Layout 1 Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 Thyroid gland is situated at the base of the throat adjacent to the trachea. It is usually in the shape of a butterfly with the left and right lobes connected by the isthmus. Thyroid cancer can be either differentiated or medullary.1 Differentiated thyroid tumours are usually cured whether they are papillary or follicular based thyroid cancers. In contrast, Censi et al.2 suggested that medullary thyroid cancer involves the neuroendocrine tumours found in thyroid C cells that produces calcitonin responsible for maintaining healthy calcium levels in the blood. Hajeer et al.3 showed that papillary thyroid carcinoma constitutes about 1% of all malignancies in the world and 80% of all thyroid cancer cases.4 Papillary thyroid carcinoma has an annual prevalence of 20,000 cases and forms the 8th major cancer in women with a higher occurrence in those aged less than 25 years. However, an individual can contract papillary thyroid carcinoma at any age, most cases are usually reported in patients of below 40 years.5 The risk factors for papillary thyroid carcinoma involves exposure to radioactive substances and genetic history of thyroid cancer. Several studies have reported good prognosis of papillary thyroid carcinoma with effective treatment and recovery in cases of early detection and diagnosis 5. More than 20% of patients of papillary thyroid cancer involves lymph nodes during diagnosis, however, compared to other malignancies involving the lymph nodes implies a poor prognosis which is contrary to thyroid cancers where the Abstract Papillary thyroid carcinoma is an epithelial malignancy with follicular cell differentiation and sets of defined nuclear features and appearance of an irregular solid mass. The main objective of our study is to research on the expression of miR-218-2 in the serum of patients with papillary thyroid cancer and its clinical significance. Our study involved patients with thyroid nodules were divided into a capitate cancer group (N = 100) and a benign nodule group (N=100). Lastly, 50 cases of healthy individuals were used as controls. The total sample size was 250. All cases were clinically diagnosed and underwent histopathological examinations at the Tonglu County Hospital of Traditional Chinese Medicine between January 2023 and January 2024. Quantitative RT-PCR was used to assess the expression levels of miR-218-2 and its host gene SLIT3 in normal and cancer thyroid tissues. We found that 45% of tumour sizes were less than 1 cm with 90% of tumours did not infiltrate the glandular capsule, implying a favourable prognosis. Lastly, 85% of tumours were well differentiated with about 75% showing no metastasis while 60% of TNM stage were classified as stage I. Also, miR-218-2 and its host gene SLIT3 are significantly down-regulated in papillary thyroid carcinoma. The inhibitory effects of miR-218-2 act in synergy with its host gene SLIT3 to alter the rates of cell invasion, cell migration and cell proliferation. Our findings have clinical significance on the involvement of miR-218-2 and SLIT3. There exists a functional relationship between host genes and intronic miRNAs in the tumorigenesis of thyroid cancers. Key Words: papillary thyroid carcinoma, miR-218-2, SLIT3, PDGFRA and PLCG1. Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 Research on the expression of Mir-218-2 in the serum of patients with papillary thyroid cancer and its clinical significance Haoting He,* Dingji Hao,* Linxiao Tian, Congru Zhu, Lili Guo, Keao Zhang, Siyao Zhu Department of Oncology, Tonglu County Hospital of Traditional Chinese Medicine, Tonglu County, Hangzhou City, Zhejiang Province, China. *Contributed equally to the work. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. - 21 - Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 lymph nodes have no effects on survival.5 Lymph nodes alter the rate of recurrence of thyroid cancers without affecting prognosis. Papillary thyroid carcinoma is an epithelial malignancy with follicular cell differentiation and sets of defined nu- clear features and appearance of an irregular solid mass.6 It is a commonly occurring thyroid neoplasm with a good prognosis. One of the key features of papillary thyroid car- cinoma is the capacity to invade surrounding lymphatics leading to 10% metastasis upon the initial diagnosis.6 Ito et al.7 found that papillary thyroid carcinoma is a histo- pathological subtype of thyroid cancer that has led to more than 90% of all thyroid cancer cases. The technological advancements in surgery and radiotherapy have not com- pletely improved the rates of relapse and recurrent