Upregulation of CYP1B1-AS1 correlates with aggressive phenotypes and poor prognosis in thyroid cancer

Abstract

Background

Aberrant expression of lncRNA CYP1B1-AS1 drives the pathogenesis of diverse cancers, with thyroid carcinoma (TC) being no exception.

Aim

This study was designed to clarify how CYP1B1-AS1 expression impacts prognostic outcomes in TC patients.

Methods

qRT-PCR was employed to measure CYP1B1-AS1 and miR-3127-5p expression signatures in TC tissues and cell lines. Prognostic value was analyzed by Kaplan-Meier survival and multivariate Cox regression assays. Binding affinities between CYP1B1-AS1/miR-3127-5p and miR-3127-5p/ERBB2 were validated by dual-luciferase reporter assays; RNA pull-down assays were used to confirm the CYP1B1-AS1/miR-3127-5p interaction. Flow cytometry, CCK-8, Wound Healing and Transwell assays were employed to evaluate TC cell apoptosis, growth, motility and invasiveness, respectively. Western blot was performed to detect ERBB2 protein expression.

Results

CYP1B1-AS1 was significantly upregulated in TC tissues and cell lines, with concomitant downregulation of miR-3127-5p. TC patients with elevated CYP1B1-AS1 expression exhibited markedly decreased survival rates. CYP1B1-AS1, TNM stage, local invasion, and LNM were defined as prognostic correlates for TC. Knockdown of CYP1B1-AS1 inhibited the malignant phenotypic behaviors of TC cells. CYP1B1-AS1 bound to miR-3127-5p, and the miR-3127-5p inhibitor could counteract the inhibitory effects induced by CYP1B1-AS1 knockdown. ERBB2 was identified as a downstream target gene of miR-3127-5p.

Conclusion

Increased CYP1B1-AS1 expression correlates strongly with reduced overall survival in TC patients, establishing it as a clinically applicable prognostic indicator and promising therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12957-026-04406-3.

Introduction

Among endocrine tumors, thyroid cancer (TC) ranks as the most widespread, contributing to 88% of these tumors and 3% of all human cancers [1]. Over the past few decades, its incidence has increased rapidly on a global scale [2]. The latest statistical data show approximately 600,000 new TC cases emerge yearly [3]. The standard treatment for TC patients involves surgical resection, often followed by adjuvant radioactive iodine (RAI) therapy after thyroidectomy [4]. Despite improvements in current therapeutic strategies for TC, the overall 5-year survival rate of patients remains suboptimal [5]. Long non-coding RNAs (lncRNAs) have evolved into highly viable diagnostic and prognostic biomarkers with considerable clinical utility across diverse cancer subtypes [6]. Therefore, exploring novel biomarkers is of great significance for prognostic prediction in TC patients.

LncRNAs are closely associated with the occurrence and development of cancers, and are regarded as important biomarkers for cancer diagnosis and prognostic follow-up [7, 8]. As a member of the CYP family, CYP1B1 has been identified to exhibit a potential function in carcinogenesis and tumor progression [9, 10]. The biological functions of CYP1B1-AS1 have been elucidated in a diverse array of diseases. Specifically, this lncRNA serves as an emerging therapeutic target and prognostic biomarker for glioblastoma (GBM) patients [11]. In a non-neoplastic disease context, CYP1B1-AS1 expression is markedly upregulated in patients diagnosed with sepsis [12]. However, the expression status and functional significance of CYP1B1-AS1 in this cancer type are yet to be explored.

miR-3127-5p, the miRNA targeted by CYP1B1-AS1 [13], was initially identified in human melanoma specimens through deep sequencing technology, with its first characterization reported in 2010 [14]. It exerts dual roles in cancers, acting either as an oncogenic factor or a tumor-inhibitory molecule. In non-small cell lung carcinoma, it is recognized as a tumor-suppressive miRNA, with its expression levels correlating with tumor recurrence and unfavorable clinical outcomes [15]. In gliomas, miR-3127-5p influences tumor progression by regulating the expression of ELAVL1 [16]. In lung and colorectal malignancies, it acts as a tumor-inhibitory factor [17, 18]. In contrast, miR-3127, a homologous family member to miR-3127-5p, can facilitate malignant lesion progression while acting as an oncogenic factor in hepatocellular carcinoma [19].

