| BackgroundPapillary thyroid carcinoma（PTC）is the most common malignant tumor of the endocrine system.PTC generally progresses slowly and has a favorable prognosis.However,after invasion and metastasis,the 5-year survival rate of patients with PTC decreases from 94.8%to 73.1%.In PTC,invasion and metastasis are independent risk factors for a poor prognosis and may cause recurrence,distant metastasis,treatment resistance,and low survival rate.Clinical studies have shown that approximately 30%-80%of patients with PTC have invasion and metastasis at initial diagnosis.The cervical lymph nodes are the primary sites of metastasis in PTC.However,due to the complex anatomical location of the neck,the detection rate of cervical lymph node metastasis via ultrasound is only 16.8%.At present,there are no other preoperative auxiliary examination methods to accurately assess cervical lymph node metastasis,causing controversy over the scope of lymph node dissection.This has led to two clinical dilemmas:one is cleaning lymph nodes that have not yet metastasized and the other is not cleaning the lymph nodes that have metastasized in time.Therefore,discovering diagnostic biomarkers that can accurately reflect invasion and metastasis of PTC is required.There are several risk factors for invasion and metastasis of PTC,among which excessive iodine is controllable and may be an entry point for the formulation of interventions from the perspective of public health policies.Iodine metabolism depends on the transmembrane transport of iodide ions by solute carriers.It has been found that abnormal expression of solute carriers is associated with the risk of invasion and metastasis of PTC.However,as there are numerous solute carriers,their relationship with PTC invasion and metastasis has not been fully elucidated.In addition,only a few studies have explored the relationship between excessive iodine and abnormal expression of solute carriers and PTC invasion and metastasis from a holistic perspective.Thus,starting from the relationship between excessive iodine and PTC invasion and metastasis,this study investigated the role of excessive iodine and abnormal expression of solute carriers in the invasion and metastasis of PTC and preliminarily assessed their diagnostic values.Our results can provide a reference for the construction of a better clinical diagnostic model to detect invasion and metastasis of PTC.Materials and methods1.Research design and methods（1）Population studyUrine samples of patients with PTC who had undergone thyroidectomy+lymph node resection/dissection were collected,and the urinary iodine concentration（UIC）was measured.General demographic characteristics,ultrasound findings,clinicopathological characteristics,and thyroid function test results of the patients were obtained from the hospital information management center.Confirmation of invasion and metastasis was used as the research outcome and basis for patient classification for data analysis.PASS 15.0.5 was used to estimate the sample size separately according to the research purposes.According to the standards recommended by the World Health Organization,iodine nutrition levels were divided into excessive（UIC≥300 μg/L）and non-excessive（UIC<300 μg/L）.All participants were informed of the purpose of the study,and all voluntarily participated and provided informed consent.（2）Meta-analysisWe systematically searched PubMed,Web of Science,Scopus,MEDLINE,Wan fang,and China National Knowledge Internet for observational studies on the relationship between UIC and PTC invasion and metastasis published before December 31,2022.Based on Boolean logic operations,we constructed a subject search formula.Relevant literature was selected based on the inclusion and exclusion criteria.（3）Experimental study① Animal model:Four-week-old male BALB/c nude mice were used in this study.K1 cells with good growth condition and in logarithmic growth phase were injected subcutaneously at the back of nude mice.The high iodine level（7.3 mg/L）and control（0 mg/L）groups were set up.The exposure route was drinking water.Two weeks later,the transplanted tumors were dissected and weighed,and the tumor coefficient（weight of transplanted tumor/weight of nude mice × 100）was calculated.②Cell models:PTC cell lines used in this study were K1 and KAT cells.In addition,undifferentiated carcinoma Uhth104 cells were used to compare the specificity of the effects of high iodine levels on PTC cells.The control（0 mM）,high iodine level(10-3 mM),moderate iodine level(10-5 mM),and low iodine level(10-7 mM)groups were set up,and the stimulation time was set at 24 h.Cells were collected from the high iodine level group at 6 h,12 h,and 18 h for the time-series study.Plasmid transfection was used to construct a cell model of SLC4A4 overexpression.The control（0 mM+vector）,high iodine level(10-3 mM+vector),overexpression（0 mM+overexpressing SLC4A4）,and high iodine level+overexpression(10-3 mM+overexpressing SLC4A4)groups were then set up.Metformin was used to construct a cell model to activate the JNK/P38 MAPK signaling pathway.The high iodine level + overexpression + Metformin group(10-3 mM+overexpressing SLC4A4+Metformin)was set up.