MiR-27a-5p inhibits malignant progression of differentiated thyroid cancer through SREBP1 and is associated with thyrotropin suppressive therapy

doi:10.21203/rs.3.rs-4819650/v1

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Research Article

MiR-27a-5p inhibits malignant progression of differentiated thyroid cancer through SREBP1 and is associated with thyrotropin suppressive therapy

https://doi.org/10.21203/rs.3.rs-4819650/v1

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Purpose

To detect the expression of miR-27a-5p in differentiated thyroid cancer (DTC) and to explore its correlation with SREBP1 expression, DTC malignant progression, and TSH suppression therapy.

Methods

The expression levels of SREBP1 and miR-27a-5p in DTC tissues (n = 75) were detected by qRT-PCR. The expression of miR-27a-5p and SREBP1 was statistically analyzed for correlation with patients' postoperative TSH inhibition therapy. Dual luciferase reporter gene assay was performed to verify the target-regulatory relationship between miR-27a-5p and SREBP1. qRT-PCR and Western blots were performed to detect the effect of miR-27a-5p on the expression level of SREBP1. mts, plate clone formation assay was performed to detect the effect of miR-27a-5p on the proliferative capacity of cells. Flow cytometry was performed to detect the effect of miR-27a-5p on cell cycle and apoptosis. Scratch assay and Transwell assay to detect the effect of miR-27a-5p on cell migration invasion ability.

Results

MiR-27a-5p expression was significantly downregulated in DTC cancer tissues and significantly negatively correlated with SREBP1 expression. It correlated with the outcome of postoperative TSH suppression therapy in DTC patients. The results of dual luciferase reporter gene assay showed that the 3'-UTR region of SREBP1 mRNA was the target site of action of miR-27a-5p. Overexpression of miR-27a-5p was associated with a significant reduction in cell proliferation, cell cycle arrest, increased apoptosis, and diminished cell invasive migration.

Conclusion

The miR-27a-5p expression level was negatively correlated with the progression of DTC, which may be inhibited by targeting SREBP1 and correlated with the outcome of TSH inhibitory therapy.

Differentiated thyroid cancer

SREBP1

MiR-27a-5p

TSH inhibition therapy

Malignant progression

Thyroid cancer (TC) is the most common malignant tumor in the endocrine system, among which differentiated thyroid cancer (DTC) is the most common type of thyroid cancer, and its incidence is increasing year by year [1, 2]. Current therapies are mainly thyroid surgery combined with long-term thyroid stimulating hormone (TSH) suppression therapy, which is accompanied by several complications and adverse effects that seriously affect the quality of life of patients [3]. Second, the occurrence and development of thyroid cancer involves the abnormal activation of multiple signaling pathways and mutations in genes, but the molecular mechanisms are still unclear [4]. Therefore, it is necessary to elucidate the molecular mechanisms leading to the development of thyroid cancer and to gain a comprehensive understanding of the key regulatory factors in the growth, proliferation, and invasion of cancer cells in order to provide new insights into targeted therapies.

Sterol Regulatory Element-Binding Proteins (SREBPs) are major transcription factors that regulate lipid synthesis and lipid homeostasis in cells [5].SREBP1 is an important member of the SREBP family, which is involved in the genesis and development of many types of tumor cells. In thyroid cancer, gastric cancer, hepatocellular carcinoma, prostate cancer and other tumors, SREBP1 usually presents a stable and high level of expression status, which influences the alteration of the biological characteristics of the tumor malignant cell proliferation, infiltration, metastasis and resistance to apoptosis, and is closely related to with the patient's treatment prognosis [6–9]. Recent studies have shown that changes in TSH levels in DTC patients receiving long-term TSH inhibition therapy will lead to abnormal lipid metabolism [10]. Another study showed that changes in TSH levels in vivo were associated with lipid composition and hypercholesterolemia in DTC patients [11]. In recent years, there has been a gradual increase in reports on the key role of lipid metabolism in cancer development and progression [12]. The previous study of our group found that SREBP1 could affect the onset, progression and treatment of DTC. However, the correlation between SREBP1 expression and the efficacy of postoperative TSH inhibition therapy in patients and the occurrence of adverse reactions caused by it is unknown.