The lncRNA CYP1B1-AS1 exerts pivotal functions in cancer initiation and malignant progression. However, the precise molecular mechanisms underlying CYP1B1-AS1 activity in TC have not yet been fully elucidated. Herein, we sought to characterize CYP1B1-AS1 expression profiles in TC tissues and cells, while further evaluating its clinical prognostic significance.

Materials and methods

Bioinformatics prediction

Raw data from patients with TC were obtained from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). Among the available datasets, GSE150899 and GSE192560, which include lncRNA profiling data, were selected for this study. Differential expression analysis was performed using these datasets. LncRNAs were considered differentially expressed if they met the following thresholds: |log2-fold change (log2FC) | ≥ 1 and p-value < 0.05.

Expression profiles of CYP1B1-AS1 in TC were retrieved from the ENCORI platform (http://starbase.sysu.edu.cn/index.php), encompassing 510 malignant tissue specimens and 58 non-tumor tissue samples. Downstream miRNAs for CYP1B1-AS1 were predicted using the LncRNASNP2 database (https://guolab.wchscu.cn/lncRNASNP/#!/).

The target genes of miR-3127-5p were retrieved from the ENCORI/starBase database, and only genes with a TDMDscore > 1 were retained. Genes associated with thyroid cancer were collected from the GeneCards database, and those with a Relevance score > 30 were selected. The two sets of genes were intersected. The resulting overlapping genes were subjected to PPI analysis using STRING and visualized with Cytoscape software. After analyzing the network using NetworkAnalyzer, the top five genes ranked by degree were identified, and their expression levels were detected via qRT‑PCR.

Clinical tissue samples

From 128 patients who underwent surgery at First Affiliated Hospital of Xuzhou Medical University between January 2018 and December 2019, TC and normal tissues were gathered, with all samples having complete clinicopathological records. The enrolled patients were newly diagnosed with TC and had not received any adjuvant therapy before surgery. Written informed patient consent was obtained from every participant, with the study having received approval from First Affiliated Hospital of Xuzhou Medical University Committee in compliance with the Declaration of Helsinki.

Cell culture

Normal (Nthy-ori3-1) and TC cell lines (BHT-101, TPC-1, B-CPAP, FTC133) were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Strict adherence to culture conditions for all cells was maintained, following the instructions provided by the cell bank.

Subcellular fractionation location

BHT-101 and B-CPAP cells were rinsed with ice-cold phosphate buffer and subsequently lysed. Cellular lysates were fractionated into cytoplasmic and nuclear components using the PARIS kit (Cat# AM1921, Thermo Fisher Scientific, USA). Following total RNA extraction, CYP1B1-AS1 expression in each fraction was determined by qRT-PCR, with normalization to GAPDH (cytoplasmic control) and U6 (nuclear control).

qRT-PCR

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used for the isolation of total RNA from the samples. Reverse transcription reactions were carried out to transcribe RNA into cDNA, following the guidelines of the Takara PrimeScript RT Kit (RR037Q). For qRT-PCR amplification, the TB Green Premix Ex Taq II Kit (Takara) was utilized, with reaction components and procedures adhering to the kit’s guidelines. The qRT-PCR assay was conducted employing the subsequent thermal cycling parameters: initial denaturation at 95 °C for 5 min, followed by 40 cycles encompassing denaturation at 95 °C for 20 s, annealing at 60 °C for 20 s, and extension at 72 °C for 30 s. Relative expression abundances of the target genes were computed via the 2^−ΔΔCt algorithm. GAPDH and U6 were used as endogenous reference genes.