（4）Bioinformatics analysisWe systematically searched the PTC gene microarray dataset from the Gene Expression Omnibus（GEO）database and selected the microarray based on the inclusion and exclusion criteria.The screening criteria for differentially expressed genes were P<0.05 and |log2 Foldchange|>1.0.The DAVID 6.8 online analysis platform was used for Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis.Gene expression profiling interactive analysis online platforms were used for gene mutation site analysis.Survival analyses were performed based on The Cancer Genome Atlas（TCGA）database.2.Sample collection and detection methods（1）Samples collection:①Preoperative morning urine was collected from all patients and centrifuged.Then,the supernatant was collected and UIC was detected within 6 h.②Tumor and adjacent tissues were collected by experienced pathologists,flash-frozen in liquid nitrogen,stored at-80℃,and detected within 6 months.③Preoperative fasting venous blood was collected from all patients,the plasma（upper）and blood cells（middle brown layer）were separated by centrifugation,stored at-80℃,and detected within 6 months.④Cell lines were collected and pretreated according to the corresponding experimental protocol.（2）Detection methods:①DUIC was detected using the iodine detection kit and a matching iodine detector.②mRNA expression levels of tissue samples,blood cells,and cell lines were detected using real-time quantitative reverse transcription polymerase chain reaction.③Enzyme linked immunosorbent assay was used to detect the protein expression of plasma.④Western blotting was used to detect the protein expression of tissue samples and cell lines.⑤Immunohistochemical staining was used to localize protein expression in tissue samples.⑥Flow cytometry was used to detect the cell cycle.⑦Immunofluorescence was used to localize protein expression in cell lines.⑧Cell proliferation and toxicity detection,clone formation,scratch healing,and migration chamber experiments were used to evaluate the growth,proliferation,invasion,and migration of cell lines,respectively.3.Construction and evaluation of the diagnostic modelThe training and validation sets were created based on the collection time of tissue samples.Cross-validation LASSO regression analysis was used to screen diagnostic factors.Based on the five-fold cross-validation method,logistic regression（LR）,random forest（RF）,and support vector machines（SVM）were used to construct the diagnostic model.Bootstrap resampling methods were used to verify the model stability.Receiver operating characteristic curves were used to evaluate the efficiency of the diagnostic model,calibration curve was used to evaluate the calibration ability,and decision curve analysis was used to evaluate its clinical usefulness.Net reclassification index（NRI）and improved discrimination index（IDI）were used to evaluate the improvement value of the model.4.Basic statistical analysisThe description of basic features is presented as mean ± standard deviation or percentage.Categorical variables were compared using the χ2 test or Fisher’s exact probability method.Continuous variables were compared using the t/F test or Mann-Whitney U rank sum test.Pairwise comparisons within groups were performed using the Bonferroni test or Dunnett’s ttest.The variance inflation factor was used for collinearity diagnosis.LR analysis was used to assess the odds ratio（OR）and 95%confidence interval（CI）between the independent variables and risks of outcomes.For the meta-analysis,Cochran’s test was used to evaluate heterogeneity,one-by-one elimination method was used for sensitivity analysis,and Begg and Egger’s tests were used to evaluate publication bias.The Kaplan-Meier method and log-rank test were used for survival analysis.The Spearman rank sum test was used for linear correlation analysis.All statistical analyses and visualization were conducted using SPSS 22.0（IBM Corp.）,Stata 15.1,R 4.0.1,Graphpad Prism 5.0,and Photoshop 13.0.1 software.A two-tailed P value of<0.05 was considered statistically significant.Results1.The association of excessive iodine levels with invasion and metastasis of PTC1.1.Results of the population studyOverall,300 patients with PTC were included in the study.The proportion of patients with invasive and noninvasive PTC was 73.67%and 26.33%,respectively.In addition,49.67%of patients had metastasis,whereas 50.33%did not have metastasis.The proportion of patients with excessive iodine levels was higher in those with metastasis than in those without metastasis.Excessive iodine was an independent risk factor for PTC metastasis,with an OR（95%CI）of 3.352（2.019-5.565）.However,excessive iodine levels might not be associated with the risk of PTC invasion.1.2.Results of meta-analysisA total of eight qualified articles were included.An excessive iodine level was a risk factor for PTC metastasis,with a combined OR（95%CI）of 1.444（1.088-1.916）.However,excessive iodine levels might not be associated with the risk of PTC invasion.1.3.Results of the animal and cell modelsCompared with the control group,the tumor coefficient of transplanted nude mice in the high iodine group increased,and growth,proliferation,invasion,and migration of K1 and KAT cells in the high iodine group were enhanced;however,there was no significant change in Uhth104 cells.2.The regulatory role of abnormal expression of solute carriers in excessive iodineinduced invasion and metastasis of PTC2.1.Bioinformatics analysis to screen for differential expression of solute carriers SLC34A2,SLC4A4,and SLC25A15Based on the GEO and TCGA databases,three solute carriers that were differentially expressed in PTC tumors and adjacent tissues and were significantly associated with a poor prognosis were identified:SLC34A2,SLC4A4,and SLC25A15.2.2.Abnormal expression of SLC34A2 and SLC4A4 is associated with the risk of PTC metastasis（1）The validation results of a small number of clinical samples showed that SLC34A2 and SLC4A4 were differentially expressed in PTC tumors and adjacent tissues,whereas SLC25A15 expression showed no significant changes.