MicroRNAs (MiRNAs) are a class of single-stranded non-coding RNAs with a length of 19–24 nt, which are post-transcriptional negative regulators of mRNAs and act by binding to the 3′-untranslated region (3′-UTR) of target genes, leading to mRNA degradation or translational inhibition [13, 14]. Studies have shown that about more than 50% of miRNAs are localized in cancer-associated regions on the chromosomes where the genes are located, and their expression is either down-regulated or up-regulated in malignant tumors, playing the role of oncogenes or proto-oncogenes, which are involved in tumorigenesis and tumor development, and regulating tumor cell proliferation, invasion, and metastasis, which are associated with treatment and prognosis [13]. It has been demonstrated that miR-27a is able to participate in lipid metabolism, and its mode of action is through the targeted binding of SREBP-1 and SREBP-2 [15]. Meanwhile, the role of MiR-27a-5p in a variety of cancers has been reported [16–18], while the regulation of expression in thyroid cancer has not yet been reported.

Based on these findings, we proposed the hypothesis that miR-27a-5p regulates cell proliferation, migration, and invasion by modulating SREBP1 in DTC development. To test this possibility, we compared the expression levels of SREBP1 and miR-27a-5p in cancer tissues from DTC patients and explored the clinical significance of both in thyroid cancer patients. At the same time, we analyzed the correlation between the expression of SREBP1 and miR-27a-5p and the efficacy of the patients' postoperative TSH inhibition therapy and the occurrence of the adverse reactions caused. And we further investigated the relationship between SREBP1 and miR-27a-5p in thyroid cancer by in vitro cellular experiments.

2.1. Clinical samples and ethics statement.

A total of 75 patients with DTC were included in the study, and the patients' cancer and paired paracancerous tissues were collected. The subjects were obtained from the Department of Thyroid Surgery, Xiangya Medical College Cancer Hospital, Central South University/ Hunan Provincial Cancer Hospital and the Department of Thyroid Surgery, Zhengzhou Central Hospital, Zhengzhou University. Inclusion criteria: 1) Diagnosis of DTC was confirmed by intraoperative pathology; 2) complete and available data; 3) good compliance; 4) no preoperative radiotherapy; and 5) age over 18 years old. Exclusion criteria: 1) violation of the above inclusion criteria; 2) history of other cancers and surgeries; 3) other thyroid diseases; 4) other co-morbid major or infectious diseases; 5) women who were in the period of pregnancy and breastfeeding; and 6) previous exposure to radioactive sources. According to the inclusion and exclusion criteria, all patients included signed the Informed Consent Form. The study complied with the principles outlined in the Declaration of Helsinki and was reviewed and approved by the Ethics Committee of the Cancer Hospital/Hunan Cancer Hospital, Xiangya School of Medicine, Central South University, with the ethical approval number (NO: SBQLL-2016-015).

2.2. Cell culture.

The human-derived papillary thyroid cancer cell line BCPAP and the human-derived follicular thyroid cancer cell line WRO were used in this study.BCPAP cells were purchased from the Cell Bank of the Chinese Academy of Sciences (CAS), and WRO cells were obtained from the Second Xiangya Hospital of Central South University. The differentiated thyroid cancer cell line BCPAP was treated with 10% FBS (Biological Industries, Israel) supplemented with 1% sodium pyruvate (Invitrogen, USA), 1% NEAA (Invitrogen, USA), 1% glutamine ( Invitrogen, USA), 1% NEAA (Invitrogen, USA), 1% glutamine ( Invitrogen, USA) in RPMI-1640 medium (Hyclone, USA).WRO cells were cultured in RPMI-1640 medium with 10% FBS. The cells were placed in a cell culture incubator at 37°C, 5% CO2.

2.3. Transfection.

Mimic and inhibitor were transfected using the Lipofectamine® RNA iMAX kit (Invitrogen, USA). Cell transfection was performed when the cell confluence in the culture plate was about 40–50% according to the manufacturer's instructions. miR-27a-5p mimic and mimic-NC, miR-27a-5p inhibitor and inhibitor-NC were purchased from RiboBio (Guangzhou, China). Plasmids were transfected using the Lipofectamine™ 3000 kit (Invitrogen, USA). The transfection operation was performed when the cell confluence in the culture plate was about 70–80% according to the manufacturer's instructions.