metas- tasis in papillary thyroid carcinoma patients with a 10% to 15% prevalence of relapse and distant metastases oc- curring among papillary thyroid carcinoma patients. These rates of relapse and distant metastasis is an indication of poor response to conventional therapies and adverse clin- ical outcomes.4 As a result of this, the molecular mech- anisms associated with the growth and development of papillary thyroid carcinoma constitute a significant ele- ment of research that requires further analysis for effective and efficient therapeutic techniques.7 Micro-ribonucleic acid (miRNAs) consists of small non- coding RNAs with a mean length of 22 nucleotides. miR- NAs are mainly transcribed from Deoxyribonucleic Acid (DNA) sequences into primary miRNAs (pri-miRNAs) before undergoing processing into precursor miRNAs and maturity into miRNAs. In most cases, miRNA undergo significant interactions with the 3’ untranslated regions (3’ UTRs) of their corresponding target messenger-RNA (mRNA) to suppress their expression.8 In contrast, a study by Broughton et al.9 reported significant interactions be- tween miRNA and other regions such as the 5’ UTR, gene promoters and coding sequences. Additionally, miRNAs have been found to regulate and activate the expression of genes under specific circumstances.10 miRNAs have been established as critical regulators of several patholog- ical and biological processes involved in cell migration, cell proliferation, cell invasion, tumorigenesis and apop- tosis.11 miRNAs can be classified as either tumour sup- pressors or oncogenes depending on the types of cancers.12 Previous studies13-15 have shown that a dysregulation of the miRNAs (for example, the miR-217, miR-199a-3p, miR-21) increases the progression and pathogenesis of thyroid cancers. According to Lu et al.,16 miR-218 is a miRNA that is typi- cally transcribed from the dual loci situated on the chro- mosome 4p15.31 (miR-218-1) and chromosome 5q35.1 (miR-218-2) and is significantly downregulated in numer- ous cancers linked with cell invasion. Moreover, previous studies17,18 have demonstrated that miR-218 has several anticancer properties. Guan et al.19 showed that down-reg- ulation of miR-218 and the associated host gene SLIT3 (Slit Guidance Ligand 3) increases the rate of cell inva- sion, cell proliferation and cell migration in thyroid cancer. Despite all this research into miR-218, its regu- latory mechanisms through which it achieves the biolog- ical functions in thyroid cancer cells are unclear and not fully elucidated. The secretion of SLIT glycoproteins such as SLIT1, SLIT2 and SLIT3 into the regulation of the cell environ- ment through the mediation of the roundabout receptors.20 Recent studies have suggested that the promoters of SLIT- roundabout pathway of genes are commonly hypermeth- ylated in in several cancers proposing that SLITs are crucial candidates of tumour suppression genes.21,22 In breast cancer, loss of SLIT2 and SLIT3 contributes to hy- perplastic changes in cells via the Cxcr4 and Sdf1 signal- ling.21 Furthermore, SLIT3 has been implicated in the suppression of cellular migration of melanoma cells by in- hibiting the activator of protein 1.20 SLIT2 and SLIT3 genes have been found to encode miR-218-1 and miR- 218-2 situated on their introns.23 Similar studies24-26 have demonstrated that the down-regulation of miR-218 in- volves numerous types of cancer and malignant pheno- types. There has been limited studies investigating the expression of mature miR-218 in papillary thyroid carci- noma. Tetzlaff et al.27 suggested that a comparison of the expression of miR-218 in papillary thyroid carcinoma compared to multinodular goiter is always down-regu- lated. Cahill et al.28 found that two papillary thyroid car- cinoma cell lines consisting of a RET mutation revealed extremely low levels of miR-218 compared to normal cell lines. However, the expression of miR-218-1 and miR- 218-2 and host genes (SLIT2 and SLIT3) coupled with their functional importance in papillary thyroid cancer re- mains unclear and unknown. The rationale of our study is to expound on the existing studies and investigate the expression of miR-218-2 in the serum of patients with papillary thyroid cancer and its clinical significance. We propose a reduction in the ex- pression of miR-218-2 and the host gene SLIT3 in thyroid cancer. Our main objective is to research on the expression of mir-218-2 in the serum of patients with papillary thy- roid cancer and its clinical significance. Materials and Methods Participants and study design Our prospective cohort study involved 200 cases of thy- roid surgeries who had been clinically