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. 2020 Oct 15;15(2):237–250. doi: 10.1007/s12079-020-00589-w

Long non-coding RNA DIO3OS/let-7d/NF-κB2 axis regulates cells proliferation and metastasis of thyroid cancer cells

Mingming Wang 1, Jin Li 2, Zhongkun Zuo 1, Chutong Ren 1, Tenglong Tang 1, Chen Long 1, Yi Gong 1, Fei Ye 1, Zhihong Wang 1, Jiangsheng Huang 1,✉
PMCID: PMC7990978  PMID: 33058043

Abstract

Due to the steadily rising morbidity and mortality, thyroid cancer remains the most commonly seen endocrine cancer. The present study attempted to investigate the mechanism from the perspective of long non-coding RNA (lncRNA) regulation. We identified 53 markedly increased lncRNAs in thyroid cancer samples according to TCGA data. Among them, high lncRNA DIO3OS expression was a risk factor for thyroid cancer patients’ poorer overall survival. DIO3OS showed to be considerably increased within thyroid cancer tissue samples and cells. Knocking down DIO3OS within thyroid carcinoma cells suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, as well as cell migration; besides, proliferating markers, ki-67 and PCNA, were decreased by DIO3OS knockdown. Cancer bioinformatics analysis suggested that NF-κB2 might be related to DIO3OS function in thyroid cancer carcinogenesis. NF-κB2 was positively correlated with DIO3OS, and DIO3OS knockdown decreased NF-κB2 protein levels. Knocking down NF-κB2 within thyroid carcinoma cells suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, cell migration, and the protein levels of proliferating markers. Let-7d directly targeted DIO3OS and NF-κB2; DIO3OS knockdown upregulated let-7d expression. The overexpression of let-7d suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, cell migration, as well as the protein levels of proliferating markers. Let-7d inhibition remarkably attenuated the functions of DIO3OS knockdown in NF-κB2 expression and thyroid cancer cell phenotype. In conclusion, DIO3OS/let-7d/NF-κB2 axis regulates the viability, DNA synthesis capacity, invasion, and migration of thyroid cancer cells. The clinical application of this axis needs further in vivo and clinical investigation.

Electronic supplementary material

The online version of this article (10.1007/s12079-020-00589-w) contains supplementary material, which is available to authorized users.

Keywords: Thyroid cancer, Long non-coding RNA (lncRNA) DIO3OS, Let-7d, NF-κB2

Introduction

Thyroid cancer is the most commonly seen endocrine cancer, with continuously increasing morbidity and mortality over the past decades (Zaballos and Santisteban 2017). Briefly, thyroid cancers are derived from two types of endocrine thyroid cells, follicular thyroid cells, and parafollicular C cells. PTC (papillary thyroid cancer), FTC (follicular thyroid cancer), PDTC (poorly differentiated thyroid cancer), and ATC (anaplastic thyroid cancer) are four types of cancers derived from follicular thyroid cells, accounting for the majority of thyroid malignancies (Fusco et al. 1987; Nikiforova et al. 2009). Considering the complexity and multiformity of thyroid cancer origin (Zhang et al. 2013b), to investigate the molecular mechanisms underlying the carcinogenesis of thyroid tumors has emerged as the basis of more potent strategies for diagnosing and treating.

By using high-throughput sequencing technologies, studies have found that only less than 2% of the transcripts could encode proteins; most of the rest showed to be transcribed as ncRNAs (non-coding RNAs) unable to encode proteins, mainly including miRNAs (microRNAs) and lncRNAs (long non-coding RNAs) (Kaikkonen and Adelman 2018). LncRNAs could transcriptionally and posttranscriptionally regulate gene expression, subsequently contributing to chromatin remodeling, RNA decay, epigenetic regulation, chromatin modification, and many other cell functions (Djebali et al. 2012; Wang and Chang 2011). Through exerting different cellular functions, lncRNAs participate in the formation, invasion, and metastasis of malignancies (Li et al. 2018; Mou et al. 2018). Several lncRNAs have previously been reported to modulate the capacity of thyroid cancer cells to proliferate, invade, and migrate (Du et al. 2017; Murugan et al. 2018; Sedaghati and Kebebew 2019). For example, increased lncRNA H19 expression and the decrease in miRNA let-7a expression might be linked to impaired prognosis within thyroid cancer patients (Liu et al. 2017a). LncRNA NEAT1 modulates the expression of miR-129-5p/KLK7 to regulate the development of papillary thyroid cancers (Zhang et al. 2018). Notably, by using cancer bioinformatics, a helpful method that allows researchers to confirm potentially critical cancer genes and pathways via database mining, studies revealed many deregulated lncRNAs within thyroid carcinoma, indicating that identifying lncRNAs related to thyroid cancer carcinogenesis and the potential mechanisms could lead to new treatment strategies for thyroid cancer.