Cell transfection

In transfection experiments with BHT-101 and B-CPAP cells, we utilized Lipofectamine 3000 transfection reagent (Invitrogen). The experiments were divided into two parts. The CYP1B1-AS1 knockdown assay included three groups: blank control (ctrl), transfected with non-targeting siRNA (si-NC), and transfected with si-CYP1B1-AS1 (si-CYP1B1-AS1). The rescue experiment comprised five treatment groups transfected with ctrl, si-NC, si-CYP1B1-AS1, si-CYP1B1-AS1 + miRNA negative control (NC), and si-CYP1B1-AS1 + miR-3127-5p inhibitor (inhibitor), respectively. All siRNA sequences were synthesized and provided by GenePharma (China). Sangon Biotech (China) designed, synthesized, and supplied both the pcDNA3.1-miR-3127-5p overexpression vector and the empty pcDNA3.1 vector.

Dual luciferase reporter assay

We first amplified the wild-type CYP1B1-AS1 (wt-CYP1B1-AS1) or ERBB2 (ERBB2-WT), along with the mutant CYP1B1-AS1 (mut-CYP1B1-AS1) or ERBB2 (ERBB2-MUT) corresponding fragments harboring the predicted binding sites, which were then cloned respectively into the pmiR-Report luciferase vector (GenScript, Nanjing, China). The constructed luciferase recombinant vectors were co-transfected into BHT-101 and B-CPAP cells alongside miR-3127-5p mimic or miRNA negative control oligonucleotide (ov-NC), in strict accordance with the instructions of Lipofectamine 3000 reagent (Invitrogen). Luciferase activity at 48 h post-transfection was quantified utilizing the Dual-Luciferase Reporter Assay System (Promega, USA).

RNA pull-down

Biotinylated CYP1B1-AS1 (bio-CYP1B1-AS1) and biotinylated negative control (bio-NC) were transfected into BHT-101 and B-CPAP cells, with incubation maintained for 48 h. RNA pull-down experiments were carried out with the Pierce Magnetic RNA-Protein Pull-Down Kit (Sigma-Aldrich). Streptavidin-agarose magnetic beads (50 µL, Thermo Fisher Scientific) were incorporated into lysates derived from BHT-101 and B-CPAP cells. miR-3127-5p expression was quantified via qRT-PCR.

CCK 8 assay

BHT-101 and B-CPAP cells in the logarithmic growth phase were seeded into 96-well microplates; the cells were subsequently subjected to incubation for 0, 24, 48, and 72 h, respectively. At each time point, CCK-8 solution (Beyotime, China) was dispensed into individual wells, after which the plates were incubated continuously for a preset period in accordance with the manufacturer’s protocol. Next, we measured 450 nm OD values using a Bio-Rad (USA) iMark microplate reader.

Transwell assay

We re-suspended transfected BHT-101 and B-CPAP cells in serum-deprived culture medium, inoculated them into the upper compartment, and loaded the lower compartment with complete culture medium supplemented with 10% fetal bovine serum (FBS). Following an incubation period of defined duration, cells remaining in the upper compartment without migration were discarded, while cells that had traversed the membrane were dyed using 0.1% crystal violet solution. Stained cells were visualized and enumerated under a light microscope to assess cellular migratory capacity. Regarding the cell invasion experiment, Matrigel (BD Biosciences) was initially covered on the basal membrane of Transwell inserts (BD Biosciences), with all subsequent experimental steps consistent with those adopted for the migration assay.

Wound healing assay

Cells were seeded in 6-well plates and cultured in DMEM medium supplemented with 10% FBS at 37 °C until complete confluence. A sterile 200 µL pipette tip was then used to scratch the cell monolayer. The medium was replaced with serum-free DMEM, and the cells were incubated at 37 °C for 24 h. Cell migratory distance was observed under a light microscope at 200× magnification (Olympus Corporation). The migration distance was quantitatively analyzed using ImageJ software (Version 1.49, National Institutes of Health, USA).

