Downregulating NRARP Gene Expression Influences The Oncologic Behavior Of Anaplastic Thyroid Cancer

BackgroundAnaplastic thyroid carcinoma(ATC),which accounts for 7% ~ 8% of all thyroid carcinomas,has a high degree of malignancy and displays highly invasive behavior,with lymph node metastasis or distant metastases.Compared with well differentiated papillary carcinoma and follicular carcinoma,patients with ATC usually have a median survival of less than 1 year.At present,there is no effective treatment method for anaplastic carcinoma,and there is no uniform standard for treatment.Treatment options for ATC are mainly palliative because of the aggressive and resilient nature of the disease.Studies have shown no survival benefit from resection,radiation therapy,chemotherapy or combined surgical resection of tumor and radiotherapy in patients with ATC.It is the difficulty challenge of the medical cares toward ATC around the world.Although a serials of medical trails have been carried out to improve the outcome of ATC by resection,radiation therapy or chemotherapy,no uniform and effective treatment has been set out because the disease incidence was low and almost all the clinical trials contained small sample and retrospective data.It is of great need to start a prospective study with large number of cases.So it is very important to explore new treatment method for thyroid cancer chemotherapy.With the rapid development of molecular biology and immunology technology and the deepening of the human understanding of cancer development mechanisms,biotherapy has become the fourth treatment models.Target therapy including testing target gene,has showed good application values in the treatment of tumor and achieved a certain effect.Base on the characteristics of ATC,all the clinical center has been exploring new methods to cure ATC.Among them,target therapy is becoming a promising treatment of cancer.Notch signal pathway shows vital role in carcinoma development.Disorder Notch signal could directly or indirectly induce carcinoma development.And some studies confirmed that Notch signal pathway disorder existed in many malignant tumor,for example,prostatic carcinoma,cervical carcinoma,breast carcinoma and so on.Notch signal pathway also works in thyroid tissue,and its receptors are regulated by thyroid hormone.For instance,Notch expression was higher in thyroid papillary carcinoma and follicular carcinoma than normal.NRARP(NOTCH-regulated ankyrin repeat protein)is a key factor in Notch signaling pathway and may take part in the development of anaplastic thyroid carcinoma.NRARP gene as a member of Notch signal pathway encoded 114 amino acide protein residues,and possessed two Ankyrin repeats.NRARP can hinder the activiation of CBF-1 induced by Notch,promote the degradation of intracellular segment,and inhibit Notch signal pathway activation.In turn,over-expression of Notch gene could up-regulate NRARP,which indicating NRARP as a feedback mechanism could limit excessive Notch enhancement.There has not been any clinical or experimental study concerning the Notch and NRARP gene expression in ATC.This study explored the expression of NRARP gene in thyoid carcinoma or celllines,evaluated its role in cell proliferation,apoptosis,cell cycl,migration and invasion,which may provide experimental evidence to determine the key target gene for biotherapy.MethodFirst of all,we collected the tissue samples of anaplastic thyroid carcinoma(in total34 cases of anaplastic thyroid carcinoma patients).Immunohistochemistry was used to semiquantitatively detect NRARP protein levels of the specimens of anaplastic thyroid carcinoma.According to the IHC(immunohistochemistry)level,we divided the patients into high-expression group and low-expression group.And survival analysis of the outcome results was applied to compare the the two groups.Then we synthetized NRARP-shRNA lentivirus,which successfully knocked down NRARP expression of the two kinds of anaplastic thyroid carcinoma cells(BHT-101 and 8305C).WST-1 assay was applied to detect the in vitro tumor cell proliferation levels after NRARP gene knocked down by adding Lenti-NRARP-shRNA.Tumor bearing mice assay was applied to detect the in vivo tumor cell proliferation levels after NRARP gene knocked down by adding Lenti-NRARP-shRNA.Cell apoptosis and cell cycle were analysed by flow cytometry after NRARP gene knocked down by adding Lenti-NRARP-shRNA.Cell invasion was tested using matrigel invasion assay after NRARP gene knocked down by adding Lenti-NRARP-shRNA.In addition,expressions of several cell cycle related and apoptosis related proteins such as p21,cyclin D1,bax,bcl-2,caspase-3,and so on were examined using western blot after transfected with Lenti-NRARP-shRNA.We found that NRARP gene was expressed more in anaplastic thyroid cancer tissues than adjacent noncancerous tissue.Lenti-NRARP-shRNA could significantly inhibited tumor cell lines(BHT-101 and 8305C)proliferation in vitro and in vivo;Lenti-NRARP-shRNA could induce p21 expression,hinder cyclin D1 expression and finally induce cell cycle G1arrest;Lenti-NRARP-shRNA could induce the expression of bax,hinder the expression of bcl-2,activate caspase-3 and resulte in cell apoptosis;Lenti-NRARP-shRNA could attenuate cell migration and invasion by hindering expression of MMP-9.Result1.NOTCH and NRARP were highly expressed in anaplastic thyroid cancer tissues.The NRARP high-expression group observed poorer outcome than low-expression group,which suggested NRARP a potential target for therapy.2.Knockdown of NRARP could inhibit ATC growth in vivo or vitro;induce G1 arrest by promoting p21 expression,attenuating cyclin D1 expression;and it also induced apoptosis by promoting bax expression,suppressing bcl-2 expression and motivating caspase-3.In addition,knockdown of NRAPR inhibits thyroid cancer cell invasion by suppressing MMP-9 expression.ConclusionNRARP could participate in the development of anaplastic thyroid cancer,indicating that NRARP may serve as a potential target for anaplastic thyroid cancer targeted therapy.