TRPC5 Regulates The Multifocality And Progression Of Papillary Thyroid Cancer

Background: Thyroid Carcinoma(TC),the main pathological type of which is Papillary Thyroid Carcinoma(PTC),is one of the most frequent endocrine system malignancy in the world.Most PTC can be cured by surgery,but there are problems such as surgical injury,complications and lifelong medication.More and more voices believe that regular follow-up and non-surgical treatment can be used for PTC with low malignancy degree.However,due to the failure to stratify the malignancy degree of PTC before surgery,no consensus has been reached on the non-surgical treatment plan.Therefore,stratification of malignancy of PTC is currently a bottleneck problem in the diagnosis and treatment of PTC.It is of great significance to study new molecular markers or mechanisms conducive to the stratification of malignancy.Multifocal PTC is a type of PTC with multiple isolated cancer foci,which is considered to be more malignant than unifocal PTC by guidelines and some clinical studies.TRPC5 is a non-selective calcium channel that we found and verified to be highly expressed in multifocal PTC and is closely related to the progression and metastasis of PTC,but its specific role and mechanism remain unclear.Objective: From the perspective of multifocal PTC,this study explored the internal mechanism promoting malignant progression of PTC and provided a new basis for malignant risk stratification of PTC.Methods:1.The clinicopathologic data of PTC patients were retrospectively collected.According to the number and distribution of cancer foci in the pathological reports,PTC patients were divided into multifocal PTC group(including bilateral multifocal PTC and unilateral multifocal PTC)and unifocal PTC group,and the differences and similarities of demographic and clinicopathological characteristics of patients in different groups were analyzed and compared.2.By using the TCGA database,limma analysis was used to screen out the differential genes of PTC tissue and paracancer thyroid tissue,multifocal PTC tissue and single PTC tissue,N1 stage PTC tissue and N0 stage PTC tissue,and the intersection of the differential genes of each group was used to obtain the common differential genes.Then,GEO database was used to verify the expression of codifferential genes.3.Differential expression of TRPC5 was verified in tissue samples by q RT-PCR,Western blot and immunohistochemical tests,and the correlation between Tr PC5 expression and clinicopathological features was studied.4.Western blot assay was used to verify the differential expression of TRPC5 in PTC cell lines and normal thyroid cell lines.5.The stable transfection cell lines with TRPC5 knockdown were constructed by lentivirus transfection and verified by Western blot assay.6.The effects of TRPC5 on the proliferation,migration and invasion of PTC cells were verified in vitro by CCK-8 cell proliferation assay,plate cloning assay,cell scratch assay,and Transwell migration and invasion assay.7.Subcutaneous tumor formation model and foot pad-popliteal lymph node metastasis model were established in nude mice to verify the effects of TRPC5 on PTC tumor growth and lymph node metastasis.8.Fluo-4 AM was used to detect the effect of TRPC5 knockdown on calcium ion concentration in PTC cells,and calcium ion carrier A23187 was used to restore the calcium ion concentration in TRPC5 cells.9.CCK-8 cell proliferation assay,plate cloning assay,cell scratch assay,Transwell migration and invasion assay were used to verify the effects of intracellular calcium ion concentration changes on the proliferation,migration and invasion of PTC cells,respectively.10.Measurements of glucose uptake,ATP production,lactic acid production and supernatant PH were used to investigate the effect of calcium ion concentration changes on glycolysis levels in cells.Results:1.Patients with multifocal PTC are older,more prone to CLNM and LLNM,more metastatic lymph nodes,more prone to ETE,and have a higher proportion of BRAFV600 E gene mutations.Bilateral multifocal PTC has larger tumor diameter,is more prone to CLNM and LLNM than unilateral multifocal PTC,and the number of metastatic lymph nodes is higher,but the incidence of AIT is lower.Patients with unilateral multifocal PTC are older,more likely to develop LLNM and have a higher proportion of BRAFV600E gene mutations than those with unifocal PTC.2.The expression of TRPC5 in PTC was higher than that in paracancer thyroid tissue,and in multifocal PTC was higher than that in unifocal PTC.Meanwhile,PTC with high expression of TRPC5 was more malignant.After TRPC5 knockdown,the proliferation,migration and invasion ability of PTC cells were decreased,and the tumor growth and lymph node metastasis ability were decreased.3.After TRPC5 knockdown,the intracellular calcium concentration decreased,and A23187 could restore the calcium concentration of the knockdown cells,and the proliferation,migration and invasion ability of the cells were also restored.The glycolysis function of cells was decreased after TRPC5 knockdown,and the glycolysis function was restored after A23187 knockdown calcium ion.Conclusion: Multifocal PTC has a higher degree of malignancy,and the high expression of TRPC5 is closely related to the clinicopathologic features of PTC,including multifocal features.TRPC5 affects the glycolysis level of PTC cells by regulating the transport of calcium ions,thus promoting the proliferation and metastasis of PTC.