2.4. Reverse transcriptase quantitative PCR.(qRT-PCR).

To analyze the relative expression levels of SREBP1 and miR-27a-5p, total RNA was extracted from tissues and cells using TRIzol™ Reagent (TaKaRa, Japan), and RT-qPCR was performed according to the instructions of the Fluorescence Quantification Kit (TaKaRa, Japan).Relative expression of SREBP1 and miR-27a-5p The 2®-ΔΔCT method was used, with β-actin or U6 as endogenous controls. Primer sequences are shown in Supplementary Table. 1.

Table 1

Gene Primer Sequences
Gene Name	Primer Sequences
SREBF1-F	5′-TTGCCGACCCTGGTGAGT-3′
SREBF1-R	5′-AATGGCGTTGTGGGCTGT-3′
β-actin-F	5′-CCTGGCACCCAGCACAAT-3′
β-actin-R	5′-GGGCCGGACTCGTCATAC-3′
miR-27a-5p	5′-AGGGCTTAGCTGCTTGTGAGC-3′
U6-F	5′-ATTGGAACGATACAGAGAAGAATT-3′
U6-R	5′-GGAACGCTTCACGAATTTG-3′

2.5. Western blot.

Cells were collected 24h after transfection, and the total protein of lysate cells was added for extraction, and the protein concentration was determined by BCA kit (Beyotime, China). Western-blot SDS-PAGE gels were prepared with a loading volume of 30 µg per well, and membrane transfer was performed after electrophoresis. The end of membrane transfer was closed with 5% skimmed milk. Add SREBP1 (1:500) primary antibody (Santa Cruz, USA), β-Actin primary antibody (1:3000) (Santa Cruz, USA) and incubate overnight in the refrigerator at 4°C. Wash with TBST for 3 times, and then add (1:10,000) diluted murine secondary antibody (Sigma, USA), respectively, and incubate at room temperature for 1h. Wash with TBST for 3 times, and add ECL luminescent solution (Sigma, USA) and incubate at room temperature for 1h. TBST was washed 3 times, and ECL luminescent solution (Beyotime, China) was used for luminescence visualization.

2.6. Dual luciferase reporter gene

The wild-type SREBP1 luciferase reporter gene vector (wt-SREBP1) and mutant SREBP2 luciferase reporter gene vector (mut-SREBP1) were constructed respectively. All miR-27a-5p mimic, miR-27a-5p inhibitor and dual-luciferase reporter vectors were designed and synthesized by Guangdong Ruibo Biological Company. The cells were transfected according to the experimental groups, and the luciferase activity was detected 24 h later using the Dual-Luciferase Reporter Gene Assay Kit (Promega, USA).

2.7. MTS assay.

The effect of miR-27a-5p on the viability of thyroid cancer cells was detected using MTS (Promega, USA) according to the manufacturer's instructions. Cells were seeded in 96-well plates at a density of 2.5×103 cells/well 24 h after transfection. After the cells regained their viability and growth was stabilized, the cells were incubated with 10 µL of MTS diluted in 90 µL of medium for 1.5 h, and the absorbance values of the cells were detected at 12, 24, 48, and 72 h. Three independent replicates of each cell line were performed. Each cell line was subjected to 3 independent replications, and statistical plots were made and analyzed.

2.8. Plate cloning experiment.

Thyroid cancer cells were seeded in 6-well plates at a density of 500 cells/well, and routinely cultured in an incubator at 37℃ for 2 weeks, during which the liquid was changed every 3–4 days to observe the growth status, and the culture was terminated when clonal groups of cells could be observed in the plates by naked eyes. At the end of the culture, the medium in the plate was aspirated, washed twice with PBS, 1 mL of 4% paraformaldehyde (Dingguo Biotechnology, China) was added to each well, and the plate was left for 30 min to fix the cells, the paraformaldehyde was aspirated and washed twice with PBS. Add 1 mL of crystal violet staining solution (Beyotime, China) to each well, stain for 30 min, recover the crystal violet staining solution, wash with PBS for 2 times, wait for the water to evaporate and dry, observe under the microscope and take pictures. quantitative analysis by Image J software.

