The Mechanism Of MiR-30b-5p Inhibiting Papillary Thyroid Cancer Progression Through GALNT7

In recent decades,thyroid cancer(TC)is a common endocrine malignancy with a rapidly increasing incidence,mainly due to overdiagnosis and overtreatment of differentiated thyroid cancer(DTC).The treatment of thyroid cancer is primarily based on surgery,adjuvant hormone suppression therapy and radioactive iodine therapy through real-time observation of iodine metabolism.The treatment of thyroid cancer has always been controversial.Although excessive surgical treatment limits the progress of thyroid cancer to a certain extent,it also increases the medical and economic burden of patients.Therefore,based on the differences in the genetic and immune characteristics of the tumour,individualized therapy seems to be more suitable for the current clinical management of thyroid cancer.At the same time,because the superficial treatment method of "one size fits all" is avoided,doctors and patients can benefit more in diagnosis and treatment.The BRAFV600E mutation is the conversion of thymine from thymine to adenine at position 1799,resulting in the replacement of valine with glutamic acid at codon 600.BRAF immunohistochemistry(IHC)in daily practice may serve as an alternative or initial tool for BRAFV600E mutation analysis.This mutation is associated with clinicopathological features of thyroid cancer that often predict tumor progression and recurrence.Previous studies have also shown that some miRNAs are dysregulated in BRAFV600E-mutated tissues.It makes it possible to detect BRAFV600E mutation combined with miRNAs to distinguish benign and malignant thyroid nodules and provides a direction for clinical combination therapy.MiR-30b-5p,a member of the miR-30 family,is considered a novel diagnostic marker for improving the diagnosis of papillary thyroid cancer(PTC).In anaplastic thyroid cancer(ATC),miR-30 expression was also reduced in tumour samples.Furthermore,the miR-30 expression pattern was associated with the dedifferentiation/transdifferentiation status of anaplastic thyroid cancer,and ATC had lower miR-30 expression than follicular papillary thyroid cancer.It implies that miR-30 may play a role in TC progression.However,the specific part of miR-30b-5p in TC is not fully understood.This study first determined the expression pattern of miR-30b-5p in thyroid cancer.Then the tool cell NIH-3T3 was used to verify the relationship between BRAFv600e mutation and miR-30b-5p expression.Based on the above results,we verified the role of miR-30b-5p in thyroid cancer cells through a series of in vivo and in vitro functional experiments,and combined the online database to predict the downstream target genes of miR-30b-5p,and experimentally verified the difference between the two.relationship between.Finally,GALNT7 was determined to be the direct downstream target of miR-30b-5p,and the mechanism pathway EGFR/PI3K/AKT involved in GALNT7 was further explored.However,there are still shortcomings in this experiment.First of all,the specific inhibitory mechanism of BRAF/Erk on miR-30b-5p is still unclear,which is worthy of further exploration in the future.Secondly,this study failed to determine the glycosylation substrate of GALNT7.It is still unclear whether GALNT7 plays a family function or acts as a specific substrate.There is an intermediate link in the regulation of EGFR expression by GALNT7,which is worthy of further research in the future.Overall,this study is the first to demonstrate that the miR-30b-5p/GALNT7 axis inhibits the proliferation and metastasis of PTC cells by regulating the EGFR/PI3K/AKT pathway(Figure 29),thus providing a new target for further understanding of its possible pathogenesis point.This result may help design molecular therapeutic strategies for PTC.Part One The Expression of Mir-30b-5p in Papillary Thyroid Carcinoma and its Correlation with The Clinicopathological Features of Patients were Analyzed.Objective The expression