Study On The Mechanism Of MiR-218-2/KDM1A/Hippo Signaling Pathway On The Biological Behavior Of Thyroid Cancer Cell

Thyroid cancer is one of the most common malignancies in the endocrine system.In the past few decades,the prevalence of thyroid cancer has increased significantly worldwide.There are four main types of thyroid cancer:papillary thyroid cancer,follicular thyroid cancer,undifferentiated thyroid cancer,and medullary thyroid cancer.Among them,papillary thyroid cancer accounts for more than 80%of adult and child,follicular thyroid cancer.Occupy 20%are prone to middle-aged people,the prognosis of both is relatively good,while undifferentiated thyroid cancer accounts for 15%,medullary thyroid cancer accounts for 3%,and the prognosis is poor.In recent decades,the incidence of thyroid cancer in China has multiplied,and the prevalence of young women is 2-5 times that of men,but its prognosis has not improved significantly.With the continuous development of molecular biology,the exploration of miRNAs in the treatment of diseases continues to yield results,especially cancer treatment.To investigate the role of miR-218-2 in the regulation of thyroid cancer development via the miR-218-2/KDM1A/hippo signaling pathway,we analyzed the expression of miR-218-2 in thyroid carcinoma tumor tissues and cells in this study.And carried out a series of cell and animal experiments to reveal the effect of abnormal expression of miR-218-2 on the biological behavior(proliferation,migration,invasion and apoptosis)and tumor formation of thyroid cancer cells,and to explore the mechanism of action of miR-218-2 in the development of thyroid carcinoma,which is the thyroid gland.Early diagnosis and clinical treatment of cancer provide new biological targets.Part ?:Effect of miR-218-2 on the biological behavior of thyroid cancer cellsMethods1.Real-time quantitative reverse transcription polymerase chain reaction was used to detect the expression of miR-218-2 in thyroid carcinoma tissues and cells.2.CCK8,Transwell chamber,flow cytometry assay for proliferation,migration,invasion and apoptosis of thyroid cancer cells overexpressing miR-218-23.Western blotting detection of overexpression of miR-218-2 on thyroid cancer cell proliferation,migration,invasion and apoptosis-related protein expressionResults1.Real-time quantitative reverse transcription polymerase chain reaction showed that the expression level of miR-218-2 in tumor tissues and cells of thyroid cancer patients was significantly lower than that of adjacent tissues and thyroid follicular epithelial cells.2.CCK8,Transwell chamber,flow cytometry analysis showed that overexpression of miR-218-2 significantly inhibited the proliferation,migration,invasion and apoptosis of thyroid cancer cells.3.Western blot results showed that overexpression of miR-218-2 inhibited the expression of proliferation-related proteins PCNA,CyclinEl and migration-invasive proteins FN and MMP-9,and promoted the expression of cleaved-PARP and cleaved-caspase-3..Conclusion1.miR-218-2 is lowly expressed in thyroid cancer.2.Overexpression of miR-218-2 inhibits proliferation,migration and invasion of thyroid cancer cells and promotes apoptosis.3.Overexpression of miR-218-2 inhibited the expression of PCNA,CyclinEl,FN and MMP-9,and promoted the expression of cleaved-PARP and cleavedcaspase-3.Part ?:miR-218-2 regulate the biological behavior of thyroid cancer cells throug targeting KDM1AMethod1.Real-time quantitative quantitative reverse transcription polymerase chain reaction and immunoblotting were used to detect the expression level of KDM1A in thyroid cancer cells and thyroid cancer cells overexpressing miR-218-2.2.Bioinformatics analysis,dual luciferase reporter assay and RIP verification of the interaction of miR-218-2 with KDM1A.3.CCK8,Transwell chamber,flow cytometry,knockdown and KDM1A thyroid cancer cell proliferation,migration,invasion and apoptosis4.Western blotting was used to detect the expression of KDM1A in proliferation,migration,invasion and apoptosis of thyroid cancer cells.Result1.Overexpression of miR-218-2 can down-regulate the expression level of KDM1A in thyroid cancer cells.2.KDM1A is highly expressed in thyroid cancer cells.3.KDM1A knockdown can inhibit the proliferation,migration and invasion of thyroid cancer cells,promote apoptosis,inhibit the expression of PCNA,CyclinEl,FN and MMP-9,and promote the expression of cleaved-PARP and cleaved-caspase-3.4.Bioinformatics prediction,dual luciferase reporter and RIP analysis showed that miR-218-2 interacts with KDM1A via a complementary binding site.5.Overexpression of KDM1A can reverse the overexpression of miR-218-2 on proliferation,migration,invasion and apoptosis of thyroid cancer cells and its effects on PCNA,CyclinE1,FN,MMP-9,cleaved-PARP,cleaved-caspase-3 Regulation of protein expression.Conclusion1.KDM1A is highly expressed in thyroid cancer cells,and KDM1A knockdown can inhibit the proliferation,migration and invasion of thyroid cancer cells,promote apoptosis,and also regulate PCNA,CyclinE1,FN,MMP-9,cleaved-PARP,cleaved-caspase-3 protein expression.2.KDM1A is a target gene of miR-218-2.3.Overexpression of KDM1A reverses the regulation of miR-218-2 on proliferation,migration,invasion and apoptosis of thyroid cancer cells.Part III:miR-218-2/KDM1A/hippo signaling pathway regulates biological behavior of thyroid cancer cells and tumor growthMethods1.Real-time quantitative reverse transcription polymerase chain reaction(RT-PCR)and immunoblotting were used to detect the expression of the key genes of the hippo signaling pathways MST1 and YAP in thyroid cancer cells and thyroid cancer cells overexpressing miR-218-2 and knocking down KDM1A.2.CCK8,Transwell chamber,flow cytometry,western blotting detection of proliferation,migration,invasion and apoptosis of thyroid cancer cells inhibiting hippo signaling pathway and expression of related proteins.3.Nude mouse tumor formation assay to detect the effect of overexpression of miR-218-2 on tumor growth.Result1.Transition expression of miR-218-2 and knockdown of KDM1A can up-regulate MST1 and down-regulate YAP.2.In the thyroid cancer cells,the key gene of the hippo signaling pathway,MST1,is down-regulated and YAP is up-regulated.3.Inhibition of hippo signaling pathway promotes proliferation,migration and invasion of thyroid cancer cells,inhibits apoptosis,up-regulates protein expression of PCNA,CyclinE1,FN and MMP-9,and down-regulates protein expression of cleaved-PARP and cleaved-caspase-3.4.Transitional expression of miR-218-2 can inhibit tumor growth in nude mice,down-regulate the expression levels of KDM1A and YAP in tumors,and up-regulate the expression level of MST1.ConclusionmiR-218-2 can down-regulate KDM1A in combination with KDM1A to activate hippo signaling pathway,inhibit proliferation,invasion and migration of thyroid cancer cells,promote apoptosis,inhibit tumor growth in nude mice,and exert tumor"inhibitor" function.