2.9. Wound healing experiment

Transfected BCPAP and WRO cells were inoculated in 6-well plates, respectively. When the cells grew to about 80% confluence, cell-free areas were formed by scraping vertically with the tip of a 200 µl pipette. The wells were then gently cleaned with PBS and the wounds were observed under a microscope and photographed. Serum-free medium was then added to the wells and cell culture was continued.After 24 hours, the wounds were observed again. The cells were then compared for wound healing.

2.10. Transwell assay.

In the Transwell invasion assay, the matrix gel was prepared according to the ratio of Matrigel Matrix Gel (BD, USA): pre-cooled serum-free medium = 1:8, and the matrix gel was spread in the upper chamber. After the cells were transfected for 24 h, 200 µL of cell suspension containing 1.0×105 cell number density was added to the upper chamber of each well, and 600 µL of medium containing 10% fetal bovine serum was added to the lower chamber, and the upper chamber was taken out after 24 h of incubation, and then washed, fixed, stained, observed under the microscope, and photographed. In the cell migration experiment, it was not necessary to lay matrix gel in the small chamber. After the cells were transfected for 24 h, 200 µL of cell suspension containing 2.5×104 cells per well was added to the upper chamber, and the lower chamber was incubated with 600 µL of medium containing 10% fetal bovine serum for 24 h. The rest of the operation was the same as that of the invasion experiment.

2.11. Cell cycle assay.

Cell cycle was detected by flow cytometry. 48 h after cell transfection, cells were digested and obtained in suspension for centrifugation. Cells were fixed after resuspension and washing according to the instructions of the cycle detection kit (Beyotime, China), and cycle changes were detected using flow cytometry after incubation with PI and avoiding light for 30 min. ModFit LT was used to assess the GO/G1, S, and G2/M cell fractions of the cells.

2.12. Cell apoptosis assay.

In the apoptosis assay, 48 hours after cell transfection, apoptotic changes were detected by flow cytometry according to the instructions of the Apoptosis Detection Kit (BIOTEC, China) and quantitatively analyzed using Flowjo software.

2.13. Statistical analysis.

This study was analyzed using SPSS 23.0 statistical software. Images were drawn by Graphpad Prism 9. All data were performed, three independent replicated experiments. For measurement data, expressed as mean ± standard deviation (Mean ± SD), independent samples t-test was performed for comparison between two groups; one-way ANOVA was performed for comparison between multiple groups. For count data, the paired t-test was used to analyze the differences in gene expression in DTC tissues and paired paracancerous tissues, and the Pearson chi-square test was used to analyze the correlation between gene expression and patients' clinical data. All statistical analyses were two-sided tests with α = 0.05, and P < 0.05 was considered statistically different.

3.1. SREBP1 expression in DTC and its clinical significance

To study the expression characteristics of SREBP1 in thyroid cancer tissues, bioinformatics analysis was performed using the GEPIA2 database (http://gepia2.cancer-pku.cn), which revealed that SREBP1 was differentially expressed in thyroid cancer tissues and normal tissues, and its expression was significantly higher in thyroid cancer tissues (Fig. 1a). The expression levels of SREBP1 mRNA detected in 75 pairs of thyroid cancer tissues and adjacent normal thyroid tissues were examined by qRT-PCR. The results showed that SREBP1 expression in thyroid cancer tissue samples was significantly high relative to adjacent normal thyroid tissue samples (Fig. 1b). In addition, the correlation between the expression level of SREBP1 and clinicopathological characteristics of DTC patients was analyzed using the Pearson chi-square test. The study showed that high expression of SREBP1 was significantly associated with the occurrence of lymph node metastasis as well as TNM stage in patients (Table.2).