of miR-30b-5p(miRNA-30b-5p,Hsa-miRNA30b-5p)in thyroid carcinoma tissues and cells were detected.and its correlation with the clinicopathological features of patients with thyroid cancer was analyzed.Methods 1.Download TCGA-THCA transcriptome data and SNP data,and limma R package was used to analyze the expression level of miR-30b-5p in thyroid carcinoma tissues and normal paracancerous tissues.2.Collect clinical thyroid cancer tissue samples and collect their clinical data,extract total RNA from tissues,and make tissue wax blocks.The expression of miR-30b-5p in thyroid carcinoma and paracancerous tissues was detected by real-time quantitative PCR(qRT-PCR)and DAB chromogenic in situ hybridization(CISH).3.Normal thyroid epithelial cells(Nthy-ori-3-1)and thyroid cancer cell lines(TPC-1,KTC-1,B-CPAP and K-1)were extracted from total RNA and detected the expression level of miR-30b-5p in the cell lines by qRT-PCR.3.The relationship between miR-30b-5p and clinicopathological features was analyzed with clinicopathological data.The metrological data were expressed by mean ±standard deviation(χ±s).T-test was used to compare the two groups,and a single-factor analysis of variance(One-way ANOVA)test was used to compare groups.The Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results 1.TCGA-THCA data analysis showed that the mean expression abundance of miR-30b-5p in the PTC group was 12.76,and the mean expression abundance in the normal group was 13.54.The difference was statistically significant(P<0.01,95%CI[1.015,-0.5375]),the expression abundance of miR30b-5p in the BRAFv600e mutant group was 0.02283,and that in the BRAF wildtype group was 0.05336(P=0.0657,95%CI[-0.001989,0.06305]);2.The results of qRT-PCR showed that the relative expression level of miR-30b-5p in PTC tissues was-1.355 times higher than that in adjacent tissues(P<0.01,95%CI[1.666,-1.043]);in situ hybridization experiment The results suggest that miR30b-5p is mainly localized in the cytoplasm,with a small amount in the nucleus,and its expression level in thyroid cancer tissues is significantly lower than that in paired paracancerous tissues;3.qRT-PCR results showed that the relative expression level of miR-30b-5p in BRAFv600e mutant cell line K-1 was 0.79 times higher than that in normal thyroid cells(P<0.05,95%CI[-0.3477,-0.03678]);The relative expression level of miR-30b-5p in BRAF wild-type cell line KTC-1 was 5.13 times higher than that in normal thyroid cells(P<0.01,95%CI[3.362,4.349]);The relative expression level of miR-30b-5p in BRAF wild-type cell line TPC-1 was 16.4 times higher than that in normal thyroid cells(P<0.01,95%CI[13.54,15.18]);the relative expression level of miR-30b-5p in BRAPv600e mutant cell line B-CPAP cell line is 0.25 times than that in normal thyroid cells(P<0.01,95%CI[-0.8675,-0.5225]);4.The results of the correlation analysis between the expression level of miR-30b-5p and clinicopathological characteristics showed that the relative expression of miR-30b-5p in the age group less than 55 years old was 1.32 times that of the age group greater than or equal to 55 years old,and the difference was not statistically significant.(P=ns,95%CI[-0.6032,0.03585]);The relative expression of miR-30b-5p in the female group was 1.17 times higher than that in the male group,and the difference was not statistically significant(P=ns,95%CI[-0.4815,0.1582]);The relative expression of miR-30b-5p in Stage Ⅱ group was 0.66 times higher than that in Stage Ⅰ group,and the difference was statistically significant(P<0.05,95%CI[0.7544,-0.06278]);The relative expression of miR-30b-5p in the T3 stage group was 0.45 times that of the T1 stage group,and the difference was statistically significant(P<0.01,95%CI[-1.181,-0.4882]);The relative expression of miR30b-5p in the T2 stage group was 0.74 times higher than that in the T1 stage group,and the difference was statistically significant(P<0.001,95%CI[-0.6727,-0.1313]);The relative expression of miR-30b-5p in the