diagnosed and un- derwent histopathological examinations at Tonglu County Hospital of Traditional Chinese Medicine between Janu- ary 2023 and January 2024. Patients with thyroid nodules were divided into a capitate cancer group (N = 100) and a benign nodule group (N =100). Lastly, 50 cases of healthy individuals were used as controls. The total sample size was 250. All participants provided informed consent and ethical ap- proval was obtained from the Institutional Review Board. All data were mainly used for research purposes and all personal identifiers were eliminated from the data. Eligibility criteria Our study involved any patient with thyroid nodules who underwent thyroid surgery in our hospital. The final dia- - 22 - Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 gnosis of these patients was determined based on the post- operative pathological results. The inclusion criteria in- volved patients with patients with papillary thyroid cancer, benign thyroid nodules and normal tissues. None of the normal controls had any other benign or malignant tumor diseases or blood system-related diseases in which the body’s major organs malfunction, such as the liver, kid- neys, lungs. Also, all patients with a complete clinical data were included. The exclusion criteria involved cases ac- companied by nodular goiter; accompanied by other ma- lignant tumors; with internal division, urinary diseases; Pregnant or lactating women. Clinical data collection The following data were collected: patient’s gender, age, tumor size, whether glandular capsule infiltration occurs, information such as degree of differentiation, number of lymph node metastases, and TNM staging. Follow-up tests were conducted from the date of surgery and all pa- tients were reviewed once a month in the first 3 months after surgery; from 3 to 12 months, reviewed once every 3 months; after 1 year, re-examination every 6 months while recording the patient’s recurrence status, including in situ restoration, recurrence, contralateral thyroid recur- rence, cervical lymph node recurrence, and distant metas- tasis. Cases of relapse were defined as those where the patient has undergone cervical ultrasound and whole-body scan to find tumour nodules and when thyroid hormone subsides or high levels of thyroid stimulating hormone and presence of serum thyroid proteins or thyroid particles. Preparation of plasma samples All selected candidates, regardless of group assignment, were approached by trained personnel who used a coagu- lation tube to draw a 5 mL peripheral venous blood sample from the patient’s elbow vein. For the patients in the pa- pillary thyroid cancer group (who underwent thyroid sur- gery), the same blood collection method was employed. After the 5 mL blood sample was extracted, the techni- cians carefully inverted the collection tube and gently mixed it for 4-5 seconds several times. This ensured proper mixing of the blood with the anticoagulant within the tube. Following this, the tubes were placed upright at room temperature, typically for around an hour, to allow the blood to completely clot. Once the blood had coagu- lated, the samples were centrifuged at 3,000 rpm (with a centrifugal radius of 10 cm) for 10 minutes. This process separated the serum, the liquid component of the blood, from the red and white blood cells. The collected serum was then carefully transferred, 100 microliters per tube, into separate cryovials. These aliquots were then stored in a -80°C freezer for future analysis. Real time polymerase chain reaction (RT-PCR) Initially, we carefully measured out 750 microliters of Tri- zol reagent (Invitrogen, USA) and added it directly to the serum. The mixture was then incubated at a low tempera- ture for 5 minutes to ensure proper interaction. Next, 200 microliters of pre-cooled chloroform were added, and the mixture was thoroughly mixed again. After another 10- minute incubation, centrifuge the mixture at 12,000 rpm for 10 minutes with a centrifugal radius of 8 cm and iso- lated the RNA-containing supernatant in a new sterile tube. To further purify the RNA, an equal volume of iso- propyl alcohol was added to the supernatant, mixed well, and allowed to sit at room temperature for 3 minutes. Another centrifugation step at 12,000 rpm for 10 minutes (with the same centrifugal radius) followed. The remaining RNA pellet was then washed with 1 mL of 75% ethanol and after another brief centrifugation step (2 minutes) at 4°C, and allowing the RNA pellet to air dry completely. Finally, 10 microliters of DEPC water were added to the tube to re-suspend the purified RNA. Then, a micronucleic acid protein concentration analyzer (Thermo Fisher Scientific) was used to determine the concentration and purity of the isolated total RNA.With the purified RNA, reverse transcription occurred into complementary DNA (cDNA). The TransScript II All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit (Beijing Quanshi Jin Biotechnology Co., Ltd.) was used. The reaction mixture typically included 1 microgram of total RNA, 4 microliters of the TransScript II SuperMix, and 1 microliter of gDNA Remover in a final volume of 20 microliters. The reaction conditions involved an initial in- cubation at 50°C for 15 minutes, followed by a heat inacti- vation step at 85°C for 5 seconds. The subsequent step involved quantitative detection of miR-218-2 expression using the SYBR green dye method. An internal reference gene, U6, was used for normaliza- tion purposes. The RT-PCR reaction system typically con- tained 25 microliters of the 2xTransStart Top Green qPCR SuperMix, 0.2 microliters each of the forward and reverse primers specific for miR-218-2, 10 microliters of the cDNA template, and enough nuclease-free water to make up the final volume of 50 microliters. The thermal cycling program consisted of an initial pre-denaturation step at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension/fluorescence signal collection at 72°C for 30 seconds. We used specific primers designed for miR-218-2: a for- ward primer sequence of 5’-AAGCACCGCGGAA- AGCACCGT-3’ and a reverse primer sequence of 5’-TAATACGACTCACTATAGGG-3’. Similarly, primers for the U6 reference gene were employed: a forward primer sequence of 5’-CTCGCTTCGGCAGCACA-3’ and a reverse primer sequence of 5’-AACGCTTCAC- GAATTTGCGT-3’. Finally, we employed the 2-ΔΔCt method to calculate the relative expression levels of miR- 218-2 compared to the U6 reference gene. Statistical analysis All statistical analyses were conducted in GraphPad Prism version 9.5.1 with all values representing at least three triplicates in means±SD. Spearman rank correlation coef- ficients were calculated between study variables while a student’s t-test was used to compare differences between normal and tumour tissues. A p < .05 was considered sta- tistically significant. - 23 - Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 Results Preliminary analysis The study population included 250 participants (Table 1): 100 with papillary thyroid cancer (70% female, average age 45.2 years old), 100 with benign nodules (65% fe- male, average age 50.7 years old), and 50 healthy controls (60% female, average age 48.5 years old). There were slight gender skews favouring females in all groups, with the benign nodule group having the highest average age. In Table 2, analysing papillary thyroid cancer character- istics within the study group (n=100), tumour sizes were fairly evenly distributed with 45% measuring less than 1 cm, 30% between 1-2 cm, and 25% exceeding 2 cm. In terms of local invasion, 80% of tumours hadn’t infiltrated the glandular capsule, while 20% had. The majority (85%) were well-differentiated, with the remaining 15% being poorly differentiated. Lymph node involvement also var- ied, with 75% showing no metastasis, 15% having 1-3 af- fected nodes, and 10% exceeding 3 positive nodes. Finally, the distribution of TNM stages indicated a favou- rable prognosis with 60% classified as stage I, 30% as stage II, and only 10% at stage III/IV. Analysis of miR-218-2 expression via RT-PCR revealed a - 24 - Table 1. Demographics across the three groups. Variable Papillary thyroid cancer Benign nodule Normal control (n=100) (n=100) (n=50) Gender (Female %) 70 65 60 Age (Mean±SD) 45.2±10.1 50.7±11.4 48.5±9.8 Table 2. Tumour characteristics (papillary thyroid cancer group only). Feature Frequency (%) Tumour size (cm) ≤1 45 (45) 1-2 30 (30) >2 25 (25) Glandular capsule infiltration Yes 20 (20) No 80 (80) Degree of differentiation Well-differentiated 85 (85) Poorly differentiated 15 (15) Number of lymph node metastases 0 75 (75) 1-3 15 (15) >3 10 (10) TNM stage I 60 (60) II 30 (30) III/IV 10 (10) Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 ΔCt value of 4.3 in the papillary thyroid cancer group com- pared to the U6 reference gene (Table 3). After normaliza- tion to the healthy control group, the 2-ΔΔCt value was 0.43, indicating a 2.6-fold increase in relative miR-218-2 expression in papillary thyroid cancer compared to healthy controls. Conversely, the benign nodule group displayed a ΔCt of 5.1, resulting in a negative 2-ΔΔCt value (-0.4) and a relative expression of 0.4, suggesting lower miR-218-2 expression compared to the normal control group. In Table 4, in situ recurrence shows microscopic evidence of cancer cells