was15.03%.Compared with control group,the expression of Bcl-2 in SW1353 cells and CAL78 cells in the experimental group exposed to 6 Gy total radiation dose decreased significantly,while the expression of Bax and cleaved caspase-3 had no significant difference,while the expression of cleaved caspase-9 increased significantly,and the change trend of the two cells was the same.2.The effect of ¹²⁵I radioactive seeds on tumor growth,cell proliferation and apoptosis after implantation of SW1353 human chondrosarcoma in nude mice.The main results were as follows:(1)¹²⁵I radioactive seed implantation had obvious inhibitory effect on the growth of human chondrosarcoma transplanted tumor in nude mice.15 days after seed implantation,the average tumor volume growth rate of the experimental group and the control group was 8.81%and 391.5%,respectively.(2)15 days after ¹²⁵I radioactive seed implantation,the nude mice in the experimental group and the control group were killed by cervical dislocation and weighed after peeling off the tumor.The tumor weight of the experimental group was significantly smaller than that of the control group,and the difference was statistically significant(P<0.05).The TIR of transplanted tumor of SW1353 nude mice treated with¹²⁵I radioactive seeds was 80.83%.(3)Microscopic observation after h E staining showed that the pathological mitosis of chondrosarcoma cells in the experimental group treating with ¹²⁵I seed was less than that in the control group,and the tumor necrosis rate was 5%-20%in the experimental group and 0%-1%in the control group,and the difference was statistically significant(P<0.05).(4)After immunohistochemical staining,the positive rate of Ki-67 in the experimental group was about 31%,and that in the control group was about 54%.There was difference between the two groups,P<0.05.The expression of caspase-9 in the experimental group treating with ¹²⁵I seed was higher than that in the control group,while the expression of Bcl-2 in the experimental group treating with ¹²⁵I seed was lower than that in the control group.¹²⁵I radiation particles could promote the expression of pro-apoptotic protein caspase-9 and inhibit the expression of apoptosis inhibitory protein Bcl-2 in chondrosarcoma SW1353 cells.3.The changes of miRNAs in SW1353 human chondrosarcoma cells irradiated by¹²⁵I radioactive seeds and the verification results.(1)A total of 2549 miRNA were identified by microarray analysis of SW1353 cells exposed to 6 Gy radiation.Compared with the control group,there were 189differentially expressed miRNA including 98 up-regulated miRNAs and 91down-regulated miRNAs.The cluster histogram showed the different miRNA expression profiles between the irradiated group and the control group,and the heat map showed the first 15 differentially expressed miRNA between the irradiated group and the unirradiated group.(2)Target Scan,PITA and miRNA.org databases were used to predict related miRNA target genes.We selected the intersection of three databases for target gene prediction,however,only three miRNA(miR-1224-5p,miR-492 and miR-135b-5p)had target genes in the intersection analyzing by bioinformatics and verification.According to the KEGG database,we identified the first 20 important pathways to predict the target gene,constructed the PPI network for predicting the target gene by STRING,and visualized it by Cytoscape.Through screening,a total of 299 essential genes were found.In addition,the whole PPI network was analyzed by MCODE,and the results showed that these genes had more interactions than the less studied genes in the network.These key genes mainly include VEGFA,MAPK1,CUL3,h NRNPA1 and PPP1CC.(3)In view of the fact that the target genes predicted by miRNA sequence complementarity may lead to false positive results,we carried out three q RT-PCR experiments on 10 miRNA and 10 predicted target genes based on the results of microarray,pathway and PPI network analysis.The results showed that 70%(7/10)of the genes we verified were consistent with the expression trend of microarray analysis.Among the 10 miRNA selected for verification,the expression levels of miR-4689,miR-6076 and miR1469 were not consistent with the results of microarray analysis,but the difference was not significant.4.The mechanism of miRNAs regulating the proliferation,migration,invasion and apoptosis of human chondrosarcoma cells after ¹²⁵I radiation.(1)In order to further study the effects of miR-6839-5p on the proliferation,migration,invasion and apoptosis of chondrosarcoma cells,SW1353 and CAL78 human chondrosarcoma cells were transfected with miR-6839-5p mimic NC and miR-6839-5p mimic for MTT,clone formation,scratch,Transwell and Tunel assays.The results showed that SW1353 and CAL78 cells transfected with miR-6839-5p mimic were compared with normal control cells.The proliferation and migration ability of SW1353and CAL78 cells in the experimental group were significantly lower than those in the control group.Compared with the normal control group,the number of cells passing through the basement membrane of the Transwell chamber covered with Matrigel matrix glue and the invasiveness of the cells in the experimental group were significantly lower than those in the control group.Compared with the control group,apoptosis occurred in some cells in the experimental group,showing abnormal manifestations such as nuclear fragmentation,cavity,DNA fragmentation and so on.Compared with the control group transfected with miR-6839-5p mimic,the expression of Bcl-2 in SW1353 cells and CAL78 cells transfected with miR-6839-5p mimic NC decreased significantly,while the expression of Bax and cleaved caspase-3 had no significant difference,while the expression of cleaved caspase-9 increased significantly,and the change trend of the two cells was the same.(2)The control group(M1)transfected with miR-6839-5p mimic NC and the experimental group(M2)transfected with miR-6839-5p mimic were screened by edge R software.A total of 253 differentially expressed genes were identified by transcriptome analysis,including 105 up-regulated genes and 148 down-regulated genes.The cluster histogram showed the different gene expression profiles between M2 group and M1group,and the heat map showed the differentially expressed genes between irradiated group and unirradiated group.In order to find the target genes regulated by miR-6839-5p,we crossed the 148 down-regulated differentially expressed genes identified by transcriptome with the predicted results of Target Scan,miRWalk or miRDB,and found23 down-regulated target genes.(3)The control group(M1)transfected with miR-6839-5p mimic NC and the experimental group(M2)transfected with miR-6839-5p mimic were sequenced and analyzed by transcriptome.Furthermore,eight possible target genes were selected from SW1353 cells transfected with miR-6839-5p mimic and verified by q RT-PCR experiment.The results showed that among the 8 target genes identified by selection,the expression of BST2,VEGFA,FPR3 and PPARA was significantly down-regulated,while miR-6839-5p inhibitor restored or partially restored the expression of these genes in SW1353 and CAL78 cells.Conclusion:1.¹²⁵I radioactive seed irradiation can inhibit the proliferation,invasion and migration of human chondrosarcoma cells and promote the apoptosis of human chondrosarcoma cells.2.¹²⁵I radioactive seeds implanted into human chondrosarcoma xenografts in nude mice can inhibit tumor growth by inhibiting the proliferation of tumor cells and promoting their apoptosis and necrosis.3.After ¹²⁵I radioactive seeds irradiate human chondrosarcoma SW1353 cells and CAL78 cells,miR-6839-5p may exert anti-tumor effect on SW1353 human chondrosarcoma cells through targeted regulation of BST2,VEGFA,FPR3 and PPARA.MiR-6839-5p may be one of the targets for the treatment of chondrosarcoma.