Long Noncoding RNA FER1L4 Promotes the Malignant Processes of Papillary Thyroid Cancer by Targeting miR-612/ Cadherin 4 Axis

doi:10.21203/rs.3.rs-483514/v1

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Long Noncoding RNA FER1L4 Promotes the Malignant Processes of Papillary Thyroid Cancer by Targeting miR-612/ Cadherin 4 Axis

https://doi.org/10.21203/rs.3.rs-483514/v1

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published 21 Jul, 2021

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Background: Long noncoding RNAs (lncRNAs) have emerged as crucial regulators in various cancers. However, the functional roles of most lncRNA in papillary thyroid cancer (PTC) are not detailly understood. This study aims to investigate the biological functions and molecular mechanisms of lncRNA Fer-1 like family member 4 (FER1L4) in PTC.

Methods: The expression of FER1L4 in PTC was determined via operating qRT-PCR assays. Meanwhile, the clinical significance of FER1L4 in PTC patients was described. The biological functions of FER1L4 on PTC cells were evaluated by gain and loss of function experiments. Moreover, animal experiments were performed to reveal the effect on tumor growth. Subcellular distribution of FER1L4 was determined by fluorescence in situ hybridization and subcellular localization assays. Luciferase reporter assay and RNA immunoprecipitation assay were applied to define the relationship between FER1L4, miR-612, and Cadherin 4 (CDH4).

Results: Upregulated expression of FER1L4 in PTC tissues was positively correlated with lymph node metastasis (P = 0.020), extrathyroidal extension (P = 0.013) and advanced TNM stages (P = 0.013). In addition, knockdown of FER1L4 suppressed PTC cell proliferation, migration and invasion, whereas ectopic expression of FER1L4 inversely promoted these processes. Mechanistically, FER1L4 could competitively bind with miR-612 to prevent the degradation of its target gene CDH4. This condition was further confirmed in the rescue assays.

Conclusions: This study first demonstrates FER1L4 plays an oncogenic role in PTC via a FER1L4-miR-612-CDH4 axis and may provide new therapeutic and diagnostic targets for PTC.

General Cell Biology & Physiology

Cancer Biology

Papillary thyroid cancer

Long noncoding RNA

FER1L4

MiR-612

Cadherin 4

Thyroid cancer (TC) is the most frequent malignancy of the endocrine system, and ranks ninth in the incidence of tumors worldwide [1]. Papillary thyroid carcinoma (PTC) is the major pathological type, accounting for more than 85% of differentiated thyroid cancer (DTC) [2]. With the improvements of diagnostic technologies such as ultrasound, the detection rate of DTC has been significantly increased during the last few decades [3]. However, patients with DTC usually have an excellent prognosis, with 10-year disease-specific survival rates over 90% [4]. Of note, about 10% of patients with DTC have distant metastases to lungs or bones at diagnosis or during follow-up, resulting in poor prognosis [5]. Meanwhile, the incidence of larger tumors also elevates, which is not likely to be explained by the screening effects [3]. And to date, the etiology of thyroid cancer remains unclear. Thus, exploring the molecular basis of PTC pathogenesis and progression is crucial for developing more effective therapeutic and diagnostic targets to PTC.

Long noncoding RNAs refer to the noncoding portions of the genome without protein-coding signature that transcript longer than 200 nucleotides [6]. LncRNAs are considered to regulate varieties of physiological and pathological functions, including the occurrence and progression of cancers [7, 8]. In recent years, aberrant expression of lncRNA has been found in multiple tumors. For example, in hepatocellular carcinoma, lncRNA-AY could promote hepatocellular carcinoma metastasis via induction of chromatin modification for ITGAV transcription [9]. In addition, it was reported that upregulated expression of lncRNA BCRT1 in breast cancer remarkably accelerated tumor growth and metastasis by acting as a sponge for miR-1303 to attenuate its repressive effect on PTBP3[10]. However, researches on the underlying role of lncRNA in PTC carcinogenesis are still lacking yet.