lymph node metastasis group was 0.59 times higher than that in the lymph node non-metastasis group,and the difference was statistically significant(P<0.01,95%CI[-0.8051,0.2864]).Conclusion 1.MiR-30b-5p is low expressed in thyroid carcinoma and plays a major role in the cytoplasm.2.The low expression of miR-30b-5p in thyroid carcinoma is related to the mutation of BRAFv600e.3.K-1 and B-CPAP can be selected for a follow-up study.4.MiR-30b-5p may be related to the poor prognosis of patients with thyroid cancer.Part Ⅱ BRAFv600e Mutation Suppresses the Expression of MiR-30b-5pObjective A series of assays were performed to clarify the relationship between miR-30b-5p and BRAFv600e mutation.Methods 1.Purchase mouse embryonic fibroblast cell line(NIH-3T3),transfect BRAF wild-type,BRAFv600e mutant and negative control plasmids into NIH-3T3 cell line,and test the transfection efficiency of the cell line according to the fluorescence abundance;2.Use a BRAF pathway inhibitor(trametinib)to inhibit the BRAF/Erk pathway and verify the expression of BRAF/Erk pathway landmark proteins by Western Blot;3.Use qRT-PCR experiment to verify the difference of miR-30b-5p expression in BRAF wild-type group,BRAFv600e mutant group and BRAFv600e mutant+trametinib group.The measurement data were expressed by mean ±standard deviation(χ±s).T-test was used to compare the two groups,and a single factor analysis of variance(One-way ANOVA)test was used to compare multiple groups.The Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results 1.The detection of fluorescence intensity showed that the transfection efficiency of the BRAF plasmid was>90%.The results of Western Blot showed that the relative expression of BRAF protein in the BRAF wild-type group was 1.58 times that of the control group(P<0.05,95%CI[0.2150,0.9473]);The relative expression of BRAF protein in the BRAFv600e mutant group was 1.85 times that of the control group(P<0.01,95%CI[0.4717,1.232]);the relative expression of p-Erk protein in the BRAF wild-type group was 1.14 times that of the control group(P=ns,95%CI[-0.2776,0.5645]);The relative expression of p-Erk protein in the BRAPv600e mutant group was 2.20 times that of the control group(P<0.05,95%CI[0.3475,2.045]);2.After the intervention of trametinib in the BRAFv600e mutant group,the relative expression of BRAF protein in the BRAFv600e mutant+trametinib group was 1.45 times that of the control group(P<0.05,95%CI[0.005553,0.8244]);The relative expression of the p-Erk protein in the BRAFv600e mutant+trametinib group was 0.80 times that of the control group(P=ns,95%CI[-0.5704,0.1791]).3.The results of qRTPCR experiments indicated that the expression of miR-30b-5p in the BRAFv600e mutant group was 0.28 times higher than that in the BRAF wild-type group(P<0.01,95%CI[-2.142,-1.594]);When trametinib inhibited the BRAF/Erk pathway,the relative expression of miR-30b-5p in the BRAFv600e mutant+trametinib group was 3.03 times that of the BRAFv600e mutant group(P<0.01,95%CI[1.856,2.639]).Conclusion 1.Trametinib can inhibit BRAF/Erk pathway;2.BRAFv600e negatively regulates the expression of miR-30b-5p.Part Ⅲ The role of MiR-30b-5p on Proliferation,Apoptosis,Invasion and Migration of Thyroid cancer cellsObjective A series of assays were performed to explore the effects of miR30b-5p on proliferation,apoptosis,invasion and migration of thyroid cancer cells in vivo and in vitro.Methods 1.MiR-30b-5p overexpression plasmid and lentivirus were constructed and packaged by the Shanghai Jikai gene.2.Lentivirus and mimic were transfected into K-1 and B-CPAP thyroid cancer cells,and the effect of overexpression was verified by fluorescence intensity and qRT-PCR experiment.3.Online database DAVID predicted miR-30b-5p molecular function;4.CCK8,EdU and tumor formation test in nude mice to detect the proliferation of thyroid cancer cells.5.Flow cytometry was used to detect the apoptosis rate of thyroid cancer cells.6.Wound healing and Transwell chamber test was used