Although lncRNAs function through diverse mechanisms, there is increasing evidence that lncRNAs could competitively bind to miRNAs via their MRE (miRNA response elements) to act as ceRNAs (competing endogenous RNAs), thereby modulating the expression of target RNAs (Salmena et al. 2011). Through this ceRNA mechanism, there has been a lot of interest in the occurrence and development of several malignant tumors, including gastric carcinoma (Song et al. 2018), thyroid carcinoma (Zhao et al. 2018), and hepatoblastoma (Liu et al. 2017b). Moreover, multiple potential ceRNA regulatory interactions have been identified in thyroid cancer. For example, within papillary thyroid carcinoma, lncRNA ABHD11-AS1 has been reported to enhance the capacity of cancer cells to proliferate, migrate, and invade, inhibit cell apoptosis in vitro, promote the occurrence of tumors in vivo through sponging miR-199a-5p, subsequently inducing the activation of SLC1A5 (Zhuang et al. 2019). Since online data also report a large amount of differentially-expressed protein-coding mRNAs, we performed more bioinformatic analyses and experiments attempting to identify the lncRNA-miRNA-mRNA axis that could modulate the thyroid cancer carcinogenesis.

In the present study, we analyzed mRNA (including ncRNA) sequencing data reporting differentially-expressed genes in thyroid cancer from The Cancer Genome Atlas (TCGA) database to identify lncRNAs related to thyroid cancer patient’s overall survival and lncRNA DIO3OS was selected. Next, the expression and functions of DIO3OS in thyroid cancer cells were examined. Then, protein-coding mRNAs related to DIO3OS were analyzed, and NF-κB2 was selected. The specific roles of NF-κB2 modulating the capacity of thyroid carcinoma cells to proliferate, invade, and migrate were investigated. Furthermore, miRNAs that might simultaneously target DIO3OS and NF-κB2 were analyzed, and three candidates were selected. The expression of four candidate miRNAs was examined in thyroid cancer tissues, and let-7d was selected. The predicted let-7d binding to DIO3OS and NF-κB2, as well as the specific effects of let-7d on thyroid cancer cells, were investigated. Finally, the dynamic effects of DIO3OS and let-7d combination on NF-κB2 expression and thyroid cells were detected (Study design flow chart was shown in Fig. S1). In summary, we attempted to determine a novel lncRNA-miRNA-mRNA axis that modulates the capacity of thyroid carcinoma cells to proliferate, invade, and migrate, therefore affecting the carcinogenesis of thyroid carcinoma.

Materials and methods

Clinical tissue sampling

Ten cases of thyroid cancer tissues and ten cases of adjacent normal tissues were obtained from 10 patients who received surgical resection at The Second Xiangya Hospital of Central South University with the approval of the Ethics Committee of The Second Xiangya Hospital of Central South University. The informed consent form was signed by each patient involved. All the tissue samples were snap-frozen and stored at −80 °C.

Cell lines and cell transfection

Normal human primary thyroid follicular epithelial cell line, Nthy-ori 3–1, was obtained from Sigma-Aldrich (CB_90011609; St. Louis, MI, USA) and cultured in RPMI 1640 supplemented with 2 mM Glutamine and 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA). Human thyroid papillary carcinoma cell line, TPC-1 cell line was purchased from Sigma-Aldrich (SCC147) and cultured in RPMI-1640 medium supplemented with 10% FBS. Human thyroid carcinoma cell line 8505C was obtained from the European Collection of Authenticated Cell Cultures (ECACC 94090184; Salisbury, UK) and cultured in EMEM (HBSS) supplemented with 2 mM Glutamine, 1% non-essential amino acids, and 10% FBS. Human thyroid carcinoma cell line SW1736 was purchased from the Tumor Cell Bank of the Chinese Academy of Medical Science (Shanghai, China) and cultured in RPMI-1640 medium supplemented with 10% FBS. Human thyroid cancer papillary cell line BCPAP was purchased from the ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS. All the cell lines were cultured in the corresponding medium supplemented with streptomycin (100 U/ml) and penicillin sodium (100 U/ml) at 37 °C in 5% CO2.

LncRNA DIO3OS knockdown was generated in cells by the transfection of si1-DIO3OS or si2-DIO3OS (GenePharma, Shanghai, China). NF-κB2 knockdown in cells was generated by the transfection of si1-NF-κB2 and si2- NF-κB2 (GenePharma). let-7d overexpression or inhibition in cells was generated by the transfection of let-7d mimics or let-7d inhibitor (GenePharma, Shanghai, China). All cell transfection was performed using Lipofectamine 3000 Reagent (Thermo Fisher Scientific). The sequences were listed in Table S1.

PCR-based analysis

The expression of lncRNA, miRNA, and mRNA was determined by real-time PCR. Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen). The expression of lncRNA, miRNA, and mRNA was measured using sybr green qPCR assay (Takara, Dalian, China) following the methods described before (Liu et al. 2018). The expression of RNU6B or GAPDH served as an endogenous control. The 2-ΔΔCT method was applied for data processing. The primer sequences were listed in Table S1.