Flow cytometry

Transfected cells were trypsinized, harvested and washed twice with PBS, then resuspended in binding buffer containing 5 µL Annexin-V-FITC and 5 µL PI (Apoptosis Detection Kit Ⅰ, BD Biosciences, San Jose, California, USA). After incubation for 15 min at room temperature in the dark, the samples were subjected to flow cytometric analysis using a BD Accuri C6 flow cytometer (BD Biosciences). The proportions of early, late and total apoptotic cells were quantitatively determined.

Western blotting

Protein samples from BHT-101 and B-CPAP cell lysates were boiled for 5 min in SDS-PAGE sample buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromophenol blue, 5% β-mercaptoethanol). The samples were then separated by SDS-PAGE, and the resolved proteins were transferred electrophoretically to a PVDF membrane (Bio-Rad Laboratories). After blocking for 1 h, the membranes were incubated with primary antibodies against ERBB2 (29D8, Cell Signaling Technologies) and β-actin (Sigma-Aldrich). Primary antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, and the immune complexes were visualized with an ECL detection system (Thermo Fisher Scientific).

Statistical analysis

SPSS 27.0 and GraphPad Prism 10.1.2 were utilized for the execution of statistical analyses. The t-test was applied to perform comparisons between the two groups (TC tissues vs. normal tissues). One-way and two-way ANOVA were employed for multiple group comparisons in line with the experimental design. Kaplan-Meier (KM) survival plots were generated utilizing GraphPad Prism 10.1.2, whereas multivariate Cox proportional hazards regression analyses were implemented via SPSS 27.0. All experimental graphs were generated using Graphpad Prism 10.1.2. Significance was defined by P < 0.05.

Result

CYP1B1-AS1 upregulation in TC

The technical roadmap of the present study is illustrated in Fig. 1. Analyses of GSE150899 (Fig. 2A) and GSE192560 (Fig. 2B) revealed that CYP1B1-AS1 was one of the dysregulated antisense lncRNAs (Fig. 2C). The expression of CYP1B1-AS1 was significantly upregulated in the ENCORI database (Fig. 2D). This lncRNA also exhibited an elevated expression pattern in TC tissues versus normal ones based on the TCGA data (Fig. 2E). Using qRT-PCR, we found that CYP1B1-AS1 was significantly increased in TC cell lines compared with normal TC cell lines (P < 0.05, Fig. 2F). Moreover, CYP1B1-AS1 exhibited markedly upregulated expression within our TC tissue cohort compared with adjacent normal tissues (Fig. 2G), which demonstrates a consistent upregulation trend of this lncRNA between independent bioinformatic databases and our clinical tissue cohort.

Fig. 1.

The technical roadmap of the present study

Fig. 2.

Expression profiles of CYP1B1-AS1 and miR-3127-5p in TC tissues and cells, and survival analysis of CYP1B1-AS1. (A) Volcano map of GSE150899 dataset showing the overall differentially expressed lncRNAs in TC. (B) Volcano map of GSE192560 dataset showing the differentially expressed lncRNAs in TC. (C) The VENN diagram showing the shared lncRNAs between GSE150899 and GSE192560. (D) Expression of CYP1B1-AS1 in normal and thyroid carcinoma (THCA) samples (from the ENCORI database). (E) Expression differences of CYP1B1-AS1 in normal and thyroid carcinoma (THCA) samples based on TCGA database. (F) Expression of miR-3127-5p in TC and normal cell lines. (G) Evaluation of CYP1B1-AS1 expression in 128 pairs of matched tissues and non-tumor tissue samples. (H) Kaplan-Meier survival analysis of CYP1B1-AS1 in patients with TC. (*P < 0.05, **P < 0.01, ***P < 0.001)

Correlation of CYP1B1-AS1 expression with clinicopathological characteristics in TC patients

TC patients were stratified into subgroups with the mean value of relative CYP1B1-AS1 expression adopted as the stratification threshold. Specifically, the study cohort was categorized into two subgroups with low and high CYP1B1-AS1 expression, respectively. Elevated expression levels of CYP1B1-AS1 were significantly correlated with tumor size, TNM stage, local invasion, lymph node metastasis (LNM), and distant metastasis (P < 0.05) (Table 1).