Table 2

Relationship between SREBP1 expression levels and clinical features of thyroid cancer in 75 patients
Factor	Number of cases(n = 75)	SREBP1 expression levels		P value
Factor	Number of cases(n = 75)	Low expression group(n = 38)	High expression group(n = 37)	P value
Age (years)	75
< 45	35	17 (44.74%)	18 (55.26%)	0.734
≥ 45	40	21 (55.26%)	19 (44.74%)
Sex
Male	22	10 (44.00%)	12 (56.00%)	0.561
Female	53	28 (52.94%)	25 (47.06%)
WHO stage
I-II	61	29 (47.54%)	32 (52.46%)	0.387
III-IV	15	9 (60.00%)	6 (40.00%)
Tumor size (cm)
≤ 2cm (T1)	33	18 (47.37%)	15 (52.63%)	0.551
> 2cm (T2-T4)	42	20 (57.89%)	22 (42.11%)
Lymph node metastasis (N)
NO (N0)	32	21 (41.67%)	11 (58.33%)	0.025*
YES (N1)	43	17 (37.50%)	26 (62.50%)
Metastasis (M)
NO (M0)	68	36 (48.53%)	32 (51.47%)	0.219
YES (M1)	7	2 (62.50%)	5 (37.50%)
TNM stage
I-II	55	32 (52.94%)	22 (47.06%)	0.007**
III-IV	20	5 (44.00%)	6 (56.00%)
LDL (mmol/L)^#	75	2.81 ± 0.74	2.83 ± 0.74	0.979
HDL (mmol/L)^#	75	1.23 ± 0.27	1.24 ± 0.27	0.980
TCHO(mmol/L)^#	75	4.75 ± 1.09	4.76 ± 1.07	0.986
TG (mmol/L)^#	75	1.65 ± 1.30	1.64 ± 1.29	0.998

^#Measurements were analyzed by independent samples t-test and expressed as Mean ± SD; the rest of the data were analyzed by chi-square test. LDL: Low-density lipoprotein; HDL: High-density lipoprotein; TCHO: Total Cholesterol; TG: Thyroglobulin.*, P < 0.05; **, P < 0.01.

3.2. Targeting relationship between SREBP1 and miR-27a-5p in DTC

Our group pre-screened miR-27a-5p associated with SREBP1 expression by miRNA-seq sequencing [19]. In order to explore whether miR-27a-5p was associated with SREBP1 expression in DTC. First, we transfected BCPAP and WRO cell lines with miR-27a mimic, control mimic, miR-27a inhibitor, and control inhibitor synthesized by our company. qRT-PCR was performed to detect the transfection efficiency. qRT-PCR showed that the designed miR-27a-5p overexpression and inhibitor were effective in altering the expression of miR-27a-5p expression (Fig. 2a), which can be used for the next experiments. western blot detected the changes of SREBP1 expression after altering miR-27a-5p expression. The results showed that the level of SREBP1 protein expression in cells was significantly decreased after transfection with miR-27a mimic compared with control, while the level of SREBP1 protein expression in cells was significantly increased after transfection with miR-27a inhibitor. It indicates that miR-27a-5p can affect SREBP1 expression in differentiated thyroid cancer (Fig. 2b, c). To further evaluate the relationship between SREBP1 and miR-27a-5p in DTC, luciferase reporter vectors containing predicted binding sites and their mutants were constructed (Fig. 2d), and one-generation sequencing detected the success of two-vector construction (Fig. 2e). Dual luciferase reporter gene assay showed that in BCPAP and WRO cells, miR-27a mimic and pmiR-RB-REPORT-SREBF1-WT transfected groups were similar to miR-27a mimic and pmiR-RB-REPORT-SREBF1-MUT transfected groups, control mimic and pmiR- RB-REPORT-SREBF1-WT transfected group and control mimic with pmiR-RB-REPORT-SREBF1-MUT transfected group showed a significant decrease in fluorescence intensity (Fig. 2f). It indicates that miR-27a-5p is not only associated with SREBP1 expression, but also by targeting SREBP1 and then regulating its expression.

