Association Between Hypercholesterol And Risk Of Differentiated Thyroid Cancer And Its Mechanism

Part ?Association between hypercholesterol and risk of differentiated thyroid cancerPurposeThis study aims to explore the relationship between hypercholesterolemia and differentiated thyroid cancer(DTC)in Chinese population by analyzing the clinical data of 3748 patients who underwent thyroid surgery in Qianfoshan Hospital Affiliated to Shandong University from January 2013 to Dec 2019,so as to provide data support for the epidemiological analysis of such a high incidence of thyroid cancer nowadaysBackgroundA large number of clinical studies and basic experiments had devoted to the epidemiological analysis and mechanism research of such a high incidence of thyroid cancer,in which metabolic risk factors have become the research hotspot in recent years.Recently,epidemiological studies have found that metabolic factors,including obesity,diabetes and insulin resistance,are closely related to the increased incidence of thyroid cancer.Previous studies have hypothesized that hyperlipidemia may be one of the risk factors for thyroid cancer.However,the relationship between hypercholesterolemia and thyroid cancer is not clear.Materials and Methods3748 patients were enrolled in the study,including 2021 DTC patients and 1727 control subjects with benign thyroid nodules.All patients underwent thyroid surgery and had postoperative pathological results(from January 2013 to Dec 2019,QianfOshan Hospital Affiliated to Shandong University).Demographic characteristics,medical history and clinical hematological examination were collected.The clinicopathologic features of DTC patients and thyroid benign nodule patients were summarized,and the prevalence of DTC with time was also analyzed.Large sample Z test and chi-square test were applied to explore the factors that associated with the risk of DTC.The association between hypercholesterolemia and the risk of DTC was analyzed using interaction/stratification analysis and multivariate logistic regression analysis modelsResults?Among the 3748 patients with thyroid nodules who underwent a thyroid surgery in this case control study,53.92%(2021 patients)of which were DTC,46.08%(1727 patients)were benign thyroid nodules.Among patients with DTC,60.52%(1332 patients)had single lesions,39.21%(683 patients)had multiple lesions,and the remaining 0.27%(6 patients)had lesions confined to lymph nodes.46.86%(947 patients)had lymph node metastasis,43.49%(879 patients)have no lymph node metastasis,and the rest 9.65%(195 patients)did not undergo lymph node dissection or pathological examination.41.81%(845 patients)had tumors with a maximum diameter of?1 cm,and 57.30%(1158 patients)had tumors less than 1 cm.The remaining 0.89%(18 patients)had no thyroid disease.?Chi-square test analysis was used to show that the overall prevalence difference of differentiated thyroid microcarcinoma between 2013 and 2019 was statistically significant(differentiated thyroid microcarcinoma accounted for the proportion of DTC(P=0.001),and the proportion of micro-cancer in the total number of operations(P<0.001)).However,when the difference between the proportion of each year and that of the previous year was compared and analyzed,there was no statistical increasement in the prevalence of differentiated thyroid microcarcinoma with time.In addition,the prevalence difference with time of DTC with different diameters showed that the prevalence of DTC with a diameter of less than 1 cm and 1-2 cm increased,while those with a diameter of?2 cm did not increase significantly.?The level of serum total cholesterol in patients with DTC is higher than that in control subjects(P<0.001).After adjusting hypercholesterolemia,age(P<0.001),triglyceride(P=0.003),and thyroid stimulating hormone(P<0.001)are found to be confounding factors.The risk of DTC in patients younger than 45 years old is 2.08 times than that of patients older than 45 years old(odds ratio=0.48,95%confidence interval=0.38-0.61,P<0.001).A high thyroid stimulating hormone level is highly associated with the increased risk of DTC(P<0.001).?The multivariable logistic regression analysis revealed that the absence of hypercholesterolemia could reduce the risk of thyroid nodules being malignant(odds ratio=-0.75,95%confidence interval=-1.39--0.12,P=0.02).Comparing to the higher level of serum total cholesterol(>5.7 mmol/1),the closer the serum total cholesterol level is to normal(3.17-5.7 mmol/1),the less the risk of thyroid nodules being malignant is,and this difference is statistically significant(odds ratio=-0.67,95%confidence interval=-1.31--0.03,P=0.040).However,this difference is not found in the group of patients with lower level of total cholesterol(<3.17 mmol/1,odds ratio=0.43,95%confidence interval=-1.22-2.09,P=0.068),suggesting that hypocholesterolemia is not a protective factor in the risk of thyroid nodules being malignantConclusionsThe number and prevalence of thyroid cancer increased year by year.The number of DTC with diameter<1cm and 1-2cm were increased significantly,while the number of DTC with