Table 1.

Correlation between CYP1B1-AS1 expression level and clinicopathological parameters in TC patients

Parameters			All cases (n = 128)	Low CYP1B1-AS1 (n = 58)	High CYP1B1-AS1 (n = 70)	P
Age (years)						0.363
		≤ 50	74	31	43
		> 50	54	27	27
Gender						0.173
		Male	47	25	22
		Female	81	33	48
Tumor size						0.020
		< 2 cm	81	43	38
		≥ 2 cm	47	15	32
Histological subtype						0.107
		PTC/FTC	110	53	57
		PDTC/ATC	18	5	13
Bilateral tumors						0.313
		No	108	51	57
		Yes	20	7	13
TNM stage						0.008
		I/II	96	50	46
		III/IV	32	8	24
Local invasion						0.006
		No	88	47	41
		Yes	40	11	29
LNM						0.006
		Negative	76	42	34
		Positive	52	16	36
Distant metastasis						0.013
		No	121	58	63
		Yes	7	0	7

Abbreviations: PTC Papillary thyroid carcinoma, FTC Follicular thyroid carcinoma, PDTC Poorly differentiated thyroid cancer, ATC Anaplastic thyroid carcinoma, LNM Lymph node metastasis

Prognostic value of CYP1B1-AS1 in TC patients

TC patients with elevated CYP1B1-AS1 expression displayed markedly reduced overall survival duration (P = 0.001; Fig. 2H). Multivariate Cox regression analysis identified CYP1B1-AS1 expression (HR = 5.857, 95%CI = 1.409–24.347, P = 0.015), TNM stage (HR = 3.999, 95%CI = 1.152–13.878, P = 0.029), local invasion (HR = 3.195, 95%CI = 1.052–9.699, P = 0.040), and LNM (HR = 5.729, 95%CI = 1.144–28.692, P = 0.034) as factors predictive of patient prognosis in TC (Table 2).

Table 2.

Multivariate cox analysis

Characteristics	Multivariate Analysis
	HR	95% CI	P
CYP1B1-AS1	5.857	1.409–24.347	0.015
Age	2.790	0.832–9.355	0.097
Gender	2.752	0.860–8.807	0.088
Tumor size	3.835	0.989–14.863	0.052
Histological subtype	1.814	0.202–16.320	0.595
Bilateral tumors	2.141	0.742–6.178	0.159
TNM stage	3.999	1.152–13.878	0.029
Local invasion	3.195	1.052–9.699	0.040
Lymph node metastasis	5.729	1.144–28.692	0.034
Distant metastasis	2.281	0.758–6.858	0.142

miR-3127-5p as a downstream miRNA of CYP1B1-AS1

Subcellular localization analysis showed a predominantly cytoplasmic distribution of CYP1B1-AS1 (Fig. 3A), suggesting its potential role as a competing endogenous RNA (ceRNA) for miRNAs. We first utilized bioinformatics tools to predict the downstream miRNAs for CYP1B1-AS1 and miR-3127-5p (Fig. 3B) was ranked highly due to its notably low calculated binding free energy of -18.4 kcal/mol, indicating a relatively strong interaction with CYP1B1-AS1. Notably, miR-3127-5p has been reported to be downregulated in TC tissues when compared with normal tissues [20], made it a prime candidate for initial experimental validation. The miR-3127-5p mimic triggered a substantial decline in luciferase reporter activity encoded by the wt CYP1B1-AS1 plasmid (P < 0.01), whereas such activity remained unaltered in the mut-CYP1B1-AS1 reporter plasmid (Figs. 3C). RNA pull-down assays confirmed the direct interaction between CYP1B1-AS1 and miR-3127-5p (Fig. 3D). The current findings confirmed that CYP1B1-AS1 and miR-3127-5p interact directly.

