| Objective: Osteosarcoma,as a kind of malignant tumor,is a serious threat to human health.At present,the clinical treatment of osteosarcoma mostly adopts surgery,chemotherapy and other comprehensive treatment methods.Although the five-year survival rate of osteosarcoma patients has improved in recent years,but the clinical treatment effect is still unsatisfactory.High dose chemotherapy can bring serious side effects and multidrug resistance to patients,which makes it difficult to cure osteosarcoma completely.As the focus of current research,gene therapy may bring new hope to patients and become an effective treatment in the future.The research shows that the decrease of malignant proliferation and death of tumor cells is closely related to the occurrence and development of tumor,and makes the cells resistant to radiotherapy and chemotherapy.Therefore,inducing tumor cell death has become a new strategy of tumor cell killing therapy.miRNA is a new kind of non coding microRNA developed in recent years.More and more studies show that miRNA participates in many important biological processes.miRNA can be used as oncogene to down regulate the activity of tumor suppressor gene,and can also be used as tumor suppressor gene to down regulate the activity of proto oncogene.Autophagic cell death is a kind of programmed cell death which is different from apoptosis and does not depend on caspase.Research shows that autophagy inhibition plays an important role in the development of tumor.Silencing the autophagy gene Beclin1 can lead to the occurrence of a variety of malignant tumors.Therefore,Beclin1 is considered as a candidate tumor suppressor gene.At present,scholars have reported that miR-664 is highly expressed in liver cancer,and gene chip technology shows that miR-664 is abnormally expressed in osteosarcoma,and there is a strong correlation between miR-664 and Beclin1 gene through biological pathway analysis.In this study,the expression of Beclin1 protein targeted by miR-664 in osteosarcoma and its effect on autophagy and chemosensitivity of osteosarcoma cells were analyzed.Methods:1.From April 2013 to December 2015,51 cases of osteosarcoma were selected,and the negative paracancerous tissues were used as the control group.The expression of miR-664 and Beclin1 mRNA in osteosarcoma and its paracancerous normal tissues were detected by q RT-PCR.The relationship between the expression of miR-664 and Beclin1 mRNA and the clinicopathological data and the overall survival time were analyzed.2.The targeting relationship between miR-664 and Beclin1 was detected by luciferase reporter assay.miR-664 inhibitor and miR-NC were transfected into SAOS-2 and MG-63 cells respectively by cultivating SAOS-2 and MG-63 cells.Cell proliferation was detected by CCK-8 TEST,apoptosis was detected by Flow Cytometry(FCM),and cell migration was detected by Transwell TEST.The fluorescence intensity of cells in the blank control group,the miR-NC group and the miR-664 inhibitor group was analyzed by MDC method.Results: 1.The relative expression of miR-664 in osteosarcoma was significantly higher than that in adjacent normal tissues(P<0.05),and the relative expression of Beclin1 mRNA in osteosarcoma was significantly lower than that in adjacent normal tissues(P<0.05).The expression of miR-664 in osteosarcoma was not related to gender,age and tumor size(P>0.05),the expression of miR-664 was related to the stage of Enneking and distant metastasis(P<0.05).The expression of Beclin1 mRNA was not related to the sex,age and tumor size(P>0.05),however,it was related to Enneking stage,distant metastasis and Beclin1 mRNA expression in tumor tissue(P<0.05).The overall survival of patients with high expression of miR-664 was significantly worse than that of patients with low expression of miR-664(P<0.05).The overall survival of patients with high expression of Beclin1 mRNA was significantly better than that of patients with low expression of Beclin1 mRNA(P<0.05).2.By luciferase double luciferase reporter gene system detection,it was found that miR-664 inhibitor could improve luciferase activity,but had no significant effect on the recombinant plasmid of mut UTR.There was significant statistical difference between the two groups,indicating that miR-664 could combine with the seed sequence of 3 '-UTR region of Beclin1 gene and inhibit gene expression.3.Compared with miR-NC control group,after miR-664 was down-regulated,the expression level of Beclin1 gene was significantly higher(P<0.05).On the protein level,compared with miR-NC control group,the expression level of Beclin1 in miR-664 inhibitor group was significantly higher.4.CCK-8 test results showed that the absorbance value of cells in each group at OD490 was detected.Compared with miR-NC group and blank control group,the proliferation activity of miR-664 mice group was significantly reduced(P<0.05),but there was no significant difference between miR-NC group and blank control group at OD490 of Saos-2 and MG-63 cells(P>0.05).5.The apoptosis rate of osteosarcoma cells in miR-664 inhibitor group was significantly higher than that in the blank control group and miR-NC group(P<0.05),but there was no significant difference between the blank control group and miR-NC group(P>0.05).6.Transwell test was used to detect the migration and invasion of Saos-2and MG-63 cells in miR-664,miR-NC and blank control groups.The number of SAOS-2 and MG-63 cells in the miR-NC group was similar to that in the blank control group,while the migration rate of SAOS-2 and MG-63 cells in the miR-664 inhibitor group was significantly inhibited,showing significant statistical differences between the three groups(P<0.05).7.The invasion ability of SAOS-2 and MG-63 cells was detected by scratch test.The cells in blank control group and miR-NC group had similar migration ability,while the migration ability of miR-664 inhibitor group was significantly inhibited.8.MDC fluorescent dye,as a tracer of autophagic vesicles,distributed in the interior of cells in a green dot structure.The fluorescence intensity of blank control group and miR-NC group was similar,and the expression was relatively weak,while that of miR-664 inhibitor group was the strongest.The fluorescence intensity of miR-664 inhibitor group was significantly stronger than that of the blank control group and miR-NC group,the difference was statistically significant(P<0.05),while the fluorescence intensity of miR-NC group was similar to that of the blank control group,the difference was not statistically significant(P>0.05).Conclusion: The relative expression of miR-664 in osteosarcoma tissue is significantly up-regulated,and Beclin 1 mRNA is significantly decreased.The expression of miR-664 and Beclin 1 mRNA is related to tumor stage,metastasis and prognosis;miR-664 and Beclin 1 protein have a targeted relationship,and miR-664 inhibitor is transfected into osteosarcoma cells SAOS-2 and MG-63 can effectively inhibit the proliferation,apoptosis and migration of osteosarcoma cells.At the same time,it may promote the autophagy of tumor cells by targeting the expression of Beclin 1 gene,and then play a role in tumor inhibition.miR-664 can be one of the potential targets of osteosarcoma treatment. |
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