detected in the thyroid bed after surgery, but no evidence of spread beyond the original location lo- coregional recurrence shows reappearance of cancer in the thyroid bed or nearby lymph nodes. distant metastasis rep- resents the spread of cancer to distant organs such as lungs, bones, or brain. lastly, the total recurrence rate gives the percentage of patients who experienced any type of recurrence during the follow-up period. Down-regulation of miR-218-2 and its host gene SLIT3 Our study analysed the expression of miR-218-2 and its role in the growth and development of papillary thyroid cancer. In Figure 1A, there was a statistically significant down-regulation of miR-218 in malignant lesions com- pared to normal tissues. In Figure 1B & 2A, statistical analysis of transcriptional expression levels of SLIT3, SLIT2, pre-miR-218-1, and pre-miR-218-2 were statisti- cally significantly positively correlated. Therefore, the ini- tial levels of miR-218-1 and miR-218-2 undergo a com- bined transcription with the host genes. Our findings shows that miR-218 is an intragenic miRNA that is basi- cally coded by 2 genes (miR-218-1 (intron 15 of SLIT2), and miR-218-2 (intron 14 of SLIT3)). Thus, the down- regulation of miR-218 in thyroid cancer is due to a reduc- tion in 1 or both of these genes. Additionally, these intronic miRNAs undergo a combined transcription with their host gene mRNAs. Our analysis of the down-regulation of miR-218 in papil- lary thyroid cancer as a result of the reduction in 1 or either of the 2 genes (miR-218-1n & miR-218-2) showed that the mature expression levels of miR-218 were signif- icantly and positively correlated with the initial levels of miR-218-2 and not the initial levels of miR-218-1 (see Figure 2B and 3A). Furthermore, the comparison between papillary thyroid cancer tissues and normal thyroid tissues showed that the expression levels of SLIT3 was signifi- cantly reduced (see Figure 3B). Discussion Our findings showed that miR-218-2 and its correspon- ding host gene (SLIT3) are significantly down-regulated in papillary thyroid cancer. Furthermore, there was a synergistic combined inhibition effect of SLIT3 and miR-218-2 on the rates of cell invasion, and cell prolif- eration in papillary thyroid cancer. After normalization - 25 - Table 3. ΔCt values and relative miR-218-2 expression. Group ΔCt 2-ΔΔCt Relative expression (miR-218-2 - U6) (Normalized to normal control) (Fold change) Papillary thyroid cancer 4.3 0.43 2.6 Benign nodule 5.1 -0.4 0.4 Normal control 6 Reference (1) 1 Table 4. Recurrence status after thyroid surgery. Follow-up period In situ Locoregional Distant Total recurrence recurrence recurrence metastasis rate (%) 0-3 months 0 1 0 1 3-12 months 1 2 0 3 1+ years 2 3 1 6 Total 3 6 1 10 Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 - 26 - Figure 1. Analysis of the down-regulation of miR-218-2, miR-218-1 and SLIT1. The mature miR-218-2 expression levels were analysed in normal tissues and tissues of papillary thyroid carcinoma. Figure 2. Analysis of the down-regulation of miR-218-2 and SLIT3. The mature miR-218-2 expression levels were anal- ysed using RT-PCR in normal tissues and tissues of papillary thyroid carcinoma. There were significant positive correla- tions between SLIT2 and miR-218-1, between miR-218-2 and SLIT3. Figure 3. Analysis of the down-regulation of miR-218-2 and SLIT3. The mature miR-218-2 expression levels were anal- ysed using RT-PCR in normal tissues and tissues of papillary thyroid carcinoma. There were significant positive correla- tions between SLIT2 and miR-218-1, between miR-218-2 and SLIT3. However, no significant correlations were found between the levels of miR-218-1 and miR-218. Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 to the healthy control group, the 2-ΔΔCt value was 0.43, indicating a 2.6-fold increase in relative miR-218-2 ex- pression in papillary thyroid cancer compared to healthy controls. Conversely, the benign nodule group displayed a ΔCt of 5.1, resulting in a negative 2-ΔΔCt value (-0.4) and a relative expression of 0.4, suggesting lower miR- 218-2 expression compared to the normal control group. We found that in local invasion, 80% of tumours had not infiltrated the glandular capsule while 20% had infiltrated the glandular capsule. 