Fig. 3.

miR-3127-5p is a downstream miRNA of CYP1B1-AS1. (A) Investigation of CYP1B1-AS1 subcellular localization in BHT-101 and B-CPAP cells. (B) Predicted binding sites between CYP1B1-AS1 and miR-3127-5p from the lncRNASNP2 database. (C) Luciferase activity of wild-type and mutant CYP1B1-AS1 following miR-3127-5p overexpression. (D) RNA pull-down validation of CYP1B1-AS1/miR-3127-5p interaction in BHT-101 and B-CPAP cells. (**P < 0.01, ***P < 0.001)

miR-3127-5p exhibited a notably decreased expression in TC tissues (P < 0.001, Fig. 4A). Across TC cell strains, a pronounced decrease in miR-3127-5p expression was additionally observed (P < 0.05, Fig. 4B). CYP1B1-AS1 was significantly negatively correlated with miR-3127-5p (r = − 0.7457, P < 0.001, Fig. 4C). Expressions of CYP1B1-AS1 were significantly reduced, and miR-3127-5p were increased in the si-CYP1B1-AS1 group compared against the si-NC group (P < 0.01, Fig. 4D and E). The miR-3127-5p inhibitor counteracted the miR-3127-5p expression upregulation elicited by CYP1B1-AS1 knockdown in BHT-101 and B-CPAP cells (P < 0.001, Fig. 4F).

Fig. 4.

The correlations of CYP1B1-AS1 and miR-3127-5p in TC tissues and cells. (A) Expression of miR-3127-5p in 128 pairs of matched tissues and non-tumor tissue samples. (B) Expression of miR-3127-5p in TC and normal cell lines. (C) The negative correlation of CYP1B1-AS1 with miR-3127-5p in TC tissues. (D-E) Expression levels of CYP1B1-AS1 (D) and miR-3127-5p (E) after CYP1B1-AS1 knockdown. (F) Comparison of miR-3127-5p expression levels between si-CYP1B1-AS1+-NC-transfected and si-CYP1B1-AS1 + inhibitor -transfected groups. (*P < 0.05, **P < 0.01, ***P < 0.001)

CYP1B1-AS1 regulates cellular malignant phenotype-related behaviours in TC cells by modulating miR-3127-5p

Knockdown of CYP1B1-AS1 significantly inhibited cell proliferation in cell lines (P < 0.001, Fig. 5A and B). However, this inhibitory effect induced by CYP1B1-AS1 silencing was abrogated by co-transfection with miR-3127-5p inhibitor (P < 0.05, Fig. 5A and B). In contrast, the TC cell apoptosis was increased upon CYP1B1-AS1 knockdown and reduced if miR-3127-5p was inhibited (Fig. 5C and D).

Fig. 5.

Effects of CYP1B1-AS1 on TC cell proliferation and apoptosis. (A-B) Cell proliferation in BHT-101 (A) and B-CPAP (B) cells assessed by CCK8 assay. (C-D) Cell apoptotic rates in BHT-101 (C) and B-CPAP (D) cells were determined using Annexin-V-FITC/PI staining and flow cytometry. (**P < 0.01, ***P < 0.001)

Consistently, the impaired migratory and invasive capacities of TC cells elicited by CYP1B1-AS1 downregulation were abrogated upon treatment with the miR-3127-5p inhibitor (P < 0.05, Fig. 6A and B, supplementary Fig. 1). The wound closure in the TC cells with CYP1B1-AS1 silencing increased as compared to the corresponding negative control group, while miR-3127-5p inhibitor abolished this increase (Fig. 6C and D). miR-3127-5p inhibitor counteracts the inhibitory effects on cellular malignant phenotype-related behaviours produced by knocking down CYP1B1-AS1.