LncRNAs perform its complex biological functions through multiple ways which are closely related with its subcellular localization [11]. For example, lncRNA largely localized in the cytoplasm mainly functions by acting as decoy for miRNA, in which lncRNA could control miRNA availability for its target gene, so named as competing endogenous RNAs (ceRNAs) [12]. In case of thyroid cancer, lncRNA-GAS8-AS1, localized in the cytoplasm of PTC cells, was found that could promote the autophagy of PTC cells by sponging oncogenic miR-187-3p and miR-1343-3p, and therefore respectively upregulates the expression of ATG5 and ATG7 [13]. In this study, lncRNA fer-1 like family member 4 (FER1L4) was identified as an oncogene which promoted the proliferation, migration and invasion of PTC cell via functioning as ceRNA for tumor suppressor, miR-612.

Cadherin 4, also named as retinal cadherin, is a classical cadherin from the cadherin superfamily[14]. Cadherins, consisted by epithelial-cadherin (E-cadherin, CDH1), neural-cadherin (N-cadherin, CDH2), placental-cadherin (P-cadherin, CDH3), and so on, are transmembrane glycoproteins responsible for cell-cell adhesion, tissue pattering and carcinogenesis [15]. Previous researches indicate that cadherins could bind to catenin proteins and then form the cadherin-catenin complex to activate the catenin signaling or not, as well as involve in the epithelial-mesenchymal transition (EMT) process [16]. However, the function of CDH4 in PTC remains unknown, and its roles in above processes are still controversial [17]. In the present study, CDH4 was considered as the downstream target of miR-612, which mediated the promoting role of FER1L4 in PTC cells.

Patients and tissue samples

Eighty papillary thyroid cancer tissues and adjacent normal tissues were collected from 80 patients with PTC who received operation on the First Affiliated Hospital of Nanjing Medical University (NMU). None of the patients underwent any other treatment but surgery. All collected tissue samples were immediately snap frozen in liquid nitrogen and stored at −80 °C until required. Our study was approved by the Ethics Committee of the First Affiliated Hospital of NMU.

Cell lines

Four PTC cell lines (K-1, TPC-1, B-CPAP and IHH-4) and a normal thyroid follicular epithelium cell line (Nthy-ori3-1) were purchased from the American Type Culture Collection (ATCC, Virginia, USA). K-1, B-CPAP and Nthy-ori3-1 cell were cultured in RPMI1640 medium (Gibco, Carlsbad, CA, USA), while TPC-1 cell was cultured in DMEM with high glucose (Gibco, Carlsbad, CA, USA). Mixture (1:1) of RPMI1640 and DMEM was used to culture the IHH-4 cell line. 1% antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) and 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) were added to all of the above culture media. All cell lines were incubated in a humidified atmosphere at 37 °C containing 5% CO2. All cell lines have been authenticated by short tandem repeat analysis, as well as tested for mycoplasma contamination before conducting this study.

RNA extraction and quantitative real-time PCR analysis

Total RNA was extracted from tissues and cultured cell lines using TRIzol reagent (Invitrogen, MA, USA). A PrimeScript RT reagent kit (Takara, Kyoto, Japan) was used to synthesize cDNA. MiRNAs were reverse transcribed after polyadenylation using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA). Quantitative real-time PCR (qRT-PCR) was performed with AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). Results were calculated using the 2^-ΔΔCT method and normalized to the expression of GAPDH for mRNA or U6 for miRNA. Primers used in the study were listed in Supplementary Table 1 (Additional file 1: Table S1).

Cell transfection

PTC cells were transfected with 50 nm small interfering RNAs (siRNAs) and plasmid vectors when they grew to 30-40% density using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). In this study, siRNAs, miR-612 mimics, miR612 inhibitor and correspondent negative control were purchased from Genepharma. After 48h post-transfection, the cells were harvested for performing the following experiments. Plasmid vectors encoding FER1L4 and biologically active short hairpin RNAs (shRNA) targeting FER1L4 or CDH4 were generated either (Genepharma, Shanghai, China). Stable cell lines were obtained by using 2 μg/ml puromycin (Sigma-Aldrich, St-Louis, Missouri, USA) for about three weeks. Nucleotide sequences mentioned above were listed in Supplementary Table 2 (Additional file 1: Table S2).

Cell proliferation assay

In order to investigate the effect on cellular proliferation of corresponding treatment to K-1 and TPC-1 cells, cell counting kit 8 (CCK8) assay, colony formation assay and 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay were applied. Detailed information has been described before [18].

