MicroRNA-181a-5p promotes papillary thyroid carcinoma progress via the PTEN/AKT pathway

Abstract

The objective of this investigation was to determine the expression profile and latent mechanism of microRNA-181a-5p (miR-181a-5p) in the genesis and progression of papillary thyroid cancer (PTC). MiR-181a-5p was discovered to be upregulated in PTC tissues and cells in this study, as confirmed by RT‒qPCR and The Cancer Genome Atlas database. Notably, in PTC patients, the miR-181a-5p level was linked to tumor size and thyroid capsule invasion. A series of experiments demonstrated that miR-181a-5p upregulation in PTC cells notably enhanced proliferation, motility, and invasion, whereas suppressing miR-181a-5p hindered these functions. Western blotting revealed that miR-181a-5p suppressed PTEN expression, boosting the activation of phosphorylated AKT (P-AKT). According to predictive bioinformatics research and luciferase reporter gene tests, miR-181a-5p may target a specific binding site on the PTEN 3'UTR. To sum up, this study indicated that miR-181a-5p promoted PTC progression through the PTEN/Akt pathway. This investigation reveals a potential mechanism for PTC progression and provides a foundation for clinical therapies.

Introduction

In the past few years, there has been a notable increase in the occurrence of thyroid cancer (TC) [1]. Papillary thyroid carcinoma (PTC) is the predominant histologic form of cancer, accounting for approximately 85% to 90% of thyroid cancer cases [2]. Although the majority of PTCs have a favorable prognosis, a certain percentage of PTCs still exhibit significant progression to secondary metastasis, leading to a poor outcome [3]. The greatest clinical challenge in identifying PTCs lies in accurately diagnosing them. Ultrasound diagnosis relies heavily on the examiner's experience and knowledge. Fine needle aspiration biopsy (FNAB) is also limited because it screens only a small volume of tissue and cellular components and may not fully represent the nature of the lesion [4]. Approximately 15%−30% of lesions cannot be diagnosed [5]. Molecular testing can enhance diagnostic accuracy by identifying patients with uncertain cytology as PTCs [6]. Hence, it is paramount to uncover the molecular mechanisms underpinning PTC to identify more potent therapeutic targets for managing this disease.

MicroRNAs (miRNAs) are small, noncoding RNAs that influence gene expression in a variety of ways, ranging from 19 to 25 nucleotides in length [7]. According to a substantial body of research, miRNAs may be involved in a number of cancers, including carcinogenesis, metastasis, and treatment resistance [8]. Approximately half of the human miRNA-coding genes that have been identified are found in tumor-associated fragile chromosomal regions [9]. MicroRNAs (miRNAs) offer potential for the identification, diagnosis, and prognosis of cancers, both within tumor tissue and in the circulatory system [10]. According to certain studies, miRNAs can be utilized as indicators and treatment targets for PTC [11, 12]. Additionally, miRNAs can be used for prognostic assessment of PTC patients [13], providing clinicians with more precise treatment options.

Despite the identification of microRNA-181a-5p as being significantly upregulated in aggressive cancers, such as lung [14], osteosarcoma [15], and breast cancer [16], limited investigations have been undertaken on papillary thyroid carcinoma. According to a 2021 study, miR-181a-5p might decrease the effectiveness of postoperative radioiodine therapy in PTC patients [17]. Nevertheless, the relevance of miR-181a-5p for the prognosis and underlying mechanisms of PTC remains obscure.

Therefore, the purpose of this study was to examine miR-181a-5p expression and investigate its implications for clinicopathology in patients with PTC. Additionally, we explored the biological functionality and pertinent functions of miR-181a-5p in PTC through an array of experiments.