to detect the invasion and migration ability of thyroid cancer cells.7.Western Blot test was used to detect the effect of miR-30b-5p on EMT of thyroid cancer.Some measurement data were expressed by mean±standard deviation(χ±s).T-test was used for comparison between the two groups,and a single-factor analysis of variance(One-way ANOVA)test was used for comparison between multiple groups.The Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results 1.The infection efficiency of the K-1 and B-CPAP cell lines was 100%observed under the fluorescence microscope;2.The quantitative qRTPCR results indicated that the lentivirus was successfully constructed,and the K-1 cell line results indicated that the expression of miR-30b-5p in the miR-30 group was 549 times higher than that of the NC group(P<0.01,95%CI[515,603]);The results of B-CPAP cell line indicated that the expression of miR-30b5p in mir-30b group was 443 times higher than that in NC group(P<0.01,95%CI[425,482]);3.The miRNA mimic was successfully transfected,and the results of the K-1 cell line indicated that the expression of miR-30b-5p in the miR-30b-5p mimic group was 3624 times higher than that in the NC mimic group(P<0.01,95%CI[3417,3830]);B-CPAP cell line results suggested that the expression of miR-30b-5p in the miR-30b-5p mimic group was 4808 times higher than that in the NC mimic group(P<0.01,95%CI[4310,5305]);4.GO(Gene Ontology)enrichment analysis suggests that miR-30b-5p is associated with protein binding,transcriptional regulation,RNA regulation,ubiquitination regulation,and membrane proteins.The KEGG enrichment analysis results suggest that miR-30b-5p is involved in multiple cancer-related pathways;5.The results of CCK8 suggest that overexpression of miR-30b-5p inhibits the proliferation of two cell lines;then,EdU staining was used to detect the proliferation ability of PTC cell lines.The results of the K-1 cell line indicated that the number of EdU-positive cells in the miR-30b-5p mimic group was 0.66 times that of the NC mimic group(P<0.01,95%CI[-0.5233,0.1586]);B-CPAP cell line The results indicated that the number of EdU-positive cells in the miR30b-5p mimic group was 0.62 times that of the NC mimic group(P<0.01,95%CI[-0.5702,-0.1832]);6.The in vivo experimental data showed that compared with the negative control(NC)group,the tumor proliferation rate in the miR-30b-5p agomir group was significantly decreased,and the tumor volume in the miR-30b-5p group was significantly smaller than that in the control(NC)group(P<0.01).7.The results of flow cytometry showed that the average apoptosis rate of the K-1 cell line miR-30b-5p mimic group was 19.4%,and the average apoptosis rate of the NC group was 11.1%,and the difference was statistically significant(P<0.01,95%CI[0.6215,0.8414]);the average apoptotic rate of B-CPAP cell line miR-30b-5p mimic group was 15.8%,and the average apoptotic rate of NC group was 7.58%,and the difference was statistically significant(P<0.01,95%CI[0.7655,1.395]);8.The wound healing experiment showed that the results of the K-1 cell line indicated that the scratch area in the miR-30b-5p mimic group was 1.44 times that of the NC mimic group(P<0.01,95%CI[0.2860,0.5858]);B The results of-CPAP cell line indicated that the scratch area in the miR-30b-5p mimic group was 1.50 times that of the NC mimic group(P<0.01,95%CI[0.3372,0.6717]).The results of Transwell migration and invasion assay showed that the cell migration rate in the miR-30b5p mimic group in the K-1 cell line was 0.64 times higher than that in the NC mimic group(P<0.01,95%CI[-0.3966,-0.3249]);the cell invasion rate of the miR-30b-5p mimic group in K-1 cell line was 0.57 times that of the NC mimic group(P<0.01,95%CI[-0.4959,-0.3565]);The B-CPAP cell migration rate in the miR-30b-5p mimic group was 0.60 times higher than that in the NC mimic group(P<0.01,95%CI[-0.4573,-0.3499]);The cell invasion rate in the miR30b-5p mimic group was 0.62 times higher than that in the NC mimic