Animal experiment

Four weeks old female BALB/c nude mice were purchased from the Animal Center of NMU, and all experiments were approved by the Committee on the Ethics of Animal Experiments of the Nanjing Medical University. For the tumorigenicity studies, a total of 20 mice were randomly assigned, and stable transfected cells (1×10⁶ cells/100 μl of phosphate buffer saline) were subcutaneously injected into the flank of nude mice. The tumor volume was measured every week and calculated by the formula: volume = (length × width²)/2.

Immunohistochemical (IHC) analysis

All specimens were fixed in 4% formalin and then embedded in paraffin. After blocking endogenic peroxides and proteins, these sections were incubated with primary antibodies specific for Ki-67 (Abcam, Cambridge, MA, USA) or CDH4 (Abclonal, Wuhan, China) at 4°C overnight, lastly were counterstained with hematoxylin after incubating with the secondary antibodies at 37 °C for 1 h. Random images were obtained using a light microscope (Olympus Corp. Tokyo, Japan).

Cell migration and invasion assays

Transwell chambers (Corning, New York, NY, USA) coated with or without Matrigel (BD Bioscience, Franklin Lakes, NJ, USA) were used to evaluate the function of genes on cellular migration and invasion. Meanwhile, cell motility was also examined by wound healing assay. Detailed information has been described before [18].

Flow cytometric analysis

Treated cells were collected for Flow cytometric analysis. According to the protocol (MultiSciences, Hangzhou, China), APC-Annexin V and Propidium Iodide (PI) were used to stain cells, and the rate of apoptosis was analyzed by a flow cytometer (FACScan, BD Biosciences, USA). For cell cycle analysis, treated cells were stained by PI-staining solution, and then the percentages of cells in G₀–G₁, S, and G₂–M phase were counted.

Western blot assay and antibodies

Western blot assay was performed following previous protocol. The primary antibody used were listed in Supplementary Table 3 (Additional file 1: Table S3).

Subcellular fractionation and Fluorescence in situ hybridization (FISH)

The separation and purification of cytoplasmic and nuclear RNA were implemented using the PARIS Kit (Life Technologies, USA) according to the manufacturer’s instructions. For the FISH assay, the Cy3-labeled FER1L4 probes used in our study were synthesized (RiBo Ltd, Guangzhou, China). Briefly, the prepared cells were incubated with specific probes at 37 °C overnight after fixation and permeabilization. Finally, the nuclei were stained by DAPI and observed using a confocal laser scanning microscope (Zeiss LSM5 Live, Oberkochen, German).

Dual-luciferase reporter assay

The sequences of FER1L4 and CDH4 3′-UTR containing wild-type or mutated miR-612 binding sites were synthesized and loaded into a pGL3 luciferase reporter vector (Promega, Wisconsin, USA). Then, TPC-1 cells (4×10⁵) were co-transfected with miRNA mimics and luciferase reporter vectors. After 48h of incubation, the luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega, Wisconsin, USA). Relative luciferase activity was normalized to Renilla luciferase.

RNA immunoprecipitation (RIP) assay

To detect whether FER1L4 influences miR-612-dependent RNA-induced silencing complex, RIP assays were conducted using the Magna RIPTM RNA-binding protein immunoprecipitation kit (Millipore, USA). Briefly, the prepared cells were lysed and incubated with anti-Ago2 (Abcam, Cambridge, MA, USA) or IgG antibody at 4 °C overnight. Then, cell lysates were incubated with the protein A magnetic beads for 4h. Finally, the coprecipitated RNAs were collected for qRT-PCR analysis.

Bioinformatic analyses

Gene expression data of PTC was extracted from TCGA database and used to explore abnormally expressed lncRNA. MiRNAs targeted by FER1L4 were predicted using starbase (http://starbase.sysu.edu.cn/), miRcode (http://www.mircode.org/) and RegRNA2.0 (http://regrna2.mbc.nctu.edu.tw/). Meanwhile, miRWALK (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/) was applied to determine the downstream targets of miR-612.

Statistics analysis

The data was described by the mean ± standard deviation (SD) in three independently experiments. For comparing statistical differences, Student’s t-tests, Pearson Chi-square test, Wilcoxon test were performed as appropriate using SPSS v22.0 and GraphPad Prism 6. Spearman’s correlation analysis was selected to analyze the correlations among FER1L4, miR-612 and CDH4. P value less than 0.05 was considered statistically significant.