Materials and methods

Tissue samples

111 individuals diagnosed with PTC and 52 individuals with benign thyroid nodules (BTNs) at Luoyang Central Hospital, a subsidiary of Zhengzhou University, were recruited for this study between August 2021 and February 2022. All enrolled patients underwent initial surgery without any preexisting anticancer interventions. Pathologically, the tumors were identified and verified. Inclusion criteria: (1) Aged between 18 and 80 years; (2) Underwent surgical resection of thyroid nodules with complete postoperative pathological reports available; (3) Had complete preoperative thyroid ultrasound examination data; (4) Voluntarily provided signed informed consent.

Exclusion criteria: (1) History of radiotherapy or chemotherapy prior to surgery; (2) Received any other preoperative treatments; (3) Presence of severe cardiopulmonary diseases, hepatic or renal failure; (4) Comorbidities including malnutrition, autoimmune diseases, infectious diseases, or other malignancies. A total of 111 PTC tissues and their respective adjacent noncancerous tissues (ANTs) and 52 BTNs obtained from patients were immediately treated with RNAlater (Thermo Fisher Scientific, USA) and then immediately followed by − 80 °C pending further analysis. Informed consent was obtained from all participants. The Ethics Committee of Luoyang Central Hospital, which is associated with Zhengzhou University, has granted approval for this study in compliance with the Helsinki Declaration.

Cell lines and transfection

Thyroid cells, including a human normal thyroid cell line (Nthy-ori3-1) and PTC cells (KTC-1, TPC-1), were obtained from Procell Biotech (Wuhan, China). The cells were incubated in RPMI 1640 (Gibco, USA) enriched with 10% fetal bovine serum (FBS; Gibco) at 37 °C in 5% CO2.

Genepharma Co., Ltd., synthesized the miR-181a-5p inhibitor and mimics and their negative controls, the NC inhibitor and the NC. The plasmid was transfected into cells using Lipofectamine 2000 (Invitrogen).

RT‑qPCR

Utilizing TRIzol reagent (Invitrogen, USA), extracts from tissues or cells total RNA were carried out. Using the First-strand Synthesis Kit Mir-X miRNA (Takara Bio, Japan), reverse transcription was carried out. RT‒qPCR reactions were performed on the TB Green Advantage RT‒qPCR Premix Kit (Takara Bio, Japan). The 2^−ΔΔCt method was employed to determine the relative expression of miR-181a-5p, U6 served as the endogenous control. Each sample needs three technical repetitions (that is, each sample needs to set three multiple holes in a 96 well plate), and three biological repetitions (different samples or individuals) need to be set at the same time.

The following primers (Sangon Biotech Co., Ltd., China) were used in this work. MiR-181a-5p, forward, 5'- ATTCAACGCTGTCGGTGAGT-3'; U6, forward, 5'- GGAACGATACAGAGAGAAGATTAGC-3', reverse, 5'-TGGAACGCTTCACGAATTTGCG-3'.

TCGA dataset analysis

From TCGA (https://portal.gdc.cancer.gov/), mature miR-181a-5p (normal: 67, tumor: 506) was obtained. Then, differentially expressed miR-181a-5p between tumors and normal tissues and between tumors and ATNs was subsequently assessed via the Wilcoxon test and Wilcoxon signed-rank test.

CCK-8 assay

Culture plates (96-well) were seeded with 3 × 10³ KTC-1 or TPC-1 cells per well. Then, 10 μL CCK-8 solution was injected at predetermined time points, and the cells were cultured at 37 °C for 2 h. A multifunctional enzyme marker (Thermo Fisher Scientific, USA) was tested at 450 nm.

Colony formation assay

A 6-well plate was used for transfected PTC cells culture. The cells were cultivated with complete media for a duration of one week to allow for the formation of visible colonies. Next, the clones were washed, fixed using 4% formaldehyde (Solarbio, China), and colored with 2.5% crystal violet (Beyotime, China).

Wound healing assay

A 6-well plate was used for cell culture. The transfected PTC cells were cultured until they reached 90% confluence. Subsequently, using a 200 μL pipette, the cell monolayer was scraped, and any floating cells were removed using PBS. Photomicrographs of the cell cultures were captured at predetermined time points after scratching.