group(P<0.01,95%CI[-0.4549,-0.3103]);9.Western Blot and immunofluorescence(IF)results showed that overexpression of miR-30b-5p decreased the expression of mesenchymal markers,including snail and vimentin,while up-regulated the expression of the epithelial marker E-cadherin.Conclusion 1.MiR-30b-5p has the ability to inhibit the proliferation of thyroid cancer cells in vivo and in vitro;2.miR-30b-5p positively regulates the apoptosis of K-1 and B-CPAP cells.3.Overexpression of miR-30b-5p impairs the metastatic ability of thyroid cancer cells by inhibiting the EMT process.Part Ⅳ MiR-30b-5p regulate proliferation,migration and invasion of papillary thyroid cancer cells through GALNT7Objective A series of assays were performed to predict and verify the downstream molecular targets of miR-30b-5p in thyroid carcinoma.Methods 1.The downstream targeting mRNA of miR-30b-5p was predicted by the online database,and the target expression pattern was further verified in GSE33630 and GSE60542 data sets.2.The qRT-PCR and Western Blot experiments were used to verify the regulatory relationship between miR30b-5p and the target.3.Double luciferase reporter gene experiments verified the direct regulatory relationship between miR-30b-5p and target molecules.4.EdU and Transwell experiments showed that GALNT7 was the molecular functional target of miR-30b-5p.All measurement data were expressed by mean±standard deviation(χ±s).T-test was used for comparison between the two groups,and a single factor analysis of variance(One-Way ANOVA)test was used for comparison between multiple groups.The Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results 1.The Venn diagram shows that GALNT7 and RUNX2 were included in the follow up study.The differential gene analysis results after log2 processing indicated that the expression of GALNT7 in the PTC group in the GSE33630 data set was higher than that in the normal group,and the difference was statistically significant(P<0.01,logFC=1.31);the expression of RUNX2 in the PTC group was higher than that in the normal group,and the difference was statistically significant(P<0.01,logFC=1.19).In the GSE60542 dataset,the expression of GALNT7 in the PTC group was higher than that in the normal group,and the difference was statistically significant(P<0.01,logFC=1.32);the expression of RUNX2 in the PTC group was higher than that in the normal group,and the difference was statistically significant(P<0.01,logFC=1.32).P<0.01,logFC=1.16);2.The results of qRT-PCR showed that the results of the K-1 cell line indicated that the expression of GALNT7 in the miR-30b-5p mimic group was 0.589 times higher than that in the NC mimic group(P<0.01,95%CI[0.5741,0.3484]);the expression of GALNT7 in the miR-30b-5p inhibitor group was 1.11 times higher than that in the NC inhibitor group(P<0.01,95%CI[0.08351,0.2277]);B-The results of CPAP cell line indicated that the expression of GALNT7 in the miR-30b-5p mimic group was 0.52 times higher than that in the NC mimic group(P<0.01,95%CI[-0.6792,-0.3625]);miR-30b5p inhibitor The expression of GALNT7 in the group was 1.27 times higher than that in the NC inhibitor group(P<0.01,95%CI[0.1892,0.3188]);the results of the K-1 cell line suggested that the expression of RUNX2 in the miR-30b-5p mimic group was higher than that in the NC inhibitor group.The expression of RUNX2 in the miR-30b-5p inhibitor group was 1.09 times higher than that in the NC inhibitor group(P=ns,95%CI[-0.1802,0.3841]);B-CPAP cell line results indicated that the expression of RUNX2 in the miR-30b-5p mimic group was 0.53 times higher than that in the NC mimic group(P<0.01,95%CI[-0.7657,0.4036]);the expression of RUNX2 in the miR-30b-5p inhibitor group was 1.03 times that of the NC inhibitor group(P=ns,95%CI[0.02407,0.2213]);Quantitative Western Blot results suggested that K-1 The expression of GALNT7 in the cell line miR-30b-5p mimic group was 0.62 times higher than that in the NC mimic group(P<0.01,95%CI[-0.4583,-0.3114]);The