diameter?2cm didnot.The overall prevalence of differentiated thyroid microcarcinoma showed a statistically significant difference during the year of 2013 to 2019,but the difference between each year and the previous year did not show a statistically significant increasement in the prevalence of differentiated thyroid microcarcinoma over time.Further data analysis suggested that hypercholesterole was associated with the risk of DTC in the Chinese population,but hypocholesterolemia is not a protective factor in the risk of thyroid nodules being malignantPart ?Study on the mechanism of the association of high cholesterol and papillary thyroid carcinoma.PurposeBioinformatics technology was applied to explore the mechanism between hypercholesterolemia and papillary thyroid carcinoma.At the same time,it was verified that high cholesterol was a risk factor for papillary thyroid carcinoma and disease progression through basic research.And the possible molecular mechanisms were analyzed to provide ideas for targeted therapy of papillary thyroid cancer.BackgroundIn recent years,more and more studies have found that thyroid cancer also has other risk factors besides radioactive radiation,which can be interfered.Among them,metabolic factors have become hotspots and difficulties in recent years.Studies have reported that obesity,hyperinsulinemia,hyperglycemia,etc.were positively related to the risk of thyroid cancer.The specific mechanism was not clear,and maight be related to several potential mechanisms,such as hyperglycemia increasing oxidative stress,hyperinsulinemia related pathways and non-hyperinsulinemia-related pathways,the latter one mainly include estrogen/androgen imbalance,adipokines,chronic "low-grade" inflammation,immune response changes,and DNA damage caused by oxidative stress.In addition,elevated thyroid-stimulating hormone and thyroid insufficiency may accelerate the growth of papillary thyroid cancer cells and/or increase cell subtype variation,and interact with other growth factors and molecular changes.However,these mechanisms were mostly speculations based on other cancers,and further exploration was still needed in thyroid cancer.Therefore,based on the clinical research that high cholesterol was related to the risk of differentiated thyroid carcinoma,we will conduct in-depth research on the mechanism.Materials and Methods?Bioinformatics analysis was used to obtain the common target genes of hypercholesterolemia and papillary thyroid carcinoma,and use Cytoscapse to construct a"Papillary Thyroid Carcinoma-Hypercholesterolemia Common Target Gene Network Diagram".In order to further expound the underlying mechanism of high cholesterol leading to an increased risk of thyroid carcinoma.We put the relevant genes into the DAVID database for GO and KEGG enrichment analysis.According to the number of genes enriched in the relevant pathways and the P value,top ten KEGG pathways and GO entries were selected for mapping to explore and analyze pathways and functions that promote cancer risk and disease progression.?The CCK8 experiment was used to screen the best intervention concentration and intervention time of cholesterol.Immunofluorescence staining was used to qualitatively and semi-quantitatively detected the expression of thyroglobulin(Tg)in Nthy-ori 3-1 cells(human normal thyroid follicular epithelial cell lines).At the same time,the expression of Tg mRNA in Nthy-ori 3-1 cells and thyroid tumor stem cell specific marker molecule stage-specific embyronic antigen-1(SSEA-1)mRNA were detected by Real-time PCR.In addition,the Edu method was used to detect cancer cell proliferation for human papillary thyroid carcinoma cell lines(BHP 10-3 cells),and the Transwell migration test and Western Blot were used to detect the effect of cholesterol on BHP 10-3 cell migration and migration protein(matrix metalloproteinase 9,MMP9).?Use Real-time PCR to detect the expression of common target genes in Nthy-ori 3-1 cells and BHP 10-3 cells,and analyze their differences.At the same time,cholesterol intervention was given to the two cell lines to detect the expression of common target genes.?The Kaplan Meier plotter online website was used to analyze the impact of 10 common target genes on the overall survival of 502 patients with thyroid cancer included in the website.Results?Bioinformatics analysis revealed that the common target genes of papillary thyroid carcinoma and hypercholesterolemia were:ABCC3,AGTR1,CD36,CHI3L1,CXCL12,DPP4,FN1,HBA1,MMRN1,PPARGC1A.For the biological function analysis in GO enrichment analysis,target genes were mainly enriched in the signal pathway mediated by chemokines(GO:0070098).Chemokine-mediated signaling pathways,proteoglycans in tumors,focal adhesions,and regulation of actin cytoskeleton were the main pathways of target gene enriched by KEGG pathway analysis.?The survival rate of Nthy-ori 3-1 cells increased with the increase of cholesterol concentration within a certain concentration.When the cholesterol concentration was 25umol/L,it had the greatest impact on the survival rate of Nthy-ori 3-1 cells;when the cholesterol concentration