Cell viability examined by CCK-8 assay

Cell viability examination was performed using a CCK-8 kit (Beyotime, Shanghai, China). After transfection or treatment, cells were seeded into 96-well plates at a density of 5 × 103 cells/well. 20 μl of CCK-8 solution was added to each well followed by the incubation for 2 h at cell culture incubator. The Optical density (OD) value was determined at the wavelength of 450 nm on a microplate reader.

DNA synthesis assays

The ability of DNA synthesis was detected by EdU (5-ethynyl-2′-deoxyuridine) assays using an EdU assay kit (RiboBio, Guangzhou, China), according to the manufacture’s instruction. Nuclei were stained with DAPI. Cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan). The EdU positive cells rate were measured from three random fields of each group using the ImageJ software (NIH, USA).

Transwell assay

Cells (5 × 105) were planted on the top side of polycarbonate Transwell filters coated with Matrigel for invasion examination. For Transwell invasion assays, cells were suspended in medium without serum, and medium with serum was used in the bottom chamber. The cells were incubated at 37 °C for 48 h. The non-invasive cells in the top chambers were removed with cotton swabs. The invaded cells on the lower membrane surface were fixed in 100% methanol for 10 min, air-dried, stained with crystal violet solution, and then counted under a microscope.

Wound healing assay

Cells were seeded in 6-well plates at 5 × 105 cells/ml until the cell monolayer emerged, and then the cell wound healing assay was performed. Briefly, cells were pretreated with 10 μg/mL mitomycin C (Sigma, USA) for 2 h. Then, a yellow pipette tip was used to make a straight scratch in the well. Then, the culture media were changed to 1% FBS RPMI-1640. The scratch area was measured under the microscope (Olympus, Japan) at 0 h and 24 h. The relative distance of cell migration to scratch area was also measured under the microscope and analyzed by ImageJ software (NIH, USA).

Immunoblotting

The protein levels of ki-67, proliferating cell nuclear antigen (PCNA), and NF-κB2 were determined by performing Immunoblotting analyses following the methods described before (Liu et al. 2018). Protein blots were incubated with the primary antibodies against ki-67 (27309–1-AP, Proteintech, Rosemont, IL, USA), PCNA (ab29, Abcam, Cambridge, UK), NF-κB2 (#4882, Cell Signaling, Danvers, MA, USA) followed by another incubation with proper HRP-conjugated secondary antibodies. β-actin (6008–1-Ig, Proteintech) was used as an endogenous control. All the antibodies were obtained from Abcam unless otherwise noted. Signals were visualized using enhanced chemilumescent (ECL) substrates (Millipore, MA, USA) normalizing to GAPDH.

Enzyme-linked immunosorbent assay (ELISA)

Cells were lysed in Cell Extraction Buffer PTR for 20 min on ice and then centrifuged 18,000 x g for 20 min at 4 °C. The supernatants were transferred into clean tubes and store at −80 °C until use. The total protein concentration in the extracts was determined by the BCA protein assay kit (Beyotime, China). The levels of VCAM1 and ICAM-1 in cells were determined by VCAM and ICAM-1 Human SimpleStep ELISA kits (ab223591 and ab174445, Abcam, USA) according to the manufacture’s instruction.

Immunohistochemical (IHC) staining

Immunohistochemistry was performed to examine the distribution and protein contents of NF-κB2 in tissue samples. Tissue sections were incubated with anti-NF-κB2 (#4882, Cell Signaling). Image Pro-plus 6.0 software (Media Cybernetics, USA) was used to assess immunohistochemical sections by measuring the integrated optical density.

Luciferase reporter assay

To validate the binding between let-7d and DIO3OS or 3’UTR of NF-κB2, the wild-type or mutated DIO3OS or 3’UTR of NF-κB2 was cloned to the downstream of the Renilla psiCHECK2 vector by PCR (Promega, Madison, WI, USA), named wt-DIO3OS or wt-NF-κB2 3’UTR or mut-DIO3OS or mut-NF-κB2 3’UTR. The primers for reporter vectors construction were listed in Table S1. Next, 293 T cells were co-transfected with two types of luciferase reporter vectors and let-7d mimics/let-7d inhibitor. Forty-eight hours later, cells were lysed and examined for the luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) following the manufactory’s instruction. Renilla luciferase activity served as a normalization control.

Data processing and statistical analysis

All experiments were implemented at least three independent times. The data were analyzed with GraphPad software. The measurement data were expressed as mean ± standard deviation (SD). All data were analyzed by the Kolmogorov-Smirnov test for normal distribution. Among-group and intra-group data comparisons were performed with the ANOVA and Student’s t-tests. P < 0.05 indicated a statistically significant difference.