expression of GALNT7 in the B-CPAP cell line miR-30b-5p mimic group was 0.66 times higher than that in the NC mimic group(P<0.01,95%CI[-0.3436 to-0.2348]);the expression of RUNX2 in the K-1 cell line miR-30b-5p mimic group 0.47 times that of the NC mimic group(P<0.01,95%CI[-0.8107,-0.3616]);The expression of RUNX2 in the B-CPAP cell line miR-30b-5p mimic group was 0.33 times higher than that in the NC mimic group(P<0.01,95%CI[-1.006,0.6795]);3.The dual-luciferase reporter gene results suggested that the relative luciferase activity of miR-30b-5p and GALNT7-WT co-transfected cells was significantly lower than the control group(P<0.01).The relative luciferase activity of miR-30b-5p and RUNX2-WT co-transfected cells was not significantly different from the control group(P=ns).4.Nude mice transplanted tumour experiments to verify the expression changes of GALNT7 in the tumors of the two groups of mice.The immunohistochemistry results indicated that the protein expression of GALNT7 in the miR-30b-5p agomir group decreased compared with that in the NC agomir group.The quantitative Western Blot results indicated that the expression of GALNT7 in the miR-30b-5p agomir group was the same as that in the NC agomir group.0.64 times(P<0.05,95%CI[-0.5778,-0.1447]);5.The results of qRT-PCR indicated that the overexpression efficiency of GALNT in K-1 cell line was 94 times(P<0.01,95%CI[62.06,125.3]),the overexpression efficiency of GALNT in the B-CPAP cell line was 50 times(P<0.01,95%CI[43.73,69.06]);quantitative Western Blot results indicated that GALNT in the K-1 cell line The relative overexpression efficiency was 2.03 times(P<0.01,95%CI[0.7868,1.282]),and the relative overexpression efficiency of GALNT in B-CPAP cell line was 1.47 times(P<0.01,95%CI[0.2608,0.8189]);when miR-30b-5p mimics were transfected into K-1 and B-CPAP cells,decreased mRNA and protein levels of GALNT7 were observed,whereas co-expression of pcDNA-GALNT7 and miR30b-5p mimics transfection restored the mRNA and protein levels of GALNT7;6.EdU staining showed that transfection of pcDNA-GALNT7 could antagonize the decrease in cell proliferation induced by miR-30b-5p mimic.Transwell invasion and migration assay results showed that transfection of miR-30b-5p mimic attenuated the metastatic ability of thyroid cancer cells,while cotransfection of pcDNA-GALNT7 and miR-30b-5p mimic could reverse this effect.Conclusion 1.GALNT7 and RUNX2 may play a role as oncogenes in thyroid cancer.2.GALNT7 is the direct downstream target of miR-30b-5p,miR30b-5p has a negative regulation on the expression of GALNT7 in thyroid cancer cells,and RUNX2 is not the direct downstream target of miR-30b-5p.3.GALNT7 is the functional target of miR-30b-5p.MiR-30b-5p inhibits the malignant behaviour of thyroid cancer cells by down-regulating GALNT7.Part Ⅴ GALNT7 regulates the EGFR pathway to promote the progress of thyroid cancerObjective A series of assays were performed to explore the ability of GALNT7 to mediate proliferation,invasion and migration of thyroid cancer cells and the regulatory mechanism of related signal pathways.Methods 1.QRT-PCR and Western Blot assay was used to detect the silencing efficiency of GLANT7.2.CCK8 and EdU were used to detect the effect of GALNT7 on the proliferation of thyroid cancer cells.3.Flow cytometry was used to detect the effect of GALNT7 on the apoptosis of thyroid cancer cells.4.Tranwell and wound healing assays were used to detect the effect of GALNT7 on the invasion and migration of thyroid cancer cells.5.GSEA was used to analyze the pathway involved in GALNT7.6.BMS-599626 was used to inhibit the EGFR pathway,and combined with Western Blot experiment to verify the effect of GALNT7 and BMS-599626 on the EGFR pathway,all measurement data were expressed by mean ±standard deviation(χ±s),t-test was used for comparison between the two groups,and one-way analysis of variance(Oneway ANOVA)test was used for comparison between groups.The Chi-square test was used for