FER1L4 was upregulated and associated with later stage diseases in PTC

To identify potential drivers in PTC tumorigenesis, expression signatures of genes were screened based on TCGA database. In this study, we observed preferential upregulation of lncRNA FER1L4 in thyroid cancer tissues (n = 513), compared with normal thyroid tissues (n = 58; Fig. 1A, B). Moreover, FER1L4 also showed concordance in a pattern of expression in PTC cell lines compared with an immortalized thyroid follicular epithelium cell line Nthy-ori-1 (Fig. 1C). Meanwhile, identical results were detected via measuring the expression of FER1L4 in 80 paired PTC tissues and adjacent normal tissues, in which 79 percent of patients exhibited higher expression of FER1L4 (Fig. 1D). Furthermore, through investigating the clinical significances of FER1L4 in these 80 PTC patients, it showed enhanced expression of FER1L4 was correlated with higher lymph node metastasis rate (P = 0.020), higher potential of extrathyroidal extension (P = 0.013), and advanced TNM stages (P = 0.013) (Table 1). In addition, analysis of TCGA cohort suggested that upregulation of FER1L4 also predicted higher lymph node metastasis rate (N stage) in patients with PTC (Fig. 1E-H). Herein, these results indicated upregulation of FER1L4 was associated with later stage diseases in PTC.

Table 1. Correlation between the expression level of FER1L4 and clinicopathological features in 80 PTC patients.

Characteristics	Cases (%)	FER1L4 expression		P-value*
	n = 80	Low (n = 40)	High (n = 40)
Age (year)
<45	43 (53.75)	23	20	0.327
>45	37 (46.25)	17	20
Gender
Male	18 (22.50)	8	10	0.395
Female	62 (77.5)	32	30
Tumor size (cm)
<2	69 (86.25)	37	32	0.096
>2	11 (13.75)	3	8
Lymph node metastasis
N0	32 (40.00)	21	11	0.020
N1a + N1b	48 (60.00)	19	29
Extrathyroidal extension
Yes	12 (15.00)	2	10	0.013
No	68 (85.00)	38	30
TNM stage
I + II	63 (78.75)	36	27	0.013
III + IV	17 (21.25)	4	13

*, Chi-square detection.

Significant results were in bold.

FER1L4 promoted cellular growth and motility of PTC

To elucidate potential functions of FER1L4 in PTC, TPC-1 and K-1 cells were transfected with siRNAs targeting FER1L4 (si-FER1L4 1#, si-FER1L4 2#) (Fig. 2A). However, only siRNA 2# was chosen for following experiments because of the best efficiency to knockdown FER1L4. First, CCK8 assays demonstrated that knockdown of FER1L4 significantly suppressed cellular growth of TPC-1 and K-1 cells (Fig. 2B). Meanwhile, overexpression of FER1L4 showed enhanced proliferative capacity of PTC cells (Fig. 2C, D). Additionally, colony formation assays indicated that silencing FER1L4 significantly reduced the colony numbers (Fig. 2E), while upregulation of FER1L4 drastically enhanced them (Fig. 2F). Moreover, EdU incorporation assays showed an analogous mode as above, in which EdU positive cell numbers were progressively reduced following depletion of FER1L4 (Fig. 2G), whereas overexpression of FER1L4 resulted in an increase of these cells (Fig. 2H). Importantly, it is successfully identified that decreased apoptosis and cell-cycle promotion are two factors that could contribute to the outgrowth of cancer cell. Herein, we performed flow cytometric assays to examine the role of FER1L4 in these properties. Notably, knockdown of FER1L4 showed a growing number of apoptotic cells in TPC-1 and K-1 cells treated with siRNA targeting FER1L4 (Fig. 3A; Additional file 2: Fig. S1A). Apart from increased cell apoptosis, FER1L4-silenced PTC cells had higher percentage of cells in the S phase with detriment to the G0/1 phase than did cells expressed a negative control RNA, which indicated the induction of cell arrest at S phase after silencing FER1L4 (Fig. 3B). Remarkably, Western blot analysis showed depletion of FER1L4 was accompanied with reduced expression of apoptotic marker like Bcl2, along with enhanced expression of pro-apoptotic protein like Bax. Meanwhile, the expression level of S-phase checkpoint proteins such as CyclinA2 and CDK2 were also decreased in FER1L4-silenced PTC cells (Fig. 3C; Additional file 2: Fig. S1B). In parallel, in order to investigate the function of FER1L4 in vivo, TPC-1 cells transduced with vectors carrying FER1L4 shRNA or scrambled sequence were subcutaneously injected into the flank of 4 weeks nude mice. It showed that depletion of FER1L4 delayed the tumor growth of TPC-1 cells (Fig. 3D), and the tumor size and final tumor weight in implanted tumors significantly decreased in FER1L4 knockdown group either (Fig. 3E, F). Furthermore, immunohistochemistry analysis of orthotopically implanted tumors showed diminished numbers of Ki67-positive cells by inhibition of FER1L4 (Fig. 3G). Thus, these data indicated that FER1L4 has a critically promoting role on PTC cell growth.

