The Significant Role And Mechanism Of Long Non-coding RNA DGUOK-AS1 In Papillary Thyroid Carcinoma

Part I A lnc RNA-clinicopathological model for the prediction of central lymph node metastasis in papillary thyroid carcinoma.Objective: The incidence of papillary thyroid carcinoma(PTC)is increasing constantly.Although PTC has a good prognosis,neck lymph node metastasis is common,especially in the central lymph nodes.Prophylactic central lymph node dissection remains controversial.The detection of central lymph node metastasis(CLNM)before operation is of great significance for treatment strategies.However,with the existing medical examination technology,it is difficult to accurately determine whether PTC patients develop CLNM before operation.Long non-coding RNA(lnc RNA)epigenetically regulates expressions of many salient genes involved in vital cellular biological processes such as cell apoptosis,proliferation,migration,and invasion,and affects tumor development as an oncogene or tumor suppressor by directly or indirectly regulating tumor-related signaling pathways.Moreover,lnc RNA can be easily obtained from tissues and body fluids and thus can be used as diagnostic tools.This part of the study aims to construct a prediction model for CLNM in PTC patients based on clinical characteristics and the identified lnc RNAs to facilitate an individualized decision for surgical plans.Methods: 1.RNA-sequencing datasets and clinical data of PTC patients from the Cancer Genome Atlas(TCGA)database were downloaded and analyzed to identify differentially expressed lnc RNAs(DElnc RNAs).2.DElnc RNAs were put into univariate,Lasso,and then multivariate analyses to find potential risk lnc RNAs related to CLNM.The coefficients of the risk lnc RNAs from multivariate logistic regression analysis were used to calculate lnc RNA-based risk score of CLNM.The diagnostic performance of lnc RNAbased risk score tool was evaluated by receiver operating characteristic(ROC)curve analysis.3.The potential clinical risk factors of CLNM and the lnc RNA-based risk score were analyzed by univariate and multivariate analyses.A nomogram to predict CLNM was established based on the multivariate logistic analysis.ROC curves,calibration curves,and decision curve analysis were used to evaluate the performance of the prediction model for CLNM.Results: 1.A total of 1583 lnc RNAs were significantly dysregulated in PTC tissues compared to normal tissues.2.A total of 234(75% of the 312 patients)PTC patients in the training set and 78(25% of the 312 patients)PTC patients in the testing set were randomly generated with a ratio of 3:1.Testing set was used to evaluate the predictive performance of the model developed from the data in the training set.The univariate,Lasso,and multivariate analyses results showed that four lnc RNAs(AC009812.4,DGUOK-AS1,AC009549.1,and AP002358.1)can independently predict CLNM.The lnc RNA-based risk score in N0 group was significantly lower than that in N1 a group.ROC curves suggested that the risk score tool performed well in terms of CLNM prediction.The areas under the ROC curves(AUC)of training set and testing set were 0.744 and 0.713,respectively.3.A prediction model for CLNM including age,ETE,stage T,pathological subtypes,and risk score was constructed.ROC curves,calibration curves,and decision curve analysis showed the great predictive performance of CLNM.The AUCs of training set and testing set were0.78 and 0.785,respectively.Conclusion: This is the first prediction model for CLNM in PTC patients based on lnc RNA sequencing data and clinical characteristics.The model has a strong ability to predict CLNM,which is helpful for clinicians to formulate surgical strategies and lays a foundation for the clinical application of this research.Part II Expression of DGUOK-AS1 in papillary thyroid carcinoma and correlation between DGUOK-AS1 and clinicopathological factors Objective: Among the four lnc RNAs significantly related to CLNM,DGUOK-AS1 was selected for further study.Previous studies have found that DGUOK-AS1 plays a carcinogenic role in cervical cancer,breast cancer,and lung squamous cell carcinoma,while the role of DGUOK-AS1 in PTC has not been reported.In this part,the study aims to explore the expression of DGUOK-AS1 in paired PTC tissues and adjacent tissues,as well as peripheral blood of PTC patients and normal controls,and analyze the relationship between the expression of DGUOK-AS1 and clinicopathological factors.Methods: 1.The pathological specimens of patients who underwent radical resection of PTC were collected,including cancer tissues and adjacent tissues.Peripheral blood samples were collected from PTC patients and normal subjects.2.q RT-PCR was used to detect the expression of DGUOK-AS1 in PTC tissues and adjacent tissues,as well as in PTC patients and normal controls.3.The correlation between the expression of DGUOKAS1 in tumor tissues and peripheral blood of PTC patients and the corresponding clinicopathological factors were analyzed.The diagnostic value of DGUOK-AS1 in PTC was analyzed by ROC curve.Results: 1.A total of 24 pairs of PTC tissues and adjacent tissues were included in this study.q RT-PCR showed that DGUOK-AS1 expression level was higher in tumor tissues than in adjacent healthy tissues in most PTC patients(P<0.05),but DGUOK-AS1 expression was not significantly correlated with clinicopathological factors.2.In this study,peripheral blood samples were collected from 30 PTC patients and 30 normal controls.q RT-PCR showed that the level of