Transwell cell assay

Transwell assays were carried out in 24-well chambers precoated with 8-micron pore inserts from Matrigel (Corning, USA). The upper chamber was suspended with 5 × 10⁴ cells in two hundred microliters of medium containing no FBS. Moreover, the lower chamber was injected medium containing 500 μL of 12% FBS. Twenty-four hours later, the invading cells were fasted in 4% formaldehyde (Solarbio, China) as well as stained with 2.5% crystal violet (Beyotime, China).

Western blot

PTC cells were lysed using RIPA lysis buffer (Beyotime, China). The proteins were subjected to 4%−20% SDS‒PAGE after BCA quantification (Beyotime, China) and finally transferred to PVDF membranes (Beyotime, China) [18]. After sealing the membranes with 5% skimmed milk powder, they were followed by incubation with specific primary antibodies against PTEN (1:1000; Bioss, China), AKT (1:1000; Cell Signaling, USA), P-AKT (1:1000; Cell Signaling, USA), PI3K (1:1000; Bioss, China), P-PI3K (1:1000; Bioss, China), or GAPDH (1:2000; Cell Signaling, USA) overnight at 4 °C. Secondary antibodies (Beyotime, China) were used to incubate the blots, after which the protein bands were observed to ECL reagent (Beyotime, China). The grayscale ratio of PTEN or Akt to the internal control GAPDH was calculated using the software Image Pro 6.0 (Media Cybernetics, Rockville, MD, USA). The grayscale of test protein bands was normalized to the GAPDH bands.

Bioinformatics analyses

The predictions of miR-181a-5p target genes were conducted via the miRDB, miRWalk, starBase, TarBase and TargetScan databases [19]. These five databases were selected to combine computational predictions with experimental validation. miRDB, TargetScan, and miRWalk provide comprehensive in silico predictions based on sequence complementarity and conservation. Conversely, starBase (CLIP-seq data) and TarBase (manually curated evidence) offer direct experimental support for interactions. To minimize false positives, the results are critically integrated. The most robust approach is to take the strict intersection of targets predicted by all or most databases. This consensus strategy ensures that only targets supported by multiple independent algorithms and evidence types are considered high-confidence, significantly increasing reliability for downstream experimental validation. A DrawVenn diagram was constructed to a Venn diagram to determine the intersections and screen for common miR-181a-5p target genes. TargetScan predicted putative binding between miR-181a-5p and PTEN.

Luciferase reporter assay

The pMIR-Reporter vectors comprising wild-type (WT) or mutant (MUT) PTEN were synthesized by cloning the 3'-UTR sequences into pmirGLO plasmid vectors (all from Wuhan Genecreat Co., Ltd.). Lipofectamine 2000 was used to cotransfect the luciferase reporter vectors (WT-PTEN or MUT-PTEN) together with the corresponding plasmids and miR-181a-5p mimic/miR-NC into HEK293T cells. After culturing for 48 h, a luciferase assay system (Yeasen Biotechnology Co., Ltd.) was utilized to measure luciferase activities.

Statistical analysis

All the data analyses were dissected using SPSS Version 25.0 or GraphPad Prism Version 8.0 and are presented as the means ± SDs. Two groups were compared by Student's t test. Univariate and multivariate logistic regression analyses were performed to determine the correlations between miR-181a-5p and clinicopathological characteristics. P < 0.05 was deemed to be statistically significant.

Results

MiR-181a-5p expression is markedly elevated in PTC tissues

MiR-181a-5p expression in PTC tissues, paired ANTs, and BTNs was evaluated using RT‒qPCR (Fig. 1A). The PTC tissues (n = 111) exhibited greater miR-181a-5p levels than did the BTN tissues (n = 52) (P < 0.0001). Furthermore, compared to that in ANTs (n = 111), miR-181a-5p was considerably overexpressed in PTC tissues (n = 111) (P < 0.001). Further validation of the above results was carried out in the TCGA database, as apparent in Figure, which discovered that miR-181a-5p concentrations were markedly greater in TC tissues than in normal (P < 0.001) and ANT tissues (P < 0.0001) (Fig. 1B).

