Mechanism Of Cinobufotalin Inbibits Metastasis Of Nasopharyngeal Carcinoma Through The Regulation Of ENKUR

Background and ObjectivesNasopharyngeal carcinoma(NPC)is a highly invasive malignant tumor occurring in the top and side walls of nasopharyngeal cavity.In 2018,there were 129,000 new cases of NPC worldwide,half of which came from China and mainly distributed in South China.The 5-year survival rate of early stage NPC treated with radiotherapy is more than 95%.However,due to hided and diversified clinical manifestations of NPC,most patients are already in the middle and late stage when diagnosed,for which the mortality increase sharply.At present,the combination of radiation and chemical drugs is mainly used for advanced nasopharyngeal carcinoma.The chemotherapy regimen is cisplatin combined with 5-fluorouracil,but it is prone to drug resistance and serious adverse reactions.Toad venom has a number of effects including anti-cancer and anti-viral.As one of the main components of toad venom,it has been proved that cinobufotalin has obvious antitumor effect in liver cancer,colorectal cancer,prostate cancer,ovarian cancer and other malignant tumors.However,there are few reports on the the antitumor effect and mechanism of cinobufotalin in nasopharyngeal carcinomaContents and methods1.Effect of cinobufotalin on the function of nasopharyngeal carcinoma cells(1)Nasopharyngeal carcinoma cells were treated with cinobufotalin according to its IC50,and wound healing assay and transwell assay were performed to explore the effect of cinobufotalin on the migration ability of nasopharyngeal carcinoma cells in vitro.(2)To explore the effect of cinobufagin on the metastasis ability of nasopharyngeal carcinoma cells in vivo,cinobufagin alone,cisplatin alone,cinobufagin combined with cisplatin were used to treat mice with lung metastasis established by tail vein injection of nasopharyngeal carcinoma cells.2.Molecular mechanism of cinobufotalin inhibits metastasis of nasopharyngeal carcinoma(1)The effect of cinobufotalin on the expression of ENKUR mRNA was detected by qPCR.(2)The expression of ENKUR was detected in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells;The expression of ENKUR was detected in normal nasopharyngeal epithelial tissue and nasopharyngeal carcinoma tissue.(3)Wound healing assay and transwell assay were performed to explore the effect of ENKUR over-expressed or silenced on the migration ability of nasopharyngeal carcinoma cells.(4)ENKUR was silenced in the cells treated with cinobufotalin to verify whether cinobufotalin inhibits the migration of NPC cells through ENKUR.3.Study on ENKUR interacts with β-catenin to inhibit the EMT process in nasopharyngeal carcinoma cells(1)To investigate the effects of cinobufotalin,overexpression of ENKUR and silencing of ENKUR on the expression of EMT-related proteins in NPC cells.(2)ENKUR was silenced in the cells treated with cinobufotalin to verify whether cinobufotalin regulated the expression of EMT-related proteins through ENKUR.(3)The relationship between ENKUR and β-catenin was investigated by separation of nuclear and cytoplasmic proteins assay,CoIP and IF.4.Study on the mechanism of cinobufotalin regulates ENKUR(1)Overexpression of c-Jun in nasopharyngeal carcinoma cells to explore the effect of c-Jun on the expression of ENKUR.(2)The binding sites of c-Jun in the ENKUR promoter region were predicted,and the predicted results were verified by ChIP-qPCR and DNA agarose gel electrophoresis.(3)Nasopharyngeal carcinoma cells were treated with cinobufotalin and PI3K inhibitors to verify whether cinobufotalin regulated ENKUR expression through the PI3K/AKT/c-Jun signaling pathway.Result1.Cinobufotalin significantly inhibits the migration of nasopharyngeal carcinoma cells in vitro and in vivo.2.Cinobufotalin inhibits the metastasis of nasopharyngeal carcinoma cells by regulating the expression of ENKUR.3.ENKUR inhibits EMT in nasopharyngeal carcinoma cells by interacting with β-catenin.4.Cinobufotalin regulates the expression of ENKUR through PI3K/Akt/c-Jun signaling pathway.ConclusionCinobufotalin promotes the expression of ENKUR through PI3K/Akt/c-Jun signaling pathway.ENKUR interacts with β-catenin to inhibit the EMT process,thereby inhibiting the metastasis of NPC.