3.3. MiR-27a-5p expression in DTC and its clinical significance

To investigate the expression characteristics of miR-27a-5p in thyroid cancer tissues, bioinformatics analysis was performed using the CancerMIRNome database (http://bioinfo.jialab-ucr.org/CancerMIRNome). miR-27a-5p was found to be differentially expressed in thyroid cancer tissues and normal tissues. Its expression was significantly lower in thyroid cancer tissues (Fig. 3a). Second, to explore the potential role of miR-27a-5p in DTC progression, we examined the expression of these mRNAs in 75 pairs of thyroid cancer tissues and adjacent normal tissues by RT-qPCR. The experimental results showed that miR-27a-5p was significantly under-expressed in cancer tissues compared with paired paracancer tissues (Fig. 3b). The expression levels of SREBP1 and miR-27a-5 were correlated, and the results of correlation analysis showed a significant negative correlation between miR-27a-5p and SREBP1 expression (Fig. 3c). Meanwhile, we analyzed the correlation between miR-27a-5p expression level and clinicopathological characteristics of DTC patients using Pearson chi-square test, and the results showed that low expression of miR-27a-5p was significantly correlated with malignant pathological characteristics of patients, such as tumor size and TNM stage. Secondly, low expression of miR-27a-5p may also be associated with the gender of DTC patients (Table.3).

Table.3 Relationship between miR-27a-5p expression levels and clinical features of thyroid cancer in 75 patients

Factor	Number of cases(n = 75)	miR-27a-5p expression levels		P value
Factor	Number of cases(n = 75)	Low expression group(n = 38)	High expression group(n = 37)	P value
Age (years)	75
< 45	35	15 (42.86%)	20 (57.14%)	0.206
≥ 45	40	23 (57.50%)	17 (42.50%)
Sex
Male	22	15 (68.18%)	7 (31.82%)
Female	53	23 (43.40%)	30 (56.60%)
WHO stage
I-II	61	29 (47.54%)	32 (52.46%)	0.387
III-IV	15	9 (60.00%)	6 (40.00%)
Tumor size (cm)
≤ 2cm (T1)	33	10 (30.30%)	23 (69.70%)	0.002**
> 2cm (T2-T4)	42	28 (66.67%)	14 (33.33%)
Lymph node metastasis (N)
NO (N0)	32	18 (56.25%)	14 (43.75%)	0.404
YES (N1)	43	20 (46.51%)	23 (53.49%)
Metastasis (M)
NO (M0)	68	35 (51.47%)	33 (48.53%)	0.664
YES (M1)	7	3 (42.86%)	4 (57.14%)
TNM stage
I-II	55	24 (43.64%)	31 (56.36%)	0.043*
III-IV	20	14 (70.00%)	6 (30.00%)
LDL (mmol/L)^#	75	2.83 ± 0.73	2.81 ± 0.73	0.968
HDL (mmol/L)^#	75	1.24 ± 0.27	1.23 ± 0.27	0.989
TCHO(mmol/L)^#	75	4.76 ± 1.07	4.75 ± 1.09	0.992
TG (mmol/L)^#	75	1.64 ± 1.29	1.63 ± 1.30	0.998

3.4. Correlation of SREBP1 and miR-27a-5p expression with patients' postoperative TSH suppression therapy.

In addition, based on the urgent problem of imbalance between efficacy and adverse effects of TSH suppression therapy in patients with differentiated thyroid cancer. We established the evaluation index of adverse reactions (Table.4) and the evaluation index of efficacy (Table.5) according to the thyroid ATA and NCCN guidelines. After compiling the follow-up data of the 75 differentiated thyroid cancer patients included above who received TSH inhibition therapy after surgery for 12 months, we analyzed the correlation between the expression of SREBP1 and miR-27a-5p and the efficacy of the patients' postoperative TSH inhibition therapy and the occurrence of the adverse reactions induced by the therapy. The results showed that the expression of SREBP1 was significantly correlated with the hormone level disorders that occurred in DTC patients after long-term TSH suppression therapy, and interestingly, the expression level of miR-27a-5p was also significantly correlated with the hormone level disorders that occurred in DTC patients after postoperative long-term TSH suppression therapy (Table.6). We grouped 50 patients on TSH suppression therapy according to the basics of efficacy (Table.7). In addition, we analyzed the correlation between the efficacy of TSH suppression therapy received by the patients after surgery and the levels of indicators related to thyroid function such as thyrotropic hormone (TSH), thyroglobulin (TG), and antithyroglobulin antibodies (TG-Ab) after treatment. The results showed that TSH and TG levels were significantly different in the group with satisfactory efficacy and the group with unsatisfactory efficacy, and there was a significant reduction in TSH levels in the group with satisfactory efficacy (Table.8). Further, we analyzed the correlation between the efficacy of patients treated with TSH inhibition after surgery and the expression levels of SREBP1 and miR-27a-5p, as well as the pathological characteristics of patients. Since TSH acts by binding to its receptor TSHR, we analyzed the correlation between TSHR expression levels and patients' TSH inhibition therapy. The results showed that the efficacy of patients' TSH inhibition therapy was significantly correlated with patients' tumor size, SREBP1 and miR-27a-5p expression. Compared with patients in the unsatisfactory efficacy group, patients in the satisfactory efficacy group had smaller tumor sizes, lower SREBP1 expression levels, and higher miR-27a-5p expression levels (Table.9). In addition, we used one-way logistic regression to analyze the specific roles of SREBP1 and miR-27a-5p, as well as tumor size, a factor found above to influence the efficacy of TSH inhibition therapy, in the treatment efficacy. The statistical results showed that both SREBP1 and miR-27a-5p as well as the tumor size of patients could influence the efficacy of postoperative TSH inhibition therapy in DTC patients. And, both SREBP1 and tumor size were risk factors, while miR-27a-5p was a protective factor (Table.10).