DGUOK-AS1 in peripheral blood of PTC patients was significantly higher than that of normal controls(P<0.05),and low expression of DGUOK-AS1 was associated with CLNM in PTC patients(P<0.05).3.ROC curve analysis showed that the AUC of DGUOK-AS1 in peripheral blood for the diagnosis of PTC was 0.671(95%CI=0.532-0.811,P=0.022),the sensitivity was 46.67%,and the specificity was 90.00%.The AUC for predicting CLNM was 0.790(95%CI=0.602-0.977,P=0.012).When the cut-off value of DGUOK-AS1 expression was 0.77,the sensitivity was 90.00% and the specificity was 73.68%.Conclusion: DGUOK-AS1 is highly expressed in the tumor tissues and peripheral blood of PTC patients.DGUOK-AS1 in peripheral blood may be a potential biomarker for the diagnosis of PTC and the prediction of CLNM.DGUOK-AS1 plays a carcinogenic role in PTC and affects the occurrence and progression of PTC.Part III lnc RNA DGUOK-AS1 regulates the biological function of papillary thyroid carcinoma Objective: The purpose of this part is to investigate the carcinogenic role of DGUOK-AS1 in PTC and the effect of its expression on the proliferation,migration,invasion,and apoptosis of PTC cells.Methods: 1.q RT-PCR was used to verify DGUOK-AS1 expression in normal thyroid cell(Nthy-ori 3-1)and PTC cells(KTC-1,BCPAP,K1,and TPC-1).2.The effects of DGUOKAS1 knockdown on the proliferation,apoptosis,migration,and invasion of KTC-1 and BCPAP cells were detected by CCK8 proliferation assay,colony formation assay,flow cytometry,Western Blot assay,wound healing assay,and Transwell migration and invasion assay in vitro.3.The effect of DGUOK-AS1 knockdown on the tumorigenic ability of PTC cells was detected by nude mice xenografts in vivo.Results: 1.Compared to the normal thyroid cell,DGUOK-AS1 expression was significantly higher in PTC cells,and then KTC-1 and BCPAP cells were selected for subsequent experiments.2.DGUOK-AS1 knockdown suppressed cell proliferation,migration,and invasion of PTC cells and promoted apoptosis of PTC cells in vitro.3.The nude mice xenografts showed that the tumorigenic ability of BCPAP cell was inhibited after DGUOK-AS1 knockdown in vivo.The results of immunohistochemistry showed that the proliferation of BCPAP cell was suppressed and the apoptosis was promoted.Conclusion: DGUOK-AS1 can promote the proliferation,migration,and invasion of PTC cells and suppress cell apoptosis.DGUOK-AS1 plays a carcinogenic role in PTC.Part IV Study on the mechanism of lnc RNA DGUOK-AS1 regulating the biological function of papillary thyroid carcinoma Objective: The purpose of this part is to investigate the mechanism of DGUOK-AS1 in the carcinogenesis of PTC cells.Methods: 1.The subcellular location of DGUOK-AS1 was evaluated by nuclear/cytosol fractionation assay and fluorescence in situ hybridization(FISH)assay.2.Potential target mi RNAs of DGUOK-AS1 were identified according to the ENCORI database.q RT-PCR was used to detect the expression of mi RNA in PTC tissues and PTC cells,as well as in PTC cells after DGUOK-AS1 knockdown.Dual luciferase reporter assay was used to verify the binding of mi RNA and DGUOK-AS1.3.PTC cells were transfected with siDGUOK-AS1 and mi RNA inhibitor and co-transfected with si-DGUOK-AS1 and mi RNA inhibitor.The changes of proliferation,apoptosis,migration,and invasion of PTC cells were detected by CCK8 proliferation assay,colony formation assay,flow cytometry,Western Blot assay,wound healing assay,and Transwell migration/invasion assay.4.Target Scan and mi RDB databases were used to predict the target genes of mi RNA and intersect with the DEm RNAs.The expression of target genes and their correlation with DGUOK-AS1 expression were detected in the TCGA database and clinical tissue specimens,and the significantly related target genes were screened out.5.The expressions of target genes at m RNA and protein levels were detected by q RT-PCR and Western Blot in clinical tissue specimens and PTC cell lines,respectively.6.Dual luciferase reporter assay was used to verify the targeted binding of target gene and mi RNA.7.The expression changes of target genes at m RNA and protein levels in PTC cells after transfection with siDGUOK-AS1,mi RNA inhibitor,and co-transfection with si-DGUOK-AS1 and mi RNA inhibitor were detected by q RT-PCR and Western Blot.Results: 1.DGUOK-AS1 was predominantly distributed in the cytoplasm.2.mi R-499a-5p was identified as a potential target of DGUOK-AS1.The expression of mi R-499a-5p was significantly lower in PTC tissues and PTC cells.Dual luciferase reporter assay verified that mi R-499a-5p had a binding site with DGUOK-AS1,and DGUOK-AS1 knockdown could up-regulate the expression of mi R-499a-5p.3.Suppression of mi R-499a-5p could promote proliferation,migration,and invasion and suppress apoptosis of KTC-1 and BCPAP cells.mi R-499a-5p inhibitor could partly abrogate the DGUOK-AS1 knockdownmediated suppression of proliferation,migration,and invasion and promotion of apoptosis in PTC cells.4.The expression of NOVA1 was positively associated with the expression of DGUOK-AS1 in PTC tissues,and the expression of NOVA1 was elevated in PTC cells compared with normal thyroid cells.5.While DGUOK-AS1 knockdown led to decreased expression of NOVA1,mi R-499a-5p inhibitor was able to reverse the decreased expression of NOVA1.These results suggest that DGUOK-AS1 could protect NOVA1 expression from mi R-499a-5p-mediated degradation.Conclusion: DGUOK-AS1 regulates the biological function of PTC cells and plays a carcinogenic role in PTC by sponging mi R-499a-5p to up-regulate NOVA1.