45 % of tumours were classified as microcarcinomas measuring less than 1 cm. In the degree of differentiation, 85% of the tumours were well differen- tiated with 15% being poorly differentiated. Liu et al.29 demonstrated that intronic sequences contains several functional regulatory elements besides biological functions. Examination of the intragenic miRNA consists of 408 human intronic miRNAs and through bioinformatic analyses, the expression levels of intronic miRNAs was sig- nificantly and largely correlated with transcription of host genes and miRNAs obtained from a similar transcript. The miR-218 is an intronic miRNA that has been reported to undergo down-regulation in numerous types of cancers and linked with tumorigenesis of several cancers.24-26 Our find- ings were similar with Venkataraman et al.,30 who postu- lated that miR-218 was down-regulated in cancers such as medulloblastoma and its differential expression is involved in several phenotypes of cancer. In a study by Kinoshita et al.,31 the presence of miR-218 was found to suppress the rates of cell invasion, cell proliferation and cell migration in head and neck squamous cell carcinoma by targeting the laminin-332. Alajez et al.32 found that the levels of miR- 218 inhibited the growth and development of tumours in nasopharyngeal carcinoma by targeting the regulation of the SLIT2-ROBO1 pathway and survivin. In the context of Papillary Thyroid Cancer (PTC), the anal- ysis of miR-218-2 expression through RT-PCR yielded in- triguing results. When compared to the healthy control group, the papillary thyroid cancer group exhibited a 2.6- fold increase in relative miR-218-2 expression after nor- malization. This is numerically represented by a 2-ΔΔCt value of 0.43. Conversely, the benign nodule group dis- played a lower relative expression of miR-218-2 compared to the healthy controls. This is indicated by a negative 2- ΔΔCt value (-0.4) and a relative expression of 0.4, which suggests miR-218-2 might be downregulated in benign nodules. These findings point towards a potential link be- tween miR-218-2 and PTC. While the underlying mech- anisms are yet to be fully understood, miR-218-2 might play a role in the development of PTC. Future studies could explore the functional role of miR-218-2 in PTC and deter- mine if it can serve as a biomarker for PTC diagnosis or prognosis. Additionally, PTC is likely a complex disease influenced by multiple factors beyond a single microRNA. The downregulation of miR-218-2 and SLIT3 in papillary thyroid cancer might not solely be driven by transcriptional or genetic alterations, but also by epigenetic modifications.33 These molecular mechanisms act as chemical switches on the DNA and its associated proteins regulating gene expres- sion without changing the actual DNA sequence. In the context of papillary thyroid cancer, the downregulation of miR-218-2 and SLIT3 suggests a potential link to epigenetic alterations. DNA methylation involves the addition of methyl groups to the DNA molecule, often occurring at CpG sites in the promoter regions of genes. This modification can result in the repression of gene transcription by impeding the binding of transcription factors to the affected regions. Similarly, histone modifications which encompass changes in the structure and function of histone proteins, contribute to the epigenetic regulation of gene expression. Acetylation, methylation, and phosphorylation of histones can influence chromatin structure, either promoting or inhibiting transcrip- tional activity. In the case of papillary thyroid cancer, these histone modifications may contribute to the observed down- regulation of miR-218-2 and SLIT3. We found 45% of tu- mours to be less than 1 cm indicating a significant proportion (almost half) of the tumours were classified as microcarci- nomas, which are very small tumours less than 1 cm in di- ameter. Papillary thyroid microcarcinomas are generally associated with a favourable prognosis implying that pa- tients with microcarcinomas have a high chance of success- ful treatment and a lower risk of recurrence compared to those with larger tumours. Moreover, the small size of these tumours suggests they might have been detected at an early stage. Early detection of cancer is crucial for successful treat- ment and improved patient outcomes. Early-stage cancers are often less aggressive and easier to treat compared to more advanced stages. Lastly, the favourable prognosis be beneficial for patients as it minimizes the potential side ef- fects associated with more extensive cancer treatments like surgery or radiation therapy. Our findings showed that 80% of tumours hadn’t infiltrated the glandular capsule which is a positive finding, as capsular invasion indicates a higher risk of the cancer spreading beyond the thyroid gland. The high percentage of non-invasive tumours suggest a poten- tially good prognosis for this group. The thyroid gland is surrounded by a thin connective tissue layer called the cap- sule, thus, when a papillary thyroid