Table.4 Indicators for evaluating adverse reactions

Hypermetabolic symptoms	Cardiotoxicity	Skeletal system	Hormone disorder
Tremor, anxiety, insomnia, excessive sweating, cramps, flushing, fever, vomiting, weight loss, diarrhea.	Arrhythmias, tachycardia, palpitations, angina, worsening angina in patients with myocardial ischemia, increased risk of atrial fibrillation in elderly patients	Osteoporosis, muscle weakness, spasticity; predominantly, increased risk of osteoporosis in menopausal women.	Scarcity of menstruation and disruption of the menstrual cycle in women; facial acne in men and women

Table.5 Indicators for evaluating therapeutic efficacy

Curative effectt	Satisfaction	Inexact	Dissatisfaction (biochemical)	Dissatisfaction (structural)
Imaging	No signs of tumor, negative imaging results	Nonspecific imaging, iodine weak thyroid bed uptake	No signs of tumor, positive imaging results	Presence of function or disease, distal metastasis, persistent disease
Serology	In the TSH-inhibited state, Tg levels were less than 0.2 ng/mL; in the TSH-stimulated state, Tg levels were less than 1 ng/mL.	Tg levels < 1 ng/mL in the absence of TSH stimulation and < 10 ng/mL in the presence of TSH stimulation; in the absence of structural or functional lesions, TgAb is stable or decreases slightly	TSH-inhibited state, Tg levels greater than 1 ng/mL; TSH-stimulated state, Tg levels greater than 10 ng/mL, TgAb progressively increasing	Any level of Tg, TgAb negative or positive

Table.6 Correlation analysis of SREBP1 and miR-27a-5p expression with adverse reactions to postoperative TSH inhibition therapy in DTC patients

Type of adverse reaction	Number of cases (n = 55)	SREBP1 expression		P value	miR-27a-5p expression		P value
Type of adverse reaction	Number of cases (n = 55)	High expression group(n = 30)	low expression group(n = 25)	P value	High expression group(n = 29)	low expression group(n = 26)	P value
Cardiotoxicity
Masculine	18	10	8	0.916	7	11	0.152
Feminine	37	20	17		22	15
Skeletal system injuries
Masculine	19	13	6	0.133	10	9	0.992
Feminine	36	17	19		19	17
Hormone level disorder
Masculine	20	15	5	0.027*	6	14	0.012*
Feminine	32	14	18		21	11
Hypermetabolic symptoms
Masculine	26	14	12	0.921	15	11	0.485
Feminine	29	16	13		14	15

Statistical analysis of count data was performed using chi-square test.*, P < 0.05.

