| BackgroundOsteosarcoma is the most common primary malignant bone tumor,which mainly occurs in teenagers and young people.It has a high degree of malignancy,can be metastasized in the early stage,and the prognosis is very poor.The cure rate of simple surgical treatment is only 15% to 20%.Since the use of effective adjuvant chemotherapy or neoadjuvant chemotherapy in the 1970 s,especially the development of neoadjuvant chemotherapy combined with limb salvage surgery,the 5-year survival rate and quality of life of patients with osteosarcoma have been greatly improved,and the 5-year survival rate can reach 60%.However,about 30-40% of osteosarcoma patients are drug-resistant to chemotherapy,which is not only prone to tumor metastasis,but also can not improve the prognosis even if the tumor is removed completely.Therefore,we urgently need to further study the molecular mechanism of osteosarcoma and find new targets for the treatment of osteosarcoma.micro RNA(tiny RNA,miRNA)is a kind of endogenous non-coding nucleotides with about 18-25 nucleotides in length,which does not have an open reading frame.By combining with the 3’ or 5 ’non-coding region of the target m RNA,it forms a silent complex,hinders or even degrades the proteins translated by m RNA,thus regulating the expression of the target gene.Many studies have shown that various disorders of miRNA are closely related to the occurrence,progression and recurrence of tumors.Mi R-320 b is a kind of tiny RNA with anti-tumor effect,which is low-expressed in many malignant tumors,such as pancreatic cancer,breast cancer and gastrointestinal tumors.Overexpression of miR-320 b can inhibit the proliferation and metastasis of these tumors and reduce their chemotherapy resistance.At present,there are few studies on miR-320 b and osteosarcoma,so it is necessary to study its biological function in osteosarcoma and explore its specific molecular mechanism.STAT3,as a member of the STATs family,has recently been found to play an important role in the progression of different types of cancer.The activation of STAT3 can regulate a variety of gene functions in the process of cancer cell proliferation,differentiation,apoptosis,metastasis,inflammation,immunity,cell survival and angiogenesis.STAT3 plays an important role in tumorigenesis and development,chemotherapy resistance and radiation resistance.Inhibition of STAT3 activation has become a protein target for disease cancer therapy.By searching miRDB,Targetscan,Target Miner and other databases,it is predicted that STAT3 is the binding site of miR-320 b,but the interaction between them in osteosarcoma is not clear.Therefore,it is necessary to further explore the relationship between miR-320 b and STAT3 in osteosarcoma and to study the regulatory mechanism between them.ObjectiveThe purpose of this study is to explain the regulatory role of miR-320 b on the growth and metastasis of osteosarcoma,and to explore that miR-320 b can inhibit the further progress of osteosarcoma by targeting combined with STAT3,and provide a new target for the clinical diagnosis and treatment of osteosarcoma.Methods1.The relative expression RNA of miR-320 b in osteosarcoma and adjacent tumor tissues,osteosarcoma cells and normal osteoblasts was measured by fluoresceence quantitative PCR.2.miR-320 b minics was used to transfect osteosarcoma cells,and the impact of overexpression of miR-320 b on the proliferation of osteosarcoma cells was tested by CCK8 assay,plate cloning assay and Ed U assay.3.miR-320 b minics was used to transfect osteosarcoma cells,and the effect of overexpression of miR-320 b on the metastasis of osteosarcoma cells was detected by detecting invasion and migration-related proteins,wounding healing and Transwell experiments.4.The binding relationship between miR-320 b and STAT3 was predicted by miRDB,Targetscan and Target Miner,and verified by double luciferase experiment.5.At the same time,osteosarcoma cells were transfected with miR-320 b minics,p-STAT3 or miR-320 b inhibitor,sh-STAT3.The changes of STAT3 protein expression and the results of CCK8 and Transwell were observed,and whether miR-320 b affected the growth and metastasis of osteosarcoma through STAT3.6.Osteosarcoma cells overexpressing miR-320 b were used to observe the effect of miR-320 b on the growth and metastasis of osteosarcoma in nude mice.Result1.The results of q RT-PCR showed that the RNA expression level of miR-320 b in osteosarcoma tissues was markedly lower than that in adjacent tumor tissues,while as same in osteosarcoma cells and osteoblasts.2.miR-320 b minics can significantly increase the RNA expression of miR-320 b in osteosarcoma cells,and the proliferation of osteosarcoma cells with up-regulation of miR-320 b expression is significantly inhibited.3.Up-regulating the expression of miR-320 b in osteosarcoma cells significantly inhibited the metastatic ability of osteosarcoma cells.4.The results in miRDB database show that miR-320 b binds to STAT3,and the double luciferase experiment also proves that miR-320 b and STAT3 are directly targeted.5.Transfection of up-regulated plasmid STAT3 into osteosarcoma cells can reverse the decrease of STAT3 expression and the ability of growth and metastasis caused by up-regulation of miR-320 b,and vice versa.It is suggested that miR-320 b affects the growth and metastasis of osteosarcoma through STAT3.6.Animal experiments showed that when the expression of miR-320 b was up-regulated in osteosarcoma cells,the volume and weight of transplanted tumor in nude mice were markedly smaller than those in the control group,and the incidence of lung metastasis was also markedly decreased.ConclusionMi R-320 b can inhibit the growth and metastasis of osteosarcoma cells as an important regulatory factor in the occurrence and development of osteosarcoma.Its regulatory ability can be achieved by targeting combined with down-regulation of STAT3.Our results suggest that miR-320 b is a potential target for the treatment of osteosarcoma and provides a new idea for the treatment of osteosarcoma. |
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