Results

DIO3OS expression in thyroid carcinoma and correlation with metastases of thyroid carcinoma

To identify lncRNAs involved in thyroid cancer carcinogenesis, we analyzed the mRNA (including ncRNA) sequencing data and clinical characteristics of 499 cases of thyroid cancer sample from the TCGA database and found that 53 ncRNA were significantly overexpressed in thyroid cancer (data not shown). Further, the correlation between these 53 ncRNAs and the patient’s overall survival was analyzed, respectively, and the expression of DIO3OS was significantly linked to thyroid cancer patients’ overall survival (Fig. 1a). A higher DIO3OS expression was a risk factor (CoxHP Hazard Ratio = 2.195, p = 2.113e-03).

Fig. 1.

Fig. 1

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DIO3OS expression in thyroid cancer and correlation with thyroid cancer metastases. a A total of 499 cases of thyroid cancer patients from TCGA database were grouped by DIO3OS expression. The correlation between DIO3OS expression and the overall survival in these patients was analyzed using Cox-proportional-hazards model (CoxPH). b Expression of DIO3OS was determined in 10 paired of thyroid cancer and normal non-cancerous tissues by real-time PCR. N = 10, ***P < 0.001. c Expression of DIO3OS was determined in one normal human primary thyroid follicular epithelial cell line, Nthy-ori 3–1, and four thyroid cancer cell lines, BCPAP, TPC-1, 8505C, and SW1736, by real-time PCR. N = 3, *P < 0.05, ***P < 0.001. d Expression of DIO3OS in tissue samples derived from different pathological processes. e The correlation between DIO3OS expression and the number of lymph nodes with metastasis analyzed by Pearson’s correlation analysis. f Expression of DIO3OS in tissue samples from lymph node metastasis patients and no metastasis patients

Before investigating the specific effect of DIO3OS on thyroid carcinoma, firstly, we performed real-time PCR to examine DIO3OS expression within 10 paired of thyroid carcinoma and normal healthy tissue samples. Figure 1b showed that the expression of DIO3OS was considerably increased within thyroid carcinoma tissues, in comparison with that in normal healthy tissue samples. To select proper cell line for further experiments, we then examined the expression of DIO3OS in one normal human primary thyroid follicular epithelial cell, Nthy-ori 3–1, as well as four thyroid carcinoma cells, BCPAP, TPC-1, 8505C, and SW1736, by real-time PCR. Figure 1c demonstrated that DIO3OS expression showed to be dramatically upregulated within all the four thyroid carcinoma cells, more upregulated within BCPAP and TPC-1 cells. Thus, BCPAP and TPC-1 cell lines were selected for further experiments. As further confirmation, DIO3OS expression was significantly upregulated, along with the development of thyroid cancer pathological processes (Fig. 1d). DIO3OS expression was positively correlated with the number of lymph nodes with metastasis, as analyzed by Pearson’s correlation analysis according to TCGA data (Fig. 1e). The expression of DIO3OS was upregulated in tissue samples from lymph node metastasis patients compared to no metastasis patients’ tissue samples (Fig. 1f).

Effects of DIO3OS on thyroid carcinoma cells

To validate the specific effects of DIO3OS on thyroid carcinoma cells, we transfected si1-DIO3OS/si2-DIO3OS to generate DIO3OS knockdown in BCPAP and TPC-1 cells, and performed real-time PCR to verify the transfection efficiency; si2-DIO3OS shown a better transfection efficiency, so we selected it in further experiments (Fig. 2a). Next, we transfected BCPAP and TPC-1 cell lines with si2-DIO3OS and examined for related indexes. DIO3OS knockdown significantly inhibited BCPAP and TPC-1 cell viability, the capacity of DNA synthesis, cell invasion capacity, and cell migration (Fig. 2b–e). Moreover, the protein contents of proliferating markers, ki-67, and PCNA, also showed to be significantly decreased via DIO3OS knockdown (Fig. 2f).

Fig. 2.

Fig. 2

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Effects of DIO3OS on thyroid cancer cells. a DIO3OS knockdown was generated in BCPAP and TPC-1 cells by the transfection of si1-DIO3OS or si2-DIO3OS, as confirmed by real-time PCR. Next, BCPAP and TPC-1 cells were transfected with si2-DIO3OS and examined for b cell viability by CCK-8 assay; c DNA synthesis capacity by EdU assay; d, e invasion capacity by Transwell assay and migration capacity by Wound healing assay; f the protein levels of ki-67 and PCNA by Immunoblotting. N = 3, *P < 0.05, **P < 0.01, ***P < 0.001

Expression and function of NF-κB2 in thyroid cancer

As we have mentioned, lncRNAs could competitively bind to miRNAs via their MRE (miRNA response elements) to act as ceRNAs (competing endogenous RNAs), therefore regulating the expression of target RNAs (Salmena et al. 2011). To identify the mRNAs that might be involved in DIO3OS functions in thyroid cancer, we analyzed a total of 499 samples record in the TCGA database by first grouping these samples by DIO3OS expression into a high expression group (n = 250) and a low expression group (n = 249). Pearson’s correlation analysis compared the differentially-expressed genes in these two groups, and these differentially-expressed mRNAs were then applied for KEGG signaling annotation analysis. As shown in Fig. S2, these mRNAs were enriched in pathways of Cell adhesion molecules (CAMs) (Fig. S2A), Cytokine-cytokine receptor crosstalk (Fig. S2B), and NF-κB (Fig. S2C). More importantly, key factors of these signaling pathways, ICAM-1 and VCAM (Elices et al. 1990), were significantly correlated with DIO3OS (Fig. 3a).