cancer remains confined to the thyroid gland and hasn’t breached the capsule, it’s con- sidered non-invasive. Conversely, if the cancer cells infiltrate the capsule, it’s termed capsular invasion. This finding can influence treatment decisions. For patients with non-invasive tumours, less aggressive surgical procedures like a lobec- tomy (removal of one thyroid lobe) might be considered. The favourable prognosis associated with non-invasive tu- mours can also impact post-surgical management. Decisions regarding the use of radioactive iodine ablation therapy might be tailored based on the individual’s risk profile. Our analysis identified that maturity of miR-218 is encoded by miR-218-1 and miR-218-2 and their distinct locations within the introns of SLIT2 and SLIT3. Although, the ex- pression levels of miR-218-1 and miR-218-2 has not been extensively examined in histological types of thyroid cancer. In the study, our findings have demonstrated that the expression of miR-218-2 and SLIT3 are downregulated and underexpressed in papillary thyroid cancer. Further- more, restoring the expression levels of miR-218-2 and SLIT3 inhibits the rate of cell invasion, and cell prolifera- tion of papillary thyroid cancer cells. These findings were similar to a previous study by Najafi-Shoushtari et al.,34 who found a synergistic interaction effect between host - 27 - Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 genes and intronic miRNAs. Their findings showed that miR-33a/b and their corresponding sterol control element- binding protein host genes act in synergy to regulate the ho- meostasis of cholesterol. Similarly, a study by Van Rooij et al.35 found that miR-208a/b controls the expression levels of myosin and performance of muscles in a synergistic ef- fect with their corresponding Myh6/7 host genes. In the context of papillary thyroid cancer, the dynamic relation- ship between the expression levels of miR-218-2 and the host gene SLIT3 assumes a pivotal role in regulating fun- damental cellular processes, particularly in terms of cell in- vasion and proliferation.7 Simultaneously, the host gene SLIT3 plays a critical role in this regulatory network. SLIT3 is a member of the SLIT family of secreted proteins and is implicated in various cellular processes, including cell migration and axon guidance.36 In the context of papil- lary thyroid cancer, the expression levels of SLIT3 become integral to the modulation of cell invasion and proliferation. Its interaction with miR-218-2 may further amplify or miti- gate the impact of their combined influence on these cel- lular behaviours. Our analysis showed in lymph node involvement, 75% of cases showed no metastasis. hence, it indicates the absence of cancer spread to the lymph nodes in a significant portion of the study group. the ab- sence of lymph node involvement is associated with a better prognosis. Lymph nodes are part of the body’s im- mune system and act as filters, trapping cancer cells that might spread from the tumour. The absence of cancer spread to the lymph nodes in such a large portion of the study group suggests a lower risk of distant metastasis (spread to other organs) and a more favourable prognosis. Patients without lymph node involvement typically have a better prognosis compared to those with positive lymph nodes. This can influence treatment decisions. For in- stance, individuals with no lymph node involvement might be candidates for less extensive surgical procedures or po- tentially require lower doses of radioactive iodine ablation therapy, minimizing potential side effects. Previous studies37,38 have suggested that there are series of func- tionally related genes that act in coordination of biological functions in cancer. Shih & Holland41 showed that after stimulation of the receptors, there are several cellular pro- cesses and corresponding signals that area activated. Therefore, regulating the functions of these receptors is based on a single specific regulatory effect. Certain miR- NAs are capable of controlling several target genes that are synergistically significant in controlling cellular processes. A study by Mavrakis et al.38 showed that miR-19 has been implicated in leukemogenesis by modulating the regulators of the signalling of phosphatidylinositol-3-OH kinase. Ac- cording to Rheinheimer et al.,44 miR-218-2 resides within an intron of the SLIT3 gene, implying, they’re physically connected on the same DNA strand. Our study suggests their downregulation is "concomitant," implying a possible shared mechanism, but the exact process needs further in- vestigation. One possibility is the targeting of distinct com- plementary