Fig. 6.

Effects of CYP1B1-AS1 on TC cell migration and invasion. (A) Cell migration in BHT-101 and B-CPAP cells were evaluated by Transwell assay. (B) Cell invasion in BHT-101 and B-CPAP cells were assessed by Matrigel-based Transwell assay. (C-D) Scratch (wound healing) assays were used to evaluate the effect of CYP1B1-AS1 on cell migration. (*P < 0.05, **P < 0.01, ***P < 0.001)

ERBB2 was a target of miR-3127-5p in TC

Potential target genes of miR-3127-5p were retrieved from the ENCORI/starBase database. Genes with a TDMDscore > 1 were retained, yielding 389 candidates. Separately, genes associated with TC were collected from the GeneCards database, and those with a Relevance score > 30 were kept, resulting in 671 genes. An intersection of these two gene sets yielded 23 overlapping genes (Fig. 7A). PPI analysis of these 23 genes via NetworkAnalyzer identified the top five genes with the highest degree scores: EGFR, BCL2, EZH2, ERBB2, and MAPK3 (Fig. 7B). Data mining from the TCGA database revealed that ERBB2 expression was significantly elevated in TC (Fig. 7C). The predicted target binding site between miR-3127-5p and ERBB2 is presented in Fig. 7D. Western Blot analysis showed that knockdown of CYP1B1-AS1 significantly decreased ERBB2 protein expression, whereas inhibition of miR-3127-5p increased ERBB2 protein levels (Fig. 7E). Finally, a direct interaction between miR-3127-5p and the ERBB2 transcript was confirmed using a dual-luciferase reporter assay (Fig. 7F).

Fig. 7.

ERBB2 was a target for miR-3127-5p. (A) The VENN diagram showing the shared genes between miR-3127-5p targets from ENCORI/starBase database and the TC-related genes from GeneCards database. (B) The protein-protein interactions among the 23 target genes according to the node degree. (C) The expression levels of the top five target genes in TCGA cohort. (D) Potential binding sites between miR‑3127‑5p and ERBB2. (E) The protein levels of ERBB2 in BHT-101 and B-CPAP cells after transfection or co-transfection were detected using Western blotting assays. (F) Luciferase activity of wild-type and mutant ERBB2 following miR-3127-5p overexpression

Discussion

Abnormal regulation of lncRNAs has been shown to drive malignant tumor progression in TC. For example, the lncRNAs H19 and HOTAIR are overexpressed in TC cells and exert a pro-tumorigenic effect by enhancing tumor cell malignant phenotypes [21]. According to extensive research findings, lncRNAs have emerged as reliable markers of prognosis for patients with TC [22]. The specific mechanisms underlying CYP1B1-AS1 involvement in TC progression remain largely unknown. Herein, we explored the clinical implications of CYP1B1-AS1 in the prognostic evaluation of TC. In TC patients, elevated CYP1B1-AS1 expression emerged as a factor predictive of reduced survival duration, a finding that emphasizes its prospective role as a novel biomarker for prognostic stratification of this malignancy.

We identified marked upregulation of CYP1B1-AS1 in TC tissues and cells, a result congruent with previous publications reporting robust overexpression of this lncRNA in breast cancer tissues [23]. Expression of CYP1B1-AS1 was strongly linked to tumor size, TNM stage, local invasion, LNM, and distant metastasis in TC patients. Emerging evidence indicates that lncRNAs exhibit a close correlation with LNM and TNM staging [24]. These prior findings exhibit a high degree of similarity to the correlations between CYP1B1-AS1 and clinicopathological features elucidated in the present study.