Fig. 1.

Expression of miR-181a-5p in papillary thyroid cancer (PTC). A MiR-181a-5p expression in PTC and benign thyroid nodules (BTNs), as well as in PTC and paired adjacent noncancerous tissues (ANTs), was assessed via qRT‒PCR. B PTC (tumor) tissues had higher expression of miR-181a-5p than did matched normal thyroid tissues (normal) and paired adjacent noncancerous tissues (ANTs). These data came from The Cancer Genome Atlas (TCGA) database

MiR-181a-5p is linked to the clinicopathological characteristics of PTC patients

To learn more about the associations between clinicopathological characteristics and miR-181a-5p levels in PTC, 111 patients were classified into 2 groups based on the median miR-181a-15p level. These subgroups included a group with high expression (≥ 68.3, n = 56) and a group with low expression (< 68.3, n = 55).

As demonstrated in the table (Table 1), according to univariate analysis, miR-181a-5p levels were greater in patients with larger tumors less than or equal to 1 cm than in those with larger tumors (OR = 5.25, 95% CI = 2.32–11.89; P = 0.000). Furthermore, high miR-181a-5p levels were strongly associated with thyroid capsule invasion (OR = 4.19, 95% CI = 1.52–11.54; P = 0.006), irregular shape (OR = 2.56, 95% CI = 1.13–5.80; P = 0.024) but not with age, sex, or combination with Hashimoto's thyroiditis.

Table 1.

miR-181a-5p expression in PTC: univariate associations with clinicopathological characteristics

Characteristics	miR-181a-5p expression	P
	OR* (95% CI)
Age (years)1	0.99(0.95–1.02)	0.465
Male vs. female	0.80(0.30–2.12)	0.653
Tumor size2	5.25(2.32–11.89)	0.000
Thyroid capsule invasion3	4.19(1.52–11.54)	0.006
Irregular shape4	2.56(1.13–5.80)	0.024
Combined with HT5	0.74(0.33–1.64)	0.457

*OR: odds ratio with 95% confidence interval (CI); ¹Age (≤ 45; > 45); ²Tumor size (≤ 1 cm; > 1 cm); ³Thyroid capsule invasion (Yes; No); ⁴Irregular shape (Yes; No); ⁵Combined with HT (Yes; No), HT: Hashimoto's thyroiditis. p less than 0.05 with statistical significance

According to the multivariate logistic regression analysis (Table 2), similar to the univariate analysis, high miR-181a-5p levels were related to tumor size (OR = 4.77, 95% CI = 2.04–11.16; P = 0.000) and to thyroid capsule invasion (OR = 3.15, 95% CI = 1.05–9.46; P = 0.041). Overall, these analysis results showed that high miR-181a-5p levels were associated with several poor clinical characteristics, such as tumor size and thyroid capsule invasion, which might predict poor prognosis in PTC patients.

Table 2.

miR-181a-5p expression in PTC: multivariable models for assessing tumor size, thyroid capsule invasion, and irregular shape

Characteristics	miR-181a-5p expression	p
	OR* (95% CI)
Tumor size1	4.77(2.04–11.16)	0.000
Thyroid capsule invasion2	3.15(1.05–9.46)	0.041
Irregular shape3	2.12(0.86–5.20)	0.103

^*OR odds ratio with 95% confidence interval (CI); ¹Tumor size (≤ 1 cm; > 1 cm); ²Thyroid capsule invasion (Yes; No); ³Irregular shape (Yes; No). p less than 0.05 with statistical significance