（2）In patients with invasion and metastasis,the expression of SLC34A2 was higher,whereas that of SLC4A4 was lower,compared with patients without invasion and metastasis.（3）High SLC34A2 expression and low SLC4A4 expression were both independent risk factors for PTC metastasis,with ORs（95%Cl）of 4.123（1.194-14.231）and 4.602（1.122-18.873）,respectively.2.3.Excessive iodine levels are associated with abnormal SLC34A2 and SLC4A4 expression（1）The proportions of excessive iodine levels were higher in patients with high expression of SLC34A2 and low expression of SLC4A4.（2）An excessive iodine level was an independent risk factor for both high SLC34A2 expression and low SLC4A4 expression,with ORs（95%CI）of 5.154（1.518-17.501）and 8.291（1.918-35.844）,respectively.2.4 Utilizes the MAPK signal pathway to identify the key solute carrier SLC4A4（1）Compared with adjacent tissues,the expression of p-ERK/ERK,p-JNK/JNK,and pP38/P38 was upregulated by 1.45-fold,1.72-fold,and 1.50-fold in tumor samples,respectively.（2）Negative linear correlations between SLC4A4 expression and JNK and P38 phosphorylation（r:-0.696 and-0.534,respectively）were observed.However,no significant correlations were observed between SLC34A2 and the MAPK signaling pathway.It is suggested that SLC4A4 might be a key solute carrier involved in driving PTC metastasis.In addition,there may be a holistic relationship among excessive iodine levels,low SLC4A4 expression,JNK/P38 MAPK signaling pathway activation,and PTC metastasis.2.5.The regulatory role of SLC4A4 in excessive iodine-induced cells growth,proliferation,invasion,and migration of PTC（1）Exploratory study:①Compared with the control group,SLC4A4 expression in K1 and KAT cells was downregulated in the high iodine level group.②Compared with the control group,p-JNK/JNK and p-P38/P38 expression in K1 and KAT cells was upregulated in the high iodine level group.③In addition,SLC4A4 expression was significantly downregulated at 12 h,whereas p-JNK/JNK and p-P38/P38 expression was significantly upregulated at 18 h in the high iodine level group.This suggests that SLC4A4 might be located upstream of the JNK/P38 MAPK signaling pathways.（2）Validation study:①Compared with the high iodine level group,the growth,proliferation,invasion,and migration of K1 and KAT cells were weakened in the high iodine level+overexpression group,and p-JNK/JNK and p-P38/P38 expression was downregulated.②Compared with the high iodine level+overexpression group,the growth,proliferation,invasion,and migration of K1 and KAT cells were enhanced in the high iodine level+overexpression+Metformin group.（3）Comparative studies:Compared with the control group,SLC4A4 expression and the JNK/P38 pathway showed no significant changes in the high iodine level group of Uhth104 cells.Overexpression of SLC4A4 and activation of the JNK/P38 MAPK signal pathway did not have a significant effect on the growth,proliferation,invasion,and migration of Uhth104 cells.3.Preliminarily assessed the diagnostic value of UIC and solute carriers in the metastasis of PTC3.1 Results of diagnostic factor screening,model construction,and evaluation（1）Age,UIC,TIRADS grade,tumor size,and SLC4A4 were screened as diagnostic factors for metastasis.（2）In the training and verification sets,the areas under the curve（AUC）of the LR,RF,and SVM models were 0.859 and 0.794,0.921 and 0.831,and 0.693 and 0.702,respectively.（3）The RF model performed best but had the worst stability,the SVM model was relatively stable but had the worst performance,and the LR model was relatively stable but had a compromised performance.3.2.Diagnostic value of UIC and SLC4A4 in metastasis of PTCBased on LR,adding UIC and SLC4A4 as diagnostic factors resulted in an increase in the AUC,sensitivity,specificity,positive predictive value,and negative predictive value of the model compared with when only age,TIRADS grade,and tumor size were the diagnostic factors.In the training and validation sets,the NRI and IDI were 0.269 and 0.120,and 0.454 and 0.210,respectively.Conclusion1.In this study,49.67%of patients with PTC had been cleared metastasized lymph nodes,whereas 50.33%had been cleared non-metastasized lymph nodes.2.Excessive iodine levels may increase the risk of PTC metastasis by specifically promoting the growth,proliferation,invasion,and migration of cells.3.SLC4A4 might be the key solute carrier involved in driving PTC metastasis.4.Excessive iodine levels increase the risk of PTC metastasis by downregulating SLC4A4 expression,activating the JNK/P38 MAPK signaling pathway,and promoting PTC cells growth,proliferation,invasion,and migration.5.Adding UIC and SLC4A4 as diagnostic factors can improve the efficiency of the PTC metastatic diagnostic model.In the future,UIC and SLC4A4 are expected to serve as diagnostic markers for PTC metastasis. |
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