counting data.P<0.05 indicates that the difference is statistically significant.Results 1.After transfection of si-GALNT7,the mRNA and protein levels of GALNT7 were significantly decreased.The results of qRT-PCR showed that the silencing efficiency of si-GALNT7-2 was the highest in the K-1 cell line,which was 0.32 times(P<0.001,95%CI[-0.8654,-0.5295]),and si-GALNT7-2 in the B-CPAP cell line.The silencing efficiency of GALNT7-2 was 0.31 times(P<0.01,95%CI[-0.8703,-0.5285]);the quantitative Western Blot results showed that the silencing efficiency of si-GALNT7-2 in K-1 cell line was 0.20 times(P<0.01,95%CI[-1.076,-0.5146]),the silencing efficiency of si-GALNT72 in B-CPAP cell line was 0.40-fold(P<0.01,95%CI[-0.8268,-0.3801]).siGALNT7-2 was selected for subsequent cell function experiments based on silencing efficiency.2.EdU staining showed that the proliferation of K-1 and BCPAP cells was significantly inhibited after silencing GALNT7.The results of the K-1 cell line indicated that the number of EdU positive cells in the siGALNT7-2 group was 0.61 times that of the NC group(P<0.01,95%CI[-0.5620,-0.2140]);the results of the B-CPAP cell line indicated that the number of EdU positive cells in the si-GALNT7-2 group was 0.51 times higher than that in the NC group(P<0.01,95%CI[-0.7182,-0.2668]).The results of invasion and migration experiments showed that the number of cell migration and invasion was significantly reduced in GALNT7-knockout PTC cells.The results of Transwell migration and invasion assay showed that the cell migration rate in the si-GALNT7-2 group in the K-1 cell line was 0.59 times higher than that in the NC group(P<0.01,95%CI[-0.5741,-0.2528]);In the K-1 cell line,the cell invasion rate in the si-GALNT7-2 group was 0.52 times that of the NC group(P<0.01,95%CI[-0.5886,-0.3365]);In the B-CPAP cell line,The cell migration rate in the si-GALNT7-2 group was 0.59 times that of NC group(P<0.01,95%CI[-0.5056,-0.3152]);the cell invasion rate of the si-GALNT7-2 group in B-CPAP cell line was 0.60 times that of NC group(P<0.01,95%CI[0.4821,-0.1837]);3.The down-regulation of GALNT7 led to more tumor cell apoptosis.The results of flow cytometry indicated that the average apoptotic rate the K-1 cell line si-GALNT7-2 group was 20.9%,and the average apoptotic rate in the NC group was 6.94%,and the difference was statistically significant(P<0.01,95%CI[1.876,2.145]);The average apoptosis rate in the B-CPAP cell line si-GALNT7-2 group was 13.7%,and the average apoptosis rate in the NC group was 7.34%,and the difference was statistically significant(P<0.01,95%CI[0.6191,1.112]).Pathogenic pathways,prostate cancer,glioma and cancer pathways,etc.In addition,it is worth noting that EGFR-related signalling pathways were significantly enriched in the GO and KEGG enrichment analysis results;5.TCGA-THCA transcriptome Data analysis of the correlation between GALNT7 and EGFR expression showed that there was a strong correlation between EGFR expression and GALNT7(r=0.49,P<0.01);6.We detected the EGFR/PI3K/AKT pathway expression in K-1 and B-CPAP cells.Activity and inhibited the exercise of this pathway using a selective HER1 and HER2 inhibitor(BMS-599626,10μM).The levels of major signalling molecules in the EGFR/PI3K/AKT pathway in PTC cells treated with BMS-599626 or siGALNT7-2 were significantly lower than those in the negative control group.Likewise,the expression levels of EGFR,p-PI3K and p-AKT in PTC cells transfected with pcDNA-GALNT7 were higher than those in the control group.After blocking this pathway with BMS-599626,the expression of EGFR,pPI3K,and p-AKT did not further increase in the GALNT7 group.Conclusion 1.GALNT7 promotes the proliferation and metastasis of thyroid cancer cells and inhibits the apoptosis of thyroid cancer cells.2.The inhibition of the EGFR/PI3K/AKT signal pathway reduces the proliferation and invasion of thyroid cancer cells.3.GALNT7 plays an important role in developing thyroid cancer through EGFR/PI3K/AKT pathway.