Screening Of Differential Genes And Signaling Pathways For NSUN6 To Inhibit The Proliferation Of Papillary Thyroid Cancer Cells And A Study Of Its Effect Of Inhibiting Proliferation

Objective:The incidence of thyroid cancer has been increasing year by year in the past decade,and 75%-85%of them are diagnosed as Papillary Thyroid Cancer(PTC).Given that the results of the previous bioinformatics analysis revealed that NSUN6 was differentially expressed in papillary thyroid cancer,i.e.,the expression in cancerous tissues was significantly lower than that in paracancerous tissues,it was speculated that NSUN6 might have an inhibitory effect on the proliferation of papillary thyroid cancer.The aim of this work was to investigate the inhibitory effect of RNA methyltransferase NSUN6 on the proliferation of papillary thyroid carcinoma,and to screen the genes or signaling pathways associated with the inhibitory effect of NSUN6 on the proliferation of papillary thyroid carcinoma,so as to provide new ideas for the next in-depth study of the mechanism of action of NSUN6,and to provide new directions for the future treatment of papillary thyroid carcinoma.Methods:Firstly,single gene and prognostic analysis of NSUN6 in several cancer types was performed using online bioinformatics tools Oncomine,TIMER 2.0,Kaplan-Meier Plotter,GEPIA,Prognoscan,and UALCAN to understand the differences in its role in different types of cancer,and for the subject of this study Thyroid cancer was the focus of the analysis.The m RNA expression profile files for papillary thyroid cancer were downloaded from the TCGA database(https://cancergenome.nih.gov/,accessed on November 9,2022),containing a total of 397 samples,and R software(version:4.1.0)was used to analyze and screen for differentially expressed genes and signaling pathways associated with the role of NSUN6 in papillary thyroid cancer.The correlation between NSUN6 expression and Ki67~+cell ratio was analyzed using immunohistochemical staining to detect NSUN6 expression in papillary thyroid cancer and normal thyroid tissues obtained from the Department of Pathology of the Fourth Hospital of China Medical University,since the high or low Ki67~+cell ratio is commonly used in immunohistochemical staining to reflect the proliferative capacity of tumor cells.The differential expression of NSUN6 in normal thyroid cells Nthy-ori 3-1 and papillary carcinoma cell lines BCPAP and K1 was first confirmed by Western Blot.The plasmid transfection technique was used to overexpress and knockdown NSUN6 in two cell lines of papillary thyroid carcinoma,BCPAP and K1,respectively.The plasmid with the strongest knockdown effect on the target gene was verified by q RT-PCR and Western Blot to verify the overexpression and knockdown of the target gene.The plasmid transfection was used to overexpress and knockdown NSUN6 in cancer cell lines with different proliferation status,and the CCK8 assay was used to detect whether there was a difference in the survival rate between the overexpressed and knockdown NSUN6 cells and the control group.The proliferation rate of cancer cells after overexpression and knockdown of the target gene was detected by Ed U proliferation to see if there was a significant change in the proliferation rate of cancer cells.Results:Comprehensive analysis of bioinformatics online tools showed that NSUN6expression and impact on prognosis differed in different types of cancer(Figure 2).The expression of NSUN6 was significantly lower in thyroid cancerous tissues than in normal paracancerous tissues(Figure 1B,Figure 3A-C);higher NSUN6 expression was associated with higher disease-free survival(DFS)in patients with thyroid cancer(Figure2A).R language screened significantly associated with the proliferative role of NSUN6in regulating thyroid cancer 16 genes were upregulated,namely TPO,PKNOX2,EDN3,DIO1,KCNAB1,FAM167A,FHL1,CA4,BMP8A,DUOX2,SLC26A7,KIT,FOSB,VEGFA,WSCD2,TFF3,and 4 genes were significantly downregulated,namely CHI3L1,LCN2,Pathway enrichment results showed that the five most significantly enriched upregulated pathways were Thyroid hormone synthesis,Herpes simplex virus 1 infection,Rap1 signaling pathway,morphine addiction,and basal cell carcinoma;the five down-regulated pathways are Rheumatoid arthritis,Cell adhesion molecules,phagosome,human T-cell leukemia virus 1 infection,and cytokine-cytokine receptor interaction.In addition,there were three significant enrichment pathways related to the thyroid gland,including thyroid hormone synthesis,thyroid hormone signaling pathway,and autoimmune thyroid disease.Immunohistochemical results suggested that NSUN6expression was higher in normal tissues than in papillary carcinomas,and cancer tissue samples with lower NSUN6 expression corresponded to higher Ki67~+ratios and greater cell proliferation.WB suggested that NSUN6 expression was significantly higher in the normal thyroid cell line Nthy-ori-3-1 than in the papillary carcinoma cell lines BCPAP and K1.CCK8 results suggested that NSUN6 knockdown in PTC cell lines BCPAP and K1 had a higher survival rate,and the cell survival rate of the corresponding cell lines overexpressing NSUN6 was lower,which can demonstrate that enhanced NSUN6expression inhibits the proliferation of PTC cell lines BCPAP and K1.Ed U proliferation results revealed that the cell proliferation rate was significantly lower after NSUN6overexpression on BCPAP,while K1 NSUN6 knockdown resulted in a significant increase in the proliferation rate of the cells,and the differences were both statistically significant compared to the control group.Conclusions:NSUN6 was differentially expressed in thyroid cancer,i.e.,significantly less in cancerous tissues than in normal paracancerous tissues.NSUN6 can inhibit the proliferation of papillary thyroid cancer cells,and bioinformatics analysis to screen out the differential genes and signaling pathways related to the proliferative effects of NSUN6 in regulating thyroid cancer is expected to be the direction of future in-depth research.