continued to increase,the survival rate of Nthy-ori 3-1 cells decreased.25umol/L cholesterol at different times(0,24h,48h,72h)on the Nthy-ori 3-1 cell viability test found that cholesterol has the greatest impact on cell survival at 24h.Therefore,follow-up experiments selected the above-mentioned optimal concentration and optimal action time.Taking the Tg mRNA expression level in the control group as the benchmark,after cholesterol intervention,it significantly increased(P<0.001).The SSEA-1 mRNA expression level in the control group was defined as 1,after intervention,it increased to 2.67.Compared with the control group,it was statistical differece(P=0.037).?The survival rate of BHP 10-3 cells increased with the increase of cholesterol concentration within a certain concentration.When the concentration of cholesterol was 25umol/L,it had the greatest impact on the survival rate of BHP 10-3 cells,while the concentration continued to increase,the survival rate of BHP 10-3 cells decreased.The effects of 25umol/L cholesterol on the viability of BHP 10-3 cells at different times(Oh,24h,48h,72h)were tested.The results showed that cholesterol had the greatest impact on cell survival at 24h.Therefore,follow-up experiments select the above-mentioned optimal concentration and optimal action time.Taking the Tg mRNA expression level in the control group as benchmark,after cholesterol intervention,the Tg mRNA expression level increased significantly(P=0.037).The results of EdU cell proliferation experiments showed that compared with the control group,cholesterol can significantly increase the proliferation of BHP 10-3 cells(P=0.037).Cholesterol significantly increased the migration and MMP9 protein expression in BHP 10-3 cells(P=0.037)by transwell migration experiment and Western blot detection.?Without the intervention of cholesterol,the expression of DPP4 mRNA in BHP 10-3 cells was significantly higher than that in Nthy-ori 3-1 cells.Except for ABCC3 mRNA,the expression of the remaining eight genes in BHP 10-3 cells was significantly increased.After 24 hours of intervention with 25umol/L cholesterol,the expression changes of HBA1 mRNA,CHI3L1 mRNA,CXCL12 mRNA and DPP4 mRNA in two cell lines were consistent,and the change was statistically different.The expression of HBA1 mRNA was significantly increased,while the expression of CHI3L1 mRNA,CXCL12 mRNA and DPP4 mRNA were decreased.Among them,the change trend of CH13L1 mRNA and CXCL12 mRNA is consistent with whether or not to add cholesterol intervention.?The Kaplan Meier plotter online website analyzed the impact of 10 common target genes on the overall survival of patients with thyroid cancer.The results showed that the differential expression of DPP4,HBA1,and PPARGC1A was significantly related to the overall survival of thyroid cancer,and high levels of PPARGC1A can significantly reduce the overall survival of patients with thyroid cancer,hazard ratio was 3.61,and the result was statistically significant(P=0,0063).Low levels of HBA1 and DPP4 were associated with poor prognosis of thyroid cancer and significantly reduced the overall survival of patients(P=0.027and P=0.0011).Conclusions?The common target genes of hypercholesterolemia and papillary thyroid carcinoma were:ABCC3,AGTR1,CD36,CHI3L1,CXCL12,DPP4,FN1,HBA1,MMRN1,PPARGC1A.The signal pathway mediated by chemokines,the regulation of proteoglycans,focal adhesions,and actin cytoskeleton in tumors were involved in the interaction between them.These results suggested the main mechanism of cholesterol leading to increased risk of thyroid cancer and disease progression.?In Nthy-ori 3-1 cells,cholesterol can increase cell survival rate,Tg mRNA expression,and tumor stem cell surface marker SSEA-1 mRNA expression.In BHP 10-3 cells,cholesterol can increase cell survival,Tg mRNA expression,and cancer cell proliferation and migration ability,suggesting the role of cholesterol in the increased risk of thyroid cancer and disesase progression.?The expression of DPP4 mRNA in BHP 10-3 cells was significantly higher than that in Nthy-ori 3-1 cells.Except for ABCC3 mRNA in the two cells,the expression of the remaining eight genes in BHP 10-3 cells was significantly increased.Cholesterol intervention in Nthy-ori 3-1 cells and BHP 10-3 cells can significantly reduce the expression levels of CHI3L1 mRNA,CXCL12 mRNA,and DPP4 mRNA in the two cells,while it can increase the expression level of HBA1 mRNA?Survival analysis results suggested that the differential expression of DPP4,HBA1,PPARGCIA was significantly related to the overall survival of thyroid cancer.High levels of PPARGC1A can significantly reduce the overall survival of patients with thyroid cancer,while low levels of HBA1 and DPP4 were associated with poor prognosis of thyroid cancer and significantly reduced the overall survival of patients.The mechanism by which cholesterol acts on PTC have nothing to do with HBA1,CXCL12 and CH13L1,while cholesterol may affect on cell proliferation and migration by reducing DPP4 expression,and participate in the mechanism by which cholesterol plays a role in PTC disease progression.