Fig. 3.

Fig. 3

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Expression and function of NF-κB2 in thyroid cancer. a mRNAs that are co-expressed with DIO3OS were analyzed and selected based on online TCGA data; selected mRNAs were applied for KEGG signaling annotation analysis and these mRNAs were enriched in hsa04514-Cell adhesion molecules (CAMs) and Cytokine-cytokine receptor interaction. Key factors of these signaling pathways, ICAM-1 and VCAM1, were significantly correlated with DIO3OS. b Transcription factor enrichment analysis was performed to screen for transcription factors related to DIO3OS, ICAM-1, and VCAM1. NF-κB2 was selected. c The protein content and distribution of NF-κB2 in thyroid cancer and normal tissues were examined by IHC staining. d BCPAP and TPC-1 cells were transfected with si1-DIO3OS or si2-DIO3OS and examined for the protein levels of NF-κB2 by Immunoblotting. e NF-κB2 knockdown was generated in BCPAP and TPC-1 cells by the transfection of si1-NF-κB2 or si2-NF-κB2, as confirmed by real-time PCR. Next, BCPAP and TPC-1 cells were transfected with si-NF-κB2 or si2-NF-κB2 and examined for f cell viability by CCK-8 assay; g DNA synthesis capacity by EdU assay; h, i invasion capacity by Transwell assay and migration capacity by Wound healing assay; j the protein levels of ki-67, PCNA, ICAM-1, and VCAM by Immunoblotting. N = 3, *P < 0.05, **P < 0.01

Next, transcription factor enrichment analysis was performed and DIO3OS was found to be significantly related to NF-κB signaling (Fig. 3b). Since NF-κB activation increases the expression of ICAM-1 and VCAM-1 (Kim et al. 2008; Lin et al. 2019), NF-κB2 was selected for further experiments.

We performed IHC staining to examine the protein content and distribution of NF-κB2 within both thyroid carcinoma and normal healthy tissue sample; Fig. 3c showed that NF-κB2 was enhanced within thyroid carcinoma tissues. Next, we transfected BCPAP and TPC-1 cell lines with si1-DIO3OS or si2-DIO3OS, then examined for NF-κB2 protein contents; after DIO3OS knockdown, NF-κB2 protein levels were significantly decreased. The si2-DIO3OS transfection showed better effect. (Fig. 3d). To validate the specific roles of NF-κB2 in thyroid carcinoma cells, we transfected si1-NF-κB2 or si2-NF-κB2 to generate NF-κB2 knockdown in BCPAP and TPC-1 cell lines, and performed real-time PCR to verify the transfection efficiency (Fig. 3e). Secondly, after NF-κB2 knockdown, we examined for related indexes. Similar to those of DIO3OS knockdown, NF-κB2 knockdown by si1-NF-κB2 or si2-NF-κB2 transfection significantly inhibited BCPAP and TPC-1 cell viability, the formation of DNA synthesis, cell invasion, and cell migration (Fig. 3f-i). Moreover, the protein contents of proliferating markers, ki-67, and PCNA also showed to be significantly decreased via NF-κB2 knockdown (Fig. 3j). The immunoblotting and ELISA results showed that adhesion molecules VCAM and ICAM-1 levels were also reduced by NF-κB2 knockdown (Fig. 3j and Fig. S3A). The si1- NF-κB2 transfection showed better effect on those indexes. These data suggest that NF-κB2 is related to DIO3OS in thyroid cancer and also serves as an oncogenic regulator in thyroid cancer.

Let-7d directly targets DIO3OS and NF-κB2 in their 3’UTR

Following the confirmation of the oncogenic roles of DIO3OS and NF-κB2 in thyroid cancer, next, we searched for miRNAs that might mediate the effects of these two factors. Firstly, online tool mirDIP (http://ophid.utoronto.ca/mirDIP/index.jsp#r’) was used to predict miRNAs that might simultaneously target DIO3OS and NF-κB2 3’UTR, and 35 miRNAs were selected. Secondly, among these 35 miRNAs, miRNAs that might be correlated with the overall survival of thyroid cancer patients were analyzed according to TCGA and Kaplan Meier-plotter (KMPLOT) database (Fig. 4a). Samples from these two databases were grouped by miR-23b, miR-30d, or let-7d expression into high miRNA expression group and low miRNA expression group and then applied for a Single-factor analysis (log-rank test) using Cox proportional-hazards models. As shown in Fig. S4, patients with lower miRNA expression obtained shorter overall survival. We performed real-time PCR to determine the expression of three candidate miRNAs in both thyroid cancer and normal healthy tissue samples; among the three miRNAs, let-7d was the most downregulated in thyroid cancer tissues (Fig. 4b). Moreover, in si-DIO3OS transfected TPC-1 cells, let-7d was the most upregulated (Fig. 4c). Thus, let-7d was selected for further experiments.

Fig. 4.

Fig. 4

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let-7d directly binds to DIO3OS and NF-κB2 3’UTR. a miRNAs that might simultaneously target DIO3OS and NF-κB2 3’UTR were predicted by online tool mirDIP and 35 miRNAs were selected. These 35 miRNAs correlated with the overall survival of thyroid cancer patients were analyzed according to TCGA and KMPLOT database and 3 miRNAs were selected. B The expression of three candidate miRNAs was determined in thyroid cancer tissues and normal non-cancerous tissues by real-time PCR and let-7d was selected for the downregulation. c BCPAP and TPC-1 cells were transfected with si-DIO3OS and examined for the expression of three candidate miRNAs. d let-7d overexpression or inhibition was generated in BCPAP and TPC-1 cells by the transfection of let-7d mimics or let-7d inhibitor, as confirmed by real-time PCR. e, f Wild-type and mutant-type DIO3OS and NF-κB2 3’UTR luciferase reporter vectors were constructed as described in the Materials and methods section. These vectors were co-transfected in 293 T cells with let-7d mimics or let-7d inhibitor and the luciferase activity was determined. N = 3, *P < 0.05, **P < 0.01, ***P < 0.001

Next, we transfected let-7d mimics/inhibitor to generate let-7d overexpression or inhibition in BCPAP and TPC-1 cell lines, thus investigating the predicted let-7d binding to DIO3OS and NF-κB2 3’UTR. We performed real-time PCR to confirm transfection efficiency (Fig. 4d). For luciferase reporter assay, based on the Materials and methods section, we constructed two different types of DIO3OS and NF-κB2 3’UTR luciferase reporter vectors, wild-type and mutant-type (Fig. 4e, f). We transfected these vectors in 293 T cells with let-7d mimics/inhibitor, then examined for luciferase activity. Figure 4e, f showed that wild-type DIO3OS and wild-type NF-κB2 3’UTR vectors’ luciferase activity was remarkably downregulated via the overexpression of let-7d whereas upregulated via the inhibition of let-7d; mutating the putative let-7d binding sites could eliminate the changes in luciferase activity. These data indicate that let-7d directly binds to DIO3OS and NF-κB2 3’UTR. DIO3OS serves as a microRNA sponge for t let-7d.

Effects of let-7d on thyroid cancer cells

After confirming let-7d binding to DIO3OS and NF-κB2 3’UTR, we transfected BCPAP and TPC-1 cell lines with let-7d mimics/inhibitor and examined the specific effects of let-7d on thyroid cancer cells. As shown in Fig. 5, let-7d overexpression significantly inhibited, while let-7d inhibition promoted BCPAP and TPC-1 cell viability, the ability of DNA synthesis, cell invasion, and cell migration (Fig. 5a–d). Moreover, let-7d overexpression significantly decreased, while let-7d inhibition increased the protein contents of ki-67, PCNA, ICAM-1, and VCAM (Fig. 5e and Fig. S3B). In summary, let-7d exerts a tumor-suppressive effect on thyroid cancer cells, possibly through interaction with DIS3OS and NF-κB2.

Fig. 5.

Fig. 5

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Effects of let-7d on thyroid cancer cells. BCPAP and TPC-1 cells were transfected with let-7d mimics or let-7d inhibitor and examined for a cell viability by CCK-8 assay; b DNA synthesis capacity by EdU assay; c, d invasion capacity by Transwell assay and migration capacity by Wound healing assay; e the protein levels of ki-67, PCNA, ICAM-1, and VCAM by Immunoblotting. N = 3, *P < 0.05, **P < 0.01, compared to NC mimics group. #P < 0.05, ##P < 0.01, compared to NC inhibitor group

Let-7d inhibits NF-κB2 expression to reverse the oncogenic effects of DIO3OS

To investigate the speculation that let-7d exerts a tumor-suppressive effect on thyroid carcinoma cells via interaction with DIS3OS and NF-κB2, we co-transfected BCPAP and TPC-1 cells with si-DIO3OS and let-7d inhibitor and examined related indexes. As shown in Fig. 6a, DIO3OS knockdown significantly downregulated, while let-7d inhibition upregulated NF-κB2 expression; the effects of DIO3OS knockdown were significantly reversed by let-7d inhibition. Consistently, DIO3OS knockdown significantly inhibited, while let-7d inhibition promoted BCPAP and TPC-1 cell viability, the ability of DNA synthesis, cell invasion, and cell migration; the effects of DIO3OS knockdown were significantly reversed by let-7d inhibition (Fig. 6b–e). Moreover, DIO3OS knockdown significantly decreased, while let-7d inhibition increased the protein levels of NF-κB2, ki-67, PCNA, ICAM-1, and VCAM; the effects of DIO3OS knockdown were significantly reversed by let-7d inhibition (Fig. 6f and Fig. S3C). These data indicate that DIO3OS/let-7d axis modulates downstream NF-κB2 to affect the thyroid cancer cell phenotype.

Fig. 6.

Fig. 6

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let-7d inhibits NF-κB2 expression to reverse the oncogenic effects of DIO3OS. BCPAP and TPC-1 cells were co-transfected with si-DIO3OS and let-7d inhibitor and examined for a the expression of let-7d and NF-κB2 by real-time PCR; b cell viability by CCK-8 assay; c DNA synthesis capacity by EdU assay; d, e invasion capacity by Transwell assay and migration capacity by Wound healing assay; f the protein levels of NF-κB2, ki-67, PCNA, ICAM-1, and VCAM by Immunoblotting. N = 3, *P < 0.05, **P < 0.01, ##P < 0.01

Discussion

Herein, we identified 53 increased lncRNAs within thyroid cancer samples according to TCGA data. Among them, high lncRNA DIO3OS expression was a risk factor for the overall survival of thyroid cancer patients. DIO3OS showed to be considerably increased within thyroid cancer tissue samples and cells. Knocking down DIO3OS in thyroid carcinoma cells suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, as well as cell migration; besides, proliferating markers, ki-67 and PCNA, were decreased by DIO3OS knockdown. Cancer bioinformatics analysis suggested that NF-κB2 might be related to DIO3OS function in thyroid cancer carcinogenesis. NF-κB2 was positively correlated with DIO3OS, and DIO3OS knockdown decreased NF-κB2 protein levels. Knocking down NF-κB2 in thyroid carcinoma cells suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, cell migration, and the protein levels of ki-67, PCNA, ICAM-1, and VCAM. Cancer bioinformatics analysis and experimental results indicate that let-7d directly targeted DIO3OS and NF-κB2; DIO3OS knockdown upregulated let-7d expression. The overexpression of Let-7d suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, cell migration, as well as the protein levels of ki-67, PCNA, ICAM-1, and VCAM. Let-7d inhibition remarkably attenuated the functions of DIO3OS knockdown in NF-κB2 expression and thyroid cancer cell phenotype.

lncRNAs regulate gene expression via transcription, post-transcriptional processing, chromatin modification, protein function modulation, and some other mechanisms, thus contributing to several tumorigenic processes (He et al. 2019; Liz and Esteller 2016; Yoon et al. 2013). Immediately adjacent to the DIO3 gene is a lncRNA transcript, DIO3OS (Hernandez et al. 2004). A previous co-expression analysis confirmed lncRNA DIO3OS as one of the targets of EZH2 (enhancer of zeste homolog 2), a dynamic chromatin regulatory factor within cancer that transcriptionally activate target genes linked to malignant development, and had a significant correlation with EZH2 (Li et al. 2016). Another group indicated a significant increase of lncRNA DIO3OS within pancreatic cancer tissue samples and cancer cells. Knocking down the expression of DIO3OS inhibited the capacity of cancer cells to proliferate and to invade, which could be stimulated by DIO3OS overexpression within pancreatic cancer cell lines (Cui et al. 2019). In the present study, online data and experimental results indicated that the expression of DIO3OS showed to be significantly increased in thyroid carcinoma tissue samples and cancer cells. DIO3OS knockdown in thyroid carcinoma cell lines also significantly reduced the proliferation, DNA synthesis capacity, invasion, and migration of cancer cells. Moreover, the protein levels of two critical markers of cell nuclear proliferation, ki-67 and PCNA (Na et al. 2017), showed to be downregulated by DIO3OS knockdown. Taken together, lncRNA DIO3OS serves as an oncogenic lncRNA within thyroid cancer.

The regulatory mechanisms of lncRNAs exerting their effects include transcription, post-transcriptional processing, chromatin modification, protein function regulation, and almost all other aspects of pre−/post-transcriptional processes (Huang et al. 2014; Sun et al. 2014). Based on online data, the differentially-expressed genes that co-expressed with DIO3OS were enriched in hsa04514-CAMs (Cell adhesion molecules) and cytokine-cytokine receptor crosstalk. Moreover, transfection factor analysis indicated that DIO3OS was significantly correlated with NF-κB signaling, whose activation enhances the expression of ICAM-1 and VCAM-1 (Kim et al. 2008; Lin et al. 2019). NF-κB system hyperactivation has been found in PTC3–5 (RET/PTC) cells (Zhou et al. 2017); in the meantime, the expression of the proteins associated with NF-κB, including phosphorylated NF-κB2 (p100/52), was promoted and p65 nuclear translocation was stimulated (Zhou et al. 2017), which are supported by previous studies (Neely et al. 2011). In a previous study on medullary thyroid carcinoma, 47 of 48 cases exhibited nuclear positivity for one or more NF-κB members (p50, p52, c-Rel, RelB, p65); moreover, nuclear immunostaining for NF-κB subunits was commonly seen within MTCs (p50, 19%; p65, 68%; p52, 86,6%; c-Rel, 75%; RelB, 36%) and p52 was the most frequent (Gallel et al. 2008). Herein, we also found that the expression of NF-κB2 was increased through IHC. Silencing NF-κB2 in thyroid carcinoma cell lines resulted in suppressed cell viability, DNA synthesis, cell invasion, and cell migration. Notably, in thyroid cancer cells, knocking down DIO3OS significantly downregulated NF-κB2 expression, indicating that DIO3OS might exert the role in thyroid cancer through NF-κB2.