pathways. miR-218-2, can directly silence genes like PDGFRA and PLCG1, disrupting their pro-can- cerous signalling. Nian et al.39 suggested that in papillary thyroid carcinoma, MCM3AP-AS1 & GLUT1 was up-reg- ulated due to the synergistic effects of miR-218 in increas- ing the rate of cellular proliferation. MiR-218 is a tumour- suppressive miRNA that inhibits the growth and development of cancer by altering the behaviour of cancer cells including invasion and proliferation.32,40 Down-regu- lation of miR-218 is significantly and positively correlated with the growth and development of papillary thyroid car- cinoma.19 Furthermore, the inhibitory effects of miR-218 increases the rate of cellular proliferation in papillary thy- roid carcinoma. miR-218 is capable of forming strong base pairings with MCM3AP-AS1; however, lncRNAs are ca- pable of sponging miRNAs and reducing their downstream effects on signalling cascades.41 Previous studies have re- ported that miR-218 targets the GLUT1 and limits the growth and development of bladder cancer.42 In papillary thyroid carcinoma, elevated levels of GLUT1 increases the growth of cancer cells and poor prognosis. Conclusions Our research on the expression of mir-218-2 in the serum of patients with papillary thyroid cancer and its clinical sig- nificance has showed that miR-218-2 and its corresponding host gene SLIT3 are significantly down-regulated in papil- lary thyroid carcinoma. Synergistically, the down-regula- tion of miR-218-2 enabled the increase in cell invasion and cell proliferation in papillary thyroid carcinoma. Further- more, we found that 45% of tumour sizes were less than 1 cm with 90% of tumours did not infiltrate the glandular cap- sule, implying a favourable prognosis. Lastly, 85% of tu- mours were well differentiated with about 75% showing no metastasis while 60% of TNM stage were classified as stage I. Hence, restoring and preserving the expression levels of SLIT3 and miR-218-2 could have a beneficial therapeutic effect and favourable prognosis in papillary thyroid carci- noma. Further studies should examine the functional rela- tionships between host genes and intronic miRNAs in papillary thyroid carcinoma. List of acronyms miRNA: MicroRNA. PTC: Papillary Thyroid Carcinoma. SLIT3: Slit Guidance Ligand 3. RT-PCR: Real-Time Polymerase Chain Reaction. UTR: Untranslated Region. TNM: Tumor, Node, Metastasis. cDNA: Complementary DNA. DEPC: Diethyl Pyrocarbonate. Conflict of interest The authors declare no potential conflict of interest, and all authors confirm accuracy. Funding Hangzhou Medical and Health Science and Technology Project, No. B20230443 - 28 - Non -co mmerc ial us e o nly Mir-218-2 in the serum of patients with papillary thyroid cancer Eur J Transl Myol 34 (3) 12678, 2024 doi: 10.4081/ejtm.2024.12678 Ethics approval The Ethics Committee of Tonglu County Hospital of Tra- ditional Chinese Medicine approved this study (No.2022a1809). The study is conformed with the Hel- sinki Declaration of 1964, as revised in 2013, concerning human and animal rights. Informed consent All patients participating in this study signed a written in- formed consent form for participating in this study. Patient consent for publication Written informed consent was obtained from a legally au- thorized representative(s) for anonymized patient infor- mation to be published in this article. Availability of data and materials All data generated or analyzed during this study are in- cluded in this published article. Corresponding author Siyao Zhu, Department of Oncology,Tonglu County Hos- pital of Traditional Chinese Medicine, Tonglu County, 311500 Hangzhou City, Zhejiang Province, China. ORICD ID: 0009-0001-2592-220X E-mail: m15158190098@163.com Haoting He ORCID ID:0009-0006-8030-0952 E-mail: a472905342@163.com Dingji Hao ORCID ID: 0009-0003-6120-6993 E-mail: hdj6668882022@163.com Linxiao Tian ORCID ID: 0009-0007-3752-1095 E-mail: tianlinxiao@163.com Congru Zhu ORCID ID: 0009-0003-6883-4773 E-mail: zcr920210@163.com Lili Guo ORCID ID: 0009-0004-4225-4989 E-mail: 18143468892@163.com Keao Zhang ORCID ID: 0008-0005-0493-8826 E-mail: zka88115943@163.com References 1. Khan YS, Farhana A. Histology, Thyroid Gland. 2022 Dec 5. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan–. PMID: 31869123. 2. Censi S, Manso J, Mian C. Other markers of medul- lary thyroid cancer, not only calcitonin. Eur J Endo- crinol 2023;188:R1-R13. 3. Hajeer MH, Awad HA, Abdullah NI, et al. The rising trend in papillary thyroid carcinoma: True increase or over diagnosis? Saudi Med J 2018;39:147-53. 4. Ito Y, Jikuzono T, Higashiyama T, et al. 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