The molecular mechanism behind the capacity for tumor cells to metastasize efficiently is an important topic. Therefore, it is necessary to evaluate whether FER1L4 involves in cellular migration and invasion of PTC. First, wound healing assays showed that knockdown of FER1L4 led to attenuated migration of PTC cells (Fig. 3H; Additional file 2: Fig. S1C). Transwell assays further suggested that loss of FER1L4 resulted in reduced numbers of cell getting through the chamber (Fig. 3I), whereas ectopic expression of FER1L4 showed elevated cell numbers (Fig. 3J). Consequently, our results suggested that FR1L4 essentially involves in the early metastatic process of PTC cell.

FER1L4 functioned by binding with miR-612 in PTC

Depending on its subcellular location, lncRNA plays various role in cellular physiology. In this study, we found FER1L4 mainly located in the cytoplasm of PTC cell by conducting FISH assay and subcellular fractionation assay (Fig. 4A, B; Additional file 2: Fig. S1D). Then, combining with gene expression profiling from TCGA database, along with predicted results from online tools such as regRNA2.0 [19], miRcode [20] and starbase [21], we selected five miRNAs (miR-612, miR-140-3p, miR-92a-3p, miR-196b-5p and miR-784-3p) with potential binding sites in FER1L4 sequences (Fig. 4C; Additional file 2: Fig. S2A). However, through conducting luciferase reporter assay, it showed only miR-612, miR-140-3p and miR-784-3p could bind with FER1L4 transcripts (Fig. 4D). Meanwhile, knockdown of FER1L4 also showed upregulated expression of miR-612, miR-140-3p and miR-784-3p in TPC-1 cells (Fig. 4E). Nevertheless, considering the highest binding affinity between FER1L4 and miR-612, we exclusively chosen miR-612 for further studies. As illustrated in Figure 4F, the binding sequences of miR-612 to FER1L4 were mutated and then fused into a luciferase reported vector (MUT type). Notably, mutation of miR-612 seed sequence abolished the suppressive effects on FER1L4-driven luciferase activity by miR-612 mimics (Fig. 4F). In addition, RIP assay further revealed that FER1L4 was successfully immunoprecipitated from cell extract using anti-Ago2 antibody (Fig. 4G). Importantly, silencing FER1L4 drastically enhanced the expression of miR-612 in K-1 cells, while overexpression of FER1L4 caused significant reduction of miR-612 expression (Fig. 4H, I). On the other hand, a significant inverse correlation between FER1L4 and miR-612 was acquired through analyzing of 20 PTC tissue samples (R²= 0.38, P = 0.0036) (Fig. 4J). Taken together, these findings suggested that FRE1L4 could sequester miR-612 away to its targets, therefore involves in PTC tumorigenesis.