CYP1B1-AS1 functions as a prognostic factor for TC patients, as shown in this study. Prior research has reported that in sepsis patients, elevated CYP1B1-AS1 expression signals poor prognosis and acts as a potential risk factor [12]. Moreover, the present study verified that TNM stage, local invasion, and LNM serve as prognostic factors for TC patients. Published evidence has indicated that TNM stage and LNM act as independent risk factors for prognosis in TC patients [25]. Separate studies have also demonstrated that local invasion represents a key prognostic factor for TC patients [26]. These prior observations align with our findings.

LncRNAs can exert regulatory effects by targeting and binding to miRNA [27]. Our results demonstrated a direct interaction between CYP1B1-AS1 and miR-3127-5p, a binding event that was verified by dual-luciferase reporter assays and RNA pull-down assays. Consistent with this regulatory pattern, prior studies have reported similar lncRNA-miRNA targeting mechanisms. lncRNA CYP1B1-AS1 exacerbates septic inflammatory responses by targeting miR-18a-5p [12]. Inhibition of miR-3127 expression by LINC00319 drives the malignant progression of bladder cancer [28]. Marked downregulation of miR-3127-5p was detected in TC, a finding consistent with an earlier investigation that not only uncovered repressed miR-3127-5p expression in PTC cells but also delineated its putative tumor-suppressive role in this malignancy [13].

In rescue assays, treatment with a miR-3127-5p inhibitor completely abrogated the suppressive effect exerted by CYP1B1-AS1 silencing on cellular malignant phenotypes. This regulatory pattern is consistent with previous research conclusions: in PTC, the miR-3127-5p inhibitor can rescue the reduced cell proliferation elicited by LINC00284 knockdown [13]. In colorectal cancer cells, miR-3127-5p is also found to facilitate the acquisition of proliferative, migratory, and invasive capabilities [29]. In hepatocellular carcinoma (HCC), miR-3127 can promote cell proliferation and tumorigenicity [19]. These research results are consistent with those of the present study. In summary, regulating miR-3127-5p expression may be the way CYP1B1-AS1 participates in TC progression.

The present study has only focused on the interaction between CYP1B1-AS1 and miR-3127-5p in TC. Several other miRNAs are predicted to be potential binding partners of CYP1B1-AS1 according to the LncRNASNP2 database, such as miR-193a-5p, miR-139-3p, miR-219a-1-3p and miR-26b-3p. The validity of these predicted binding associations remains to be verified by additional experimental assays in subsequent research. This inherent limitation of our current work also indicates that the ceRNA regulatory mechanism underlying the biological functions of CYP1B1-AS1 in TC has not yet been fully and thoroughly elucidated.

This is the first systematic study to investigate the clinical implications and molecular regulatory mechanisms underlying CYP1B1-AS1 in TC. However, this study has certain limitations: in vitro experiments fail to recapitulate the complexity of in vivo environments, and animal models were not incorporated due to experimental and funding restrictions. For instance, ERBB2 (also known as HER2) is known to promote the migration and invasion of TC cells through the MAPK/EMT signaling pathway [30], a classic oncogenic cascade that may be potentially regulated by CYP1B1-AS1 via its ceRNA network, yet this putative regulatory link remains unvalidated in our current work. Thus, further investigation into other signaling pathways associated with CYP1B1-AS1 is required to comprehensively elucidate its functional role in TC.

Conclusion

CYP1B1-AS1 regulates the malignant phenotypes of TC cells via the miR-3127-5p/ERBB2 axis, and this study is the first study to establish elevated CYP1B1-AS1 as a potential prognostic indicator in TC patients.

Authors’ contributions

Xiaotao Duan made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, and draft of the manuscript. Jike Wang revised the manuscript critically for important intellectual content. Xun Li, Huiling Qin and Jiayong Zhang re-evaluated the clinical data, revised the manuscript and performed the statistical analysis and revised the manuscript. All authors read and approved of the final manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of First Affiliated Hospital of Xuzhou Medical University. All patients and their families signed an informed consent form.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests