LncRNA NONHSAT162346.1 Promotes Papillary Thyroid Carcinoma Metastasis By Binding To KRT16

Objective: Thyroid cancer is one of the most common malignancies of endocrine system,and papillary thyroid carcinoma(PTC)is the most common subtype.Most low-risk patients with PTC have a good prognosis,as for PTC,relapse and metastasis after treatment are the main causes of poor prognosis.The relatively good prognosis and increasing incidence of PTC has attracted the attention of scholars around the world and led to the discussion of over-diagnosis and treatment.At the same time,TSH and Tg are commonly used to evaluate the risk and prognosis of PTC in clinical practice.By establishing the association between clinical and biochemical characteristics and molecular markers,it is helpful to more effectively evaluate and classify PTC patients with different characteristics.Therefore,exploring related molecules and mechanisms,combining molecular diagnosis with existing clinical diagnostic methods,is an effective method to distinguish low-risk PTC from high-risk PTC which is easily metastasis and develop different diagnosis and treatment schemes.Long noncoding RNA(Lnc RNA)is a class of non-coding RNA with a length of more than 200 nucleotides which is similar in structure to m RNA.Lnc RNA,which usually regulate gene expression in the form of RNA,are highly tissue and cell specific,thus showing potential as molecular diagnostics.A large number of studies have confirmed that lnc RNA can play a regulatory role in tumor biological behavior through various mechanisms,such as m RNA expression through ce RNA mechanism,modification and rearrangement of genes and chromatin,binding to specific proteins to regulate or change the activity of corresponding proteins,influence m RNA modification and degradation,and translate peptides.Studies on lnc RNAs are not as mature as m RNAs,so more studies are needed to reveal the function and mechanism of lnc RNA.Thyroid stimulating hormone(TSH)is synthesized by the pituitary gland and regulates the function and growth of thyroid cells.Abnormal activation of TSH signaling may lead to a variety of thyroid-related diseases,including thyroid cancer.In vitro studies indicated that TSH was found to stimulate tumor cell proliferation either directly or indirectly.It has also been found that this effect is bidirectional: at low concentrations of TSH,thyroid cell proliferation is significantly promoted by TSH,while at concentrations beyond physiological TSH,cell proliferation and differentiation potential are inhibited.This study aims to discover lnc RNA related to the progression and clinical characteristics of PTC,study their functions,and further explore the specific mechanism of their functions.Methods:Part Ⅰ: From July 2020 to May 2021,70 pairs of papillary thyroid cancer with paraneoplastic specimens were collected from the Department of Thyroid Surgery,The First Affiliated Hospital of China Medical University to form the clinical cohort for this study with the approval of the Ethics Committee of The First Affiliated Hospital of China Medical University.The ln RNA fraction of the whole transcriptome sequencing data from this group was selected for initial selection based on log2 Ratio and FPKM values,and then further screening was performed by q RT-PCR in PTC tissues and cell lines.The expression level of NONHSAT162346.1in tissues was detected using q RT-PCR,and the cardinality test and pearson correlation were used to analyze the expression of NONHSAT162346.1 in PTC and correlation with clinical and pathological data as well as biochemical indices.NONHSAT162346.1 cellular localization was detected using prediction sites and nucleoplasmic separation assays.PartⅡ: PTC cell lines were stimulated with a concentration gradient of TSH(0,0.1,1,10 m IU/m L)and the effect of TSH on the proliferation and metastasis of PTC cells was observed by Ed U,CCK8,Transwell,q RT-PCR and Western Blot.q RT-PCR was used to detect the effect of TSH on the expression of NONHSAT162346.1.PTC cell lines TPC1 and K1 were transfected using si RNA and overexpression lentivirus.q RT-PCR was used to verify the transfection efficiency.q RT-PCR and Western Blot were used to verify the effect of up-regulation of NONHSAT162346.1 on the expression of cell proliferation and metastasis markers PCNA,MMP2,MMP9 and E calmodulin.The effects of NONHSAT162346.1 on PTC cell proliferation and metastasis were observed by Ed U,CCK8 and Transwell in PTC cell lines TPC1 and K1 with up-and down-regulation of NONHSAT162346.1.Co-transfection of TSH and si-NONHSAT162346.1 by Ed U,CCK8,and Transwell verified the targeting and regulating effect of TSH on NONHSAT162346.1.PartⅢ: chirp-MS analysis was performed to detect proteins that may bind to NONHSAT162346.1,and possible enriched functions and pathways were analyzed by GO and KEGG.Possible correlations were explored by interactions and co-expression analysis of chirp results with the whole transcriptome data of our group in the previous phase.Exploration of co-transcription factors and differential expression of each transcription factor in transcriptome data by co-analysis of chirp results with whole transcriptome data from our group.q RT-PCR and Western Blot to verify the regulatory relationship of NONHSAT162346.1 on target proteins.Expression of KRT16 was examined by public databases,clinical tissues and PTC cell lines using q RT-PCR,immunohistochemistry,Western Blot and nucleoplasmic separation assays.Bioinformatics analysis was performed to correlate KRT16 with clinical features,analyze the effect of KRT16 expression on survival outcome,analyze co-expressed genes of KRT16 and correlation analysis.Plasmid transfection overexpression of KRT16 was performed using Ed U,CCK8,Transwell,q RT-PCR and Western Blot to detect the transfection efficiency,the effect of KRT16 on the proliferation and metastasis of PTC cells.Targeted regulation of KRT16 by NONHSAT162346.1 was demonstrated in PTC cell lines cotransfected with KRT16 and ov-NONHSAT162346.1 using CCK8 and transwell assays.Results:Part Ⅰ: There are many significantly differentially expressed lnc RNAs in PTC and normal thyroid tissue.22 differentially expressed lnc RNAs were obtained by the ploidy change analysis method,followed by validation in PTC tissue to obtain four differentially expressed lnc RNAs: NONHSAT159017.1,NONHSAT161218.1,NONHSAT162346.1,and NONHSAT162346.1.The mean age of the clinical cohort was 42 years and the mean maximum tumor diameter was 1.85 cm.All biochemical parameters except TPOAb and Tg Ab were within the normal range.NONHSAT162346.1 was significantly highly expressed in the tissues of PTC patients,and the high expression of NONHSAT162346.1 was significantly and positively correlated with TSH and Tg.NONHSAT162346.1 expression was higher in those with lymph node metastasis and in those younger than 55 years of age.NONHSAT162346.1 expression was higher in the PTC cell lines TPC1 and K1 and was mainly localized in the cytoplasm.Part Ⅱ : TSH at 0.1 m IU/m L significantly promoted the proliferation and metastasis of PTC cells in vitro,and TSH at 10 m IU/m L inhibited the promotion of PTC cell proliferation and metastasis brought by TSH at 0.1 m IU/m L.The concentration gradient of TSH brought promotion and then inhibition to NONHSAT162346.1.This promotion effect was most obvious when the TSH concentration was 0.1 m IU/m L.Transfection validation experiments on NONHSAT162346.1 demonstrated the effectiveness of silencing and overexpression.0.1 m IU/m L of TSH stimulation and upregulation of lnc RNA promoted the expression of PCNA,MMP2,MMP9,etc.Downregulation of NONHSAT162346.1inhibited the proliferation and metastasis of PTC cells,and upregulation of NONHSAT162346.1 had the opposite effect.We also found that downregulation of NONHSAT162346.1 could partially attenuate the promotion of proliferation and migration ability of PTC cells brought about by TSH stimulation.PartⅢ: The chirp assay demonstrated that NONHSAT162346.1 binds a large number of specific proteins.We found that the results of GO and KEGG analyses of the subgroups mainly focused on keratinization,neutrophil degranulation,proteins targeting estrogen receptors,endoplasmic reticulum localization of proteins,and intermediate filament organization,while the results of KEGG mainly focused on estrogen signaling pathways,ribosomes,and amino acid biosynthesis.Interactions and co-expression analysis with whole-transcriptome sequencing data from our group revealed that the expression patterns of genes in the six groups were very comparable,mainly concentrated in three clusters,respectively,ribosome-associated cluster,mitochondrial respiration-associated cluster,and keratin-associated cluster.co-transcription factor prediction of chirp data and whole-transcriptome data indicated that KRT family members and ribosome family members were enriched with a large number of co-transcription factors.Based on these results,13 alternative targets were identified.q RT-PCR and Western Blot results indicated that KRT16 was expressed in the same trend as NONHSAT162346.1.Overexpression of NONHSAT162346.1positively regulates KRT16 at the m RNA and protein levels,while silencing has the opposite effect.KRT16 is correlated with tumor staging and grading and has the ability to predict prognosis,and there are many genes co-expressed with KRT16.KRT16 is significantly upregulated in PTC tissues and cell lines,localized in the cytoplasm and nucleus,and overexpression of KRT16 significantly promotes proliferation and metastasis of PTC cells.si-KRT16 partially alleviates the promotion of proliferation and metastatic capacity of PTC cells brought about by the upregulation of NONHSAT162346.1.Conclusions: A large number of differentially expressed lnc RNAs were present in PTC,among which Lnc RNA NONHSAT162346.1 was significantly highly expressed in tumor tissues.It was positively correlated with age,lymph node metastasis,TSH and Tg.TSH can play a synergistic role with Lnc RNA NONHSAT162346.1 to promote proliferation and metastasis of papillary thyroid.The gene expression pattern of the binding protein of Lnc RNA NONHSAT162346.1 has a great number of similarities with the transcriptome results of our group.NONHSAT162346.1 can regulate KRT16 expression and promote tumor progression together with KRT16 through the mechanism of binding to KRT16 and is a potential biomarker for PTC.