miR-612 is sufficient and necessary for FER1L4

Although above data confirmed miR-612 was one of the targets of FER1L4, but the function of miR-612 in PTC has never been determined yet. Herein, in order to investigate the effect of miR-612 on PTC cells, the expression of miR-612 was successfully attenuated by miR-612 inhibitor, and was artificially enhanced by miR-612 mimics (Additional file 2: Fig. S2A-C). First, overexpression of miR-612 repressed cellular growth (Fig. 5A, B), and colony-formation ability of PTC cells (Fig. 5C; Additional file 2: Fig. S2D), whereas inhibition of miR-612 showed opposite effects. Flow cytometric assays also indicated reintroduction of miR-612 into PTC cells induced cell apoptosis (Fig. 5D), and cell-cycle arrest at S phase (Fig. 5E). Moreover, transwell assays indicated ectopic expression of miR-612 progressively inhibited PTC cell migration and invasion either (Fig. 5F; Additional file 2: Fig. S2E). We next sought to explore whether FER1L4 promotes PTC cell growth and motility via regulating miR-612. PTC cells were co-transfected with siRNA against FER1L4 and miR-612 inhibitor, then the resulting cells were subjected to CCK8 assay and transwell assay. It revealed the inhibitory effects on TPC-1 and K-1 cells by knockdown of FER1L4 were nullified by miRNA-612 inhibitor (Fig. 5G-J). Notably, qRT-PCR analysis verified that siRNA targeting FER1L4 could reverse the suppression on miR-612 expression induced by miRNA inhibitor, suggesting the binding affinity of FER1L4 to miR-612 was comparable to miR-612 inhibitor (Fig. 5K). As a result, these data unveiled that FER1L4 promoted PTC cell growth and invasion by competitively binding miR-612.

CDH4 was determined as the target of miR-612

The downstream targets of miR-612 were predicted by online tool such as miRwalk [22], and CDH4 was selected. Intriguingly, the RNA level of CDH4 was not influenced by silencing FER1L4 or overexpression of miR-612 (Fig. 6A; Additional file 2: Fig. S3A). However, the protein level of CDH4 was significantly diminished in PTC cells transfected with siRNA targeting FER1L4 or miR-612 inhibitor (Fig. 6B; Additional file 2: Fig S3B). On the other hand, PTC cells transfected with FER1L4 vectors or miR-612 inhibitor also exhibited enhanced expression of CDH4 in protein level (Fig. 6C; Additional file 2: Fig. S3C). For subsequent investigation, dual luciferase reporter assays were performed to validate that miR-612 could bind with CDH4. As presented in Figure 6D, the binding sites of miR-612 in CDH4 3′ UTR were mutated (MUT-CDH4), and then the mutated 3′ UTR sequence and the wild type 3′ UTR sequence of CDH4 (WT-CDH4) were fused into a plasmid vector respectively. As expected, the luciferase activity was significantly decreased following co-transfection with miR-612 mimics and WT-CDH4 reporter vector, while it was blocked by mutating miR-612 binding sequences (Fig. 6D). Of note, FER1L4 siRNA induced inhibition of CDH4 expression was rescued by co-transfecting miR-612 inhibitor into PTC cells (Fig. 6E; Additional file 2: Fig. S3D). Meanwhile, miR-612 mimics also successfully reversed FER1L4-mediated promotion on CDH4 expression (Fig. 6F; Additional file 2: Fig. S3E). Thus, on the basis of above observation, CDH4 was confirmed as a target of miR-612, and therefore was regulated by FER1L4.

FER1L4/miR-612/CDH4 axis promoted cellular proliferation and invasion of PTC

It was clear that the expression of CDH4 was upregulated in TC tissues and cell lines (Additional file 2: Fig. S4A, B). To delineate further the functional significances of CDH4 in PTC cell proliferation and invasion, the expression of CDH4 was efficiently knocked down in K-1 and TPC-1 cells (Fig. 6G; Additional file 2: Fig. S4C, D). First, loss of CDH4 repressed cellular proliferation as presented by EdU incorporation assay (Fig. 6H; Additional file 2: Fig. S4E) and CCK8 assay (Fig. 6I). Furthermore, silencing CDH4 also displayed significant induction of apoptosis accompanied by elevated Bax expression and reduced Bcl2 expression, as well as cell cycle arrest at S phase with reduced proteins level of CDK2 and cyclinA2 (Fig. 6J-L; Additional file 2: Fig. S4F-H). Importantly, subcutaneous tumor-formation experiments revealed that knockdown of CDH4 significantly inhibited the formation of subcutaneous tumors of PTC cells (Fig. 7A, B; Additional file 2: Fig. S4I). In addition, IHC analysis of CDH4 also showed upregulated expression of CDH4 in PTC tissue samples (Fig. 7C). As expected, wound healing assays uncovered a reduction of cell migrative ability in CDH4-depleted cells (Fig. 7D). Consistently, transwell assays displayed a sharp decline in cell numbers getting through the chamber by knockdown of CDH4 (Fig. 7E). Together, these experiments support an oncogenic role for CDH4 in promoting PTC cell proliferation and invasion.