MiR-181a-5p promotes PTC cells proliferation

To clarify the potential impact of miR-181a-5p on PTC cells, a variety of in vitro biological experiments involving gene silencing and gene overexpression were carried out. RT‒qPCR was used to measure miR-181a-5p expression in thyroid cells (Fig. 2A). The endogenous miR-181a-5p levels in KTC-1 and TPC-1 cells were increased via transfecting miR-181a-5p mimics (Fig. 2B). Results obtained by CCK-8 demonstrated overexpressed miR-181a-5p had promoted proliferation capacity in KTC-1 and TPC-1 cells. However, this ability was reduced by transfecting miR-181a-5p inhibitors (Fig. 2C). The colony formation assay demonstrated the influence of miR-181a-5p on cell growth. As indicated in Figure (Fig. 2D), after adding miR-181a-5p mimics, the capacity of cells to form colonies was enhanced; conversely, there was a decrease in colony numbers following miR-181a-5p inhibition. Overall, miR-181a-5p enhanced the ability of PTC cells to proliferate in vitro.

Fig. 2.

MiR-181a-5p overexpression stimulates PTC cells proliferation. A The abundance of miR-181a-5p was measured by qRT‒PCR in PTC cell lines (KTC-1 and TPC-1) and a normal thyroid cell line (Nthy-ori3-1). B After transfection with miR-181a-5p mimics, the expression of miR-181a-5p was measured via qRT‒PCR. C Cell proliferation was investigated using CCK8 assays after transfection with miR-181a-5p mimics/inhibitors and their corresponding control NC/NC inhibitors. D Colony formation was evaluated posttransfection. **p < 0.01, *** p < 0.001, **** p < 0.0001. Mimics, miR-181a-5p mimics; inhibitors, miR-181a-5p inhibitors; NC negative control mimic; in-NC, NC inhibitors

MiR-181a-5p induces the migration and invasion of PTC cells

By measuring the proportion of blank regions after scratching, a wound healing assay indicated the influence of miR-181a-5p on PTC cell migration. The results showed that in KTC-1 and TPC-1 cells, compared to those in the NC group, the migration of the thyroid cancer cells in the miR-181a-5p mimic group was dramatically greater (Fig. 3A). However, compared with the inhibitor NC, the miR-181a-5p inhibitor significantly inhibited migration (in-NC) (Fig. 3A, lower panel). A transwell invasion assay was then performed to determine whether miR-181a-5p impacts the invasive capacity of PTC cells. As shown in Figure (Fig. 3B), the total number of tumor cells invaded by the matrix-coated transwell inserts was greater when transfected with miR-181a-5p than NC. In contrast, the miR-181a-5p inhibitor dramatically decreased cell invasion (Fig. 3B, lower panel). These findings demonstrated that miR-181a-5p upregulation was strongly linked to the metastatic phenotype of thyroid carcinoma cells.

Fig. 3.

MiR-181a-5p overexpression promotes PTC cells migration and invasion. A Detection of cell migration in KTC-1 and TPC-1 cells transfected with miR-181a-5p mimics/inhibitors and their control NC/NC inhibitors by wound healing assay. B Transwell invasion was investigated after transfection. * p < 0.05, ** p < 0.01, ***p < 0.001. mimics, miR-181a-5p mimics; inhibitors, miR-181a-5p inhibitors; NC negative control mimic; in-NC, NC inhibitors

MiR-181a-5p influences protein expression in the PTEN/AKT pathway and directly targets PTEN

Studies have confirmed that increased activity of the PI3K/Akt pathway is most often observed in aggressive thyroid cancers[20]. To ascertain whether miR-181a-5p acts via the PI3K/Akt pathway in PTC, we amplified miR-181a-5p or inhibited it in KTC-1 and TPC-1 cells. MiR-181a-5p mimics dramatically increased pathway activity, as characterized by elevated p-Akt (Fig. 4A). However, no changes in p-PI3K or PI3K levels were observed. Next, we investigated the influence of miR-181a-5p inhibitors on these pathways. MiR-181a-5p inhibitors decreased p-Akt levels, as expected, but had no effect on p-PI3K or PI3K levels (Fig. 4B). To explore how miR-181a-5p elevates p-Akt expression, we detected the PTEN content by Western blot. Interestingly, we found that the PTEN level decreased when cells were transfected with miR-181a-5p mimics (Fig. 4A) and increased when cells were transfected with miR-181a-5p inhibitors (Fig. 4B). Thus, in PTC cells, we first demonstrated that miR-181a-5p might influence the protein expression of the PTEN/AKT pathway.

Fig. 4.

Effects of miR-181a-5p mimics/inhibitors on the expression of PTEN and Akt in PTC cells. A PTEN, AKT, and PI3K protein expression was measured through western blotting in KTC-1 and TPC-1 cells transfected with miR-181a-5p mimics/NC. B The PTEN, AKT, PI3K proteins were analyzed in KTC-1 and TPC-1 cells transfected with the miR-181a-5p inhibitors/NC inhibitors via western blotting. *p < 0.05. mimics, miR-181a-5p mimics; inhibitors, miR-181a-5p inhibitors; NC negative control mimic; in-NC, NC inhibitors

To find out whether miR-181a-5p modulates PTEN by direct interaction, we first studied bioinformatics databases. The possible target mRNAs of miR-181a-5p by databases were intersected; 79 target mRNAs were obtained, and PTEN was included among the downregulated mRNAs (Fig. 5A). The luciferase test was used to validate this binding. A luciferase assay revealed that the introduction of miR-181a-5p mimics dramatically decreased the luciferase activity of PTEN-WT contrasted with the control NC. However, the miR-181a-5p mimic had no influence on PTEN-Mut luciferase activity (Fig. 5B). These data give solid evidence for direct targeting of PTEN by miR-181a-5p.

Fig. 5.

MiR-181a-5p directly targets PTEN. A The target genes of miR-181a-5p were predicted, and the putative binding sites of miR-181a-5p with the 3ʹUTR of PTEN were identified via the TargetScan database. B The dual-luciferase reporter assay confirmed the target relationship. *** p < 0.001

Discussion

According to both the 2017 [21] and 2023 [6] European Thyroid Association guidelines, molecular testing techniques are recommended for identifying patients with indeterminate cytology, thereby improving the accuracy of thyroid nodule diagnosis. These guidelines highlight the significance of miRNA research in diagnosing and treating PTC. The relevant literature indicates that miR-181a-5p regulates the most differentially expressed genes in the miRNA-gene regulatory network of PTC [22]. These findings imply that miR-181a-5p plays a role in PTC development. Although two studies, in 2021 [17] and 2022 [23], reported differential miR-181a-5p levels in PTC patients, and miR-181a-5p could be a possible biomarker, its specific molecular mechanism is not fully understood.

Even though PTC has a decent overall prognosis, some patients—known as highly aggressive papillary thyroid cancer—still have greater rates of lymph node metastasis, recurrence, and enhanced invasion [24]. To explore the association of miR-181a-5p levels with clinicopathologic features of PTC patients, we categorized the patients into high and low miR-181a-5p groups based on the 50th percentile of miR-181a-5p levels. Our results showed that the high miR-181a-5p expression group was more likely to result in thyroid capsule invasion and irregular nodal borders. According to reports by Kazaure et al. [25] and Ohashi et al. [26], tumors with thyroid capsule invasion are characteristic of highly aggressive thyroid cancer. In our investigation, we discovered that miR-181a-5p was strongly expressed in PTC and was positively linked to tumor size and thyroid capsule invasion, both of which are indicators of poor prognosis [27–29]. In previous studies, miR-181a-5p in highly invasive colorectal cancer was found to activate hepatic stellate cells by regulating IL6/STAT3 signaling, promoting their secretion of CCL20 and further activating the ERK1/2/Elk-1 pathway via CCR6, which ultimately led to peritoneal invasion as well as liver metastasis [30]. It has been reported that miR-181a-5p serves as an indicator of bone metastasis in prostate cancer and positively correlates with the degree of malignancy of prostate cancer [31]. All these evidences suggest that miR-181a-5p may be involved in the development and progression of papillary thyroid carcinoma. These findings imply that miR-181a-5p may be a diagnostic marker for highly aggressive papillary thyroid cancer.

Moreover, our cellular function experiments verified this conjecture. MiR-181a-5p strongly increased PTC growth by promoting cell proliferation, migration, and invasion. Numerous investigations have revealed that miR-181a-5p is key for cancer progression. Specifically, miR-181a-5p acts as an oncogene in colorectal cancer, driving proliferation and metastasis [32]. Moreover, miR-181a regulates TGF-β signaling in gastric carcinoma, enhancing cellular migration and proliferation [33]. Additionally, miR-181a-5p can inhibit SRCIN1, thereby promoting angiogenesis through the SRC/VEGF pathway [34]. Angiogenesis plays a vital role in thyroid nodule formation and growth, as malignant thyroid nodules exhibit increased angiogenesis compared to normal thyroid tissue. Moreover, in peritumoural invasive and highly malignant PTCs, irregular blood vessel patterns, uneven thickness, and abundant blood flow have been observed [35]. These findings support our preclinical findings, implying that miR-181a-5p could be a valuable marker when distinguishing malignant from benign thyroid nodules.

The relationship between miRNA and mRNA is a many-to-many interaction, where a single miRNA can regulate multiple mRNAs simultaneously, and an mRNA can also be targeted by multiple miRNAs. This intricate regulatory network of miRNA and mRNA serves as a crucial mechanism in gene expression control, significantly influencing cellular development, functional maintenance, and the onset and progression of diseases. In studies related to thyroid cancer, Fei et al. confirmed that ZNF674-AS1 directly interacted with miR-181a to increase SOCS4 expression[36]. In previous studies, KDM5C was identified as the target gene of miR-181a and represses S100A2 expression through histone demethylation to diminish the migration and proliferation of PTC cells [37]. It has been reported that miR-181a-5p directly regulates SLC5A5 expression in the context of PTC and may decrease efficacy of radioiodine treatment[17].

An important part of cancer development is that the activation of proto-oncogenes is accompanied by the loss or impairment of oncogenes. Since miR-181a-5p plays a pro-carcinogenic role in PTC, the mRNAs downstream of its target in PTC should be oncogenes. We predicted the target genes of miR-181a-5p in multiple biological information libraries, took the intersecting parts and screened out the oncogenes, which were found to contain the oncogene PTEN. PTEN has been recognized as an important tumor suppressor with both lipid phosphatase and protein phosphatase bisphosphatase activities, and the inhibitory effect of PTEN on the classical PI3K/AKT signaling pathway is achieved through its lipid phosphatase function and exerts its oncostatic effects in cancer through the PI3K/AKT pathway [38].PTEN/PI3K/AKT is a key component of the regulation of cellular signaling pathway for a variety of biological processes including apoptosis, metabolism, cell proliferation, and cell growth [39, 40], and also plays a critical role in the development of PTC. It is known that two signaling pathways, MAPK/ERK and PI3K/AKT, are classical pathways in thyroid cancer development [41, 42]. Moreover, aberrant activation of the PI3K/AKT pathway has been associated with metastasis, and dissemination, and has been proposed as an independent predictor of mortality in thyroid cancer and as a new treatment for advanced thyroid cancer [43–45].