The targeting relationship between non-coding RNAs has been widely reported. For instance, it has been previously revealed that H19 could regulate miR-200, let-7, and miR-675 (Gao et al. 2014; Ma et al. 2014; Zhang et al. 2013a; Zhu et al. 2014). Another oncogenic lncRNA, HOTAIR, could target miR-200c, miR-20b, miR-34a, and miR-206 (Chang et al. 2018; Shao et al. 2019; Tang et al. 2019; Yang et al. 2019). Commonly, lncRNAs serve as sponge or ceRNA for miRNAs to inhibit miRNA expression and counteract miRNA-mediated suppression on downstream target mRNAs (Sun et al. 2014; Tan et al. 2015). In thyroid cancer, H19 competitively targets miR-17-15p to modulate the expression of YES1, thus acting as an oncogenic lncRNA (Liu et al. 2016). Herein, online tools proposed a total of 35 miRNAs that might simultaneously target DIO3OS and NF-κB2, and 4 of them were correlated with thyroid cancer patients’ overall survival according to the TCGA and KMPLOT database. In the 4 miRNAs, let-7d was the most downregulated in thyroid cancer tissues. Further experimental results indicate that let-7d was a direct target of DIO3OS, and let-7d directly targeted NF-κB2. DIO3OS knockdown significantly upregulated let-7d expression in thyroid cancer cells, suggesting that let-7d might mediate the roles of DIO3OS and NF-κB2 in thyroid cancer.

Let-7 miRNA family, which plays a tumor-suppressive role, is often inactivated in various human malignancies (Sakurai et al. 2012). In ovarian carcinoma, let-7d binds to c-Myc to enhance the sensitivity of cancer cells to a genistein analog (Ning et al. 2017). In breast cancer, let-7d binds to Jab1/Cops5 to suppress the growth and metastasis of cancer cells (Wei et al. 2018). Recently, Perdas et al. reported that the let-7 miRNA family was significantly higher in papillary thyroid cancer patients plasma than healthy controls, suggesting the prognostic potential of circulating let-7 in papillary thyroid cancer (Perdas et al. 2020). In the present study, the expression of let-7d expression showed to be significantly reduced in thyroid carcinoma tissue samples and cells. Let-7d exerts a tumor-suppressive effect on thyroid carcinoma via significantly inhibiting the cell viability, DNA synthesis, invasion, and migration of thyroid carcinoma cells. More importantly, when co-transfected with si-DIO3OS in thyroid cancer cells, the let-7d inhibitor significantly reversed the effects of si-DIO3OS on NF-κB2 expression and thyroid cancer cells, indicating that DIO3OS/let-7d axis modulates NF-κB2 expression to affect thyroid cancer cell phenotype.

In conclusion, we presented a lncRNA/miRNA/mRNA network consist of lncRNA DIO3OS, let-7d, and NF-κB2, which regulates the cell viability, DNA synthesis capacity, invasion, and migration of thyroid cancer cells. The clinical application of this axis needs further in vivo and clinical investigation.

Electronic supplementary material

Fig. 1 (782.3KB, tif)

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Fig. 3 (562.1KB, tif)

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Author contributions

MW, JL, JH made substantial contribution to the conception and design of the work; ZZ, CR were involved in in the experimental conducting; TT, CL analyzed and interpreted the data; MW, YG drafted the manuscript; FY, ZW revised the work critically for important intellectual content; JH collected grants; Final approval of the work: all authors.

Funding

This work was supported by the Natural Science Foundation of Hunan Province, (2020JJ5835) and grants from the Innovation Project Fund of Hunan Development and Reform Commission (NO. 2019412).

Data availability

Please contact the authors for data requests.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approvals

All procedures performed in studies involving human participants were in accordance with the Ethics Committee of the Second Xiangya Hospital and with the 1964 Helsinki declaration. Informed consent to participate in the study has been obtained from participants.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Fig. 1 (782.3KB, tif)

(TIF 782 kb)

High resolution image (PNG 1743 kb) (1.7MB, png)
ESM 2 (316.2KB, png)

(PNG 316 kb)

Fig. 3 (562.1KB, tif)

(TIF 562 kb)

High resolution image (PNG 734 kb) (734.8KB, png)
ESM 4 (636.6KB, png)

(PNG 636 kb)

ESM 5 (18.8KB, docx)

(DOCX 18 kb)

Data Availability Statement

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