Subsequently, we performed several functional experiments in CDH4-depleted cells with simultaneous knockdown of miR-612. First, co-transfection with miR-612 inhibitor in PTC cells resulted in a reversal of CDH4 siRNA-induced repression on CDH4 expression (Fig. 7F). Then, we further examined the effects on cellular proliferation and invasion. As indicated by CCK8 assays, CDH4 siRNA-mediated suppression in cell growth was nullified by artificially lowering the expression of miR-612 (Fig. 7G). And an analogous mode was detected in transwell assays (Fig. 7H, I; Additional file 2: Fig. S4J). In conclusion, these results suggested there may exist an axis between FER1L4, miR-612 and CDH4 which involves in the tumorigenesis and aggressiveness of papillary thyroid cancer.

The development of thyroid cancer involves multiple genetic and epigenetic alterations such as point mutation of the BRAF and RAS genes which seem to be linked to specific etiologic factors such as exposure to ionizing radiation and chemical mutagenesis [23]. In recent decades, technical advances in genome editing and high-throughput sequencing have promoted the deeply research of non-coding genomes [24]. Notably, long noncoding RNAs, the main components of non-coding genes, are considered to be essentially implicated in the process of tumor initiation or progression including thyroid cancer [25]. Fer-1 like family member 4 is a lncRNA with 6.7 kb length, located in chromosome 20 q11.22. However, intriguingly, plenty of literatures document context-dependent phenotypes of FER1L4 in human cancers. It was previously reported that FER1L4 served as tumor suppressor in colon [26] and gastric cancer [27], while it showed an oncogenic role in glioma [28]. In a human pan‐cancer analysis, it suggested FER1L4 may act as an oncogenic driver in human cancers [29]. Thus, importantly, the current study unraveled a new facet of FER1L4 which accelerates PTC cell proliferation, migration and invasion. In agreement, upregulated expression of FER1L4 in PTC tissues was closely related to advanced clinicopathological features such as lymph node metastasis, extrathyroidal extension, and advanced TNM stage. Accordingly, FER1L4 played an oncogenic role in PTC which could be a new biomarker and treatment target of PTC.

In our study, the confirmation of FER1L4 localized in cytoplasm given us a reason to hypothesize that FER1L4 may influence the progression of PTC through the mechanism model of ceRNA. Subsequently, miR-612 was validated to directly bind with FER1L4 in PTC cells. MiR-612 has been proved to suppress tumor growth and metastases in many tumors. For example, miR-612 negatively controlled the formation of invadopodia, matrix degradation and metastasis in hepatocellular carcinoma by HADHA-mediated lipid reprogramming [30]. Nevertheless, the function of miR-612 in PTC was never described before. Herein, in present study, miR-612 was also successfully identified as a tumor suppressor which inhibits cellular proliferation, migration and invasion of PTC. Meanwhile, functional experiments also suggested FER1L4-induced promotion on PTC cell was mediated by negatively regulating miR-612. Hence, our findings uncovered that FER1L4 exerts its oncogenic behavior partially via sponging miR-612 in PTC.