In this experiment, we confirmed that miR-181a-5p has a direct targeting effect with PTEN by biological information prediction, as well as dual luciferase assay. To gain a deeper understanding of the potential mechanism of miR-181a-5p in PTC, we investigated its effect on the PI3K/AKT signaling pathway in PTC. We found that the expression of PTEN/PI3K/AKT signaling pathway proteins was detected by Western blot assay after transfection of miR-181a-5p mimic/inhibitor, and the results showed that overexpression of miR-181a-5p inhibited the expression of PTEN proteins, while increasing the expression of p-AKT, and knocking down miR-181a −5p gave the opposite results to those mentioned above, while the expression of both p-PI3K and PI3K were not affected. Through reviewing the literature, we found that Guo Deyin's team at Sun Yat-sen University published a paper entitled "PTEN suppresses tumorigenesis by directly dephosphorylating Akt" in the Nature sub-magazines [46], which confirmed through mouse experiments that protein phosphatase activity but lack of lipid phosphatase activity of the PTEN variants could also dephosphorylate AKT and inhibit tumorigenesis. This study proposed that PTEN can directly interact with AKT and dephosphorylate AKT at the S473 and T308 sites, and it is the protein phosphatase activity of PTEN that plays a role. The primary anti-p-AKT antibody used in the Western blot experiments of this study is the antibody to the protein at the Ser473 site. Therefore, we hypothesized that miR-181a-5p, by targeting PTEN in the PTC in this study and possibly by inactivating the protein phosphatase properties of PTEN, would in turn cause an increase in the protein expression of p-AKT in the cascade reaction. In other related studies, which corroborate this view, Chen et al. discovered that miR-181a-5p may activate the PTEN/Akt pathway in osteosarcoma [7], while Zhai et al. discovered that miR-181a-5p promoted breast cancer through its involvement in the PTEN/Akt pathway [47]. Zhang et al. have reported that PTEN is associated with activated AKT and PTC [48], while Sayaka et al. indicated that the PTEN/Akt pathway is involved in malignant events such as envelope invasion in PTC in rats [38]. Zhao et al. [49], on the other hand, demonstrated that bittersweet could play a therapeutic role in PTC by inducing apoptosis and cell cycle arrest in G1 and S phases via the miR-21/PTEN/Akt pathway. Li et al. [50] found that LINC00893 inhibited thyroid-like carcinoma by inhibiting AKT phosphorylation through the upregulation of PTEN expression.

In summary, the outcomes of our investigation suggest that miR-181a-5p might influence PTC by modulating the PTEN/Akt signaling pathway. This study potentially introduces new biomarkers for the prognosis, therapy in PTC. miR-181a-5p levels can be assessed in formalin-fixed paraffin-embedded (FFPE) tissue blocks from biopsies or surgical resections using RT-qPCR or, more commonly for clinical pathology, by in situ hybridization (ISH). ISH assays on tissue sections would allow pathologists to directly visualize miR-181a-5p expression within the tumor cells or diseased tissue in situ. This could provide valuable prognostic information and help guide patient stratification. High intratumoral miR-181a-5p could be linked to PTEN loss and a more aggressive disease course, potentially informing therapeutic choices. However, the miR-181a-5p/PTEN/Akt pathway in PTC requires further validation through animal experiments. Additionally, future research will explore the influence of miR-181a-5p on PTC cell metastasis and investigate other genes that may be associated with this pathway.

Author contributions

YZ, LH, ZQ, and JL were responsible for the design and implementation of the meticulously planned experiments, ensuring their scientific rigor; HW and YW assessed the participants’ eligibility for the intervention. The meticulous execution of the experiments and the analysis of the resulting data were skillfully carried out by WZ and YG. HC and XL contributed to the statistical analysis. WZ drafted the manuscript. YZ guided the study and revised the manuscript.

Funding

The Henan Science and Technology Research Funds (No. 212102310193) helped fund this research; Innovative Leading Talents of Science and Technology Project of Health Commission of Henan Province (No. YXKC2021024); Medical Key Project of Luoyang City (No. 2022014 A); and Graduate Research Innovation Program of Xinxiang Medical College (No. YJSCX2022101Y).

Data availability

All the data utilized to substantiate the discoveries of this investigation can be accessed from the corresponding author upon solicitation.

Declarations

Conflict of interest

The authors assert that they possess no conflicting interests.

Ethical approval

The use of clinical tissues was endorsed by the Ethics Committee of Luoyang Central Hospital Affiliated with Zhengzhou University. The study was conducted in strict accordance with the principles outlined in the Helsinki Declaration.

Informed consent

Informed consent was obtained from the participants.