Here, Cadherin 4 was proved to be the target of miR-612. However, FER1L4 or miR-612 only influence the expression of CDH4 at the protein level, indicating miR-612 may repress the translation of CDH4 and then attenuates its expression [31]. Deregulation of CDH4 has been implicated in several human cancers. Dependent on cell context, in most epithelial cancers like breast cancer [32], colorectal and gastric cancer [33], CDH4 is epigenetically silenced by promoter hypermethylation and commonly acts as a tumor suppressor. Nevertheless, some researches also support an oncogenic function of CDH4, such as in high-grade gliomas [17] and osteosarcoma [34]. Although there are discrepancies to the functions of CDH4 in human cancers, our study identified and suggested an oncogenic role of CDH4 in PTC. Consistent with its function in cell-cell adhesion, immunohistochemical analysis indicated that CDH4 shows crisp membrane localization in PTC tissues. In particular, Gene Set Enrichment Analysis (GSEA) of CDH4 using gene expression data from TCGA database also validated that CDH4 involved in processes such as adherens junction and extracellular matrix-receptor interaction in thyroid cancer (Additional file 2: Fig. S5A, B) [35]. On the other hand, FER1L4 was also characterized with association to gene set of cell adhesion molecules (Additional file 2: Fig. S5C). However, correlation analysis of thyroid cancer cohort from TCGA database suggested FER1L4 was positively correlated with P-cadherin (R² = 0.2513, P <0.001), but not E-cadherin, N-cadherin or CDH4 (Additional file 2: Fig. S5E-H), which was consistent with the qRT-PCR results (Additional file 2: Fig. S3A). Intriguingly, the expression of CDH4 was also positively correlated with P-cadherin using TCGA database (R² = 0.1156, P <0.001; Additional file 2: Fig. S5K), indicating that FER1L4 may regulate the expression of P-cadherin via governing CDH4 or vice versa [36]. However, it needs further experimental confirmation. Meanwhile, to explore the mechanism in PTC progression of CDH4 is an important topic either. It was reported CDH4 is necessary to the activation of ERK and p27 [17], and also of c-Jun [34]. GSEA analysis suggested CDH4 was positively correlated with the Wnt/β-catenin pathway, which may indicate the underlying mechanism of CDH4 (Additional file 2: Fig. S5D). Of note, the distinct features of FER1L4 and CDH4 in cancer may be caused by the heterogeneity of tumor [37], suggesting the great potential of FER1L4 and CDH4 as diagnostic biomarkers of PTC. However, the underlying mechanism of CDH4 in PTC carcinogenesis requires thoroughly exploration.

Taken together, FER1L4 promoted PTC development via working as a ceRNA for miR-612 to attenuate the repressive effect on CDH4. These results provided a better understanding to the role of lncRNA in PTC progression, as well as suggested potential therapeutic and diagnostic targets for PTC.

Papillary thyroid cancer: PTC

Thyroid cancer: TC

Differentiated thyroid cancer: DTC

LncRNA: Long noncoding RNA

Fer-1 like family member 4: FER1L4

Cadherin 4: CDH4

Competing endogenous RNA: CeRNA

Tumor-node-metastasis: TNM

Quantitative real-time PCR: qRT-PCR

Cell counting kit 8: CCK8

Immunohistochemical: IHC

RNA immunoprecipitation: RIP

TCGA: The Cancer Genome Atlas

GSEA: Gene Set Enrichment Analysis

ECM: Extracellular Matrix

EMT: Epithelial-Mesenchymal Transition

SiRNA: Small interfering RNA

Ethics approval and consent to participate

This study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. Written informed consent was obtained from each patient. And this study was performed in accordance with the Declaration of Helsinki.

Consent for publication

Not applicable

Availability of data and materials

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Competing interests

The authors declare that they have no conflict of interest.

Funding

This study was supported by grants from the Nanjing Medical University Science and Technology Development Fund-Specialized Disease Cohort (NMUC2018019A), and the General Project of Jiangsu Provincial Department of Health (H2018057).

Author contributions

L Wu and Y Ding designed this study. L Wu, Y Ding and M Shen were responsible for the interpretation of the results. J Cai, H Tong, Y Si, H Zhang, X Wang processed the data processing and performed the statistical analysis. L Wu wrote the manuscript. X Zhuang and M Shen revised and improved the manuscript. M Shen for the supervision. All authors approved the submission.

Acknowledgements

Not applicable

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Additionalfile1.docx
Additional file 1: Supplementary Tables Supplementary Table 1 Primers used for PCR. Supplementary Table 2 siRNA and shRNA used in this study. Supplementary Table 3 Antibodies used for Western blot.
Additionalfile2.docx
Additional file 2: Supplementary Figures Figure S1 Quantitative analysis of the effect of FER1L4 on cell apoptosis and cell cycle. Figure S2 MiR-612 suppressed PTC cell migration and invasion. Figure S3 Quantitative analysis of the effect of FER1L4 and miR-612 on CDH4 expression. Figure S4 Quantitative analysis of the oncogenic effects of CDH4 on PTC cells. Figure S5 GSEA analysis of FER1L4 and CDH4, and correlation analysis between cadherins and FER1L4.

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Long Noncoding RNA FER1L4 Promotes the Malignant Processes of Papillary Thyroid Cancer by Targeting miR-612/ Cadherin 4 Axis

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