Table.7 Basic information on the efficacy of long-term TSH suppression therapy in patients

Curative effectt	Satisfied group	Dissatisfied group
Curative effectt	Satisfied group	Inexact	Dissatisfaction (biochemical)	Dissatisfaction (structural)
Number of cases	16(32%)	17(34%)	10(20%)	7(14%)

Table.8 Correlation between TSH inhibition and thyroid function indexes in DTC patients

Clinical case characteristics	Number of cases(n = 50)	Efficacy of TSH suppression therapy		P value
Clinical case characteristics	Number of cases(n = 50)	Satisfied group(n = 16)	Dissatisfied group(n = 34)	P value
TG-Ab(IU/mL)^#	50	161.34 ± 401.96	29.81 ± 47.64	0.697
TG(ng/mL)^#	50	0.09 ± 0.46	12.46 ± 46.16	0.001***
TSH(mIU/L)^#	50	9.92 ± 37.43	20.59 ± 50.47	0.031*

^#Measurements were statistically analyzed by the Mann-Whitney test and expressed as Mean ± SD. TG-Ab: Anti-thyroglobulin Antibodies; TG: Thyroglobulin; TSH: Thyroid Stimulating Hormone. *, P < 0.05; ***, P < 0.001.

Table.9 Correlation between the efficacy of TSH suppression therapy in DTC patients and basic patient characteristics and gene expression

Clinical case characteristics	Number of cases(n = 50)	Efficacy of TSH suppression therapy		P value
Clinical case characteristics	Number of cases(n = 50)	Satisfied group(n = 16)	Dissatisfied group(n = 34)	P value
Age (years)
< 45	28	10(35.71%)	18(64.29%)	0.525
≥ 45	22	6(27.27%)	16(72.73%)
Sex
Male	16	6(37.50%)	10(62.50%)	0.567
Female	34	10(29.41%)	24(70.59%)
BMI *	50	23.22 ± 3.46	23.28 ± 3.52	0.135
Tumor size (cm)
≤ 2cm (T1)	30	13(43.33%)	17(56.67%)	0.035*
> 2cm (T2-T4)	20	3(15.00%)	17(85.00%)
Lymph node metastasis (N)
NO (N0)	20	4(20.00%)	16(80.00%)	0.137
YES (N1)	30	12(40.00%)	18(60.00%)
Metastasis (M)
NO (M0)	43	12(27.91%)	31(72.09%)	0.124
YES (M1)	7	4(57.14%)	3(42.86%)
TNM stage
I-II	34	10(29.41%)	24(70.59%)	0.567
III-IV	16	6(37.50%)	10(62.50%)
L-T4 dose (µg/day)^#	50	117.31 ± 54.05	106.67 ± 55.88	0.918
TSHR expression
High expression group	24	7(29.17%)	17(70.83%)	0.680
Low expression group	26	9(34.62%)	17(65.38%)
miR-27a-5p expression
High expression group	30	13(43.33%)	17(56.67%)	0.035*
Low expression group	20	3(15.00%)	17(85.00%)
SREBP1 expression
High expression group	21	2(9.52%)	19(90.48%)	0.004**
Low expression group	29	14(48.28%)	15(51.72%)

#Measurements were statistically analyzed using the Mann-Whitney test and expressed as Mean ± SD; counts were statistically analyzed using the chi-square test.TSHR: thyrotropin receptor.*, P < 0.05; **, P < 0.01.

Table.10 One-way logistic regression analysis of factors influencing the efficacy of TSH suppression therapy in patients with DTC

	Coefficient of regression	OR value	95% CI	P value
TSHR expression	−0.085	0.918	0.370–7.580	0.500
SREBP1 expression	2.218	8.867	1.739–45.206	0.009**
miR-27a-5p expression	−1.996	0.136	0.025–0.743	0.021*
Tumor size	3.118	22.559	2.030-251.607	0.011*

Statistical analysis of count data was performed using one-way logistic regression analysis.OR: Odds Ratio; CI: Confidence Interval.*, P < 0.05; **, P < 0.01.

3.5. MiR-27a-5p inhibits DTC cell proliferation

To detect the effect of miR-27a-5p on DTC cell function. We used MTS assay and plate cloning assay to explore the effect of miR-27a-5p on DTC cell viability and proliferation ability. The results showed that cell viability was significantly inhibited after overexpression of miR-27a-5p, and this effect was more significant in BCPAP cells (Fig. 4a), and conversely, cell viability was significantly enhanced after inhibition of miR-27a-5p expression (Fig. 4b). The results of plate clone formation assay also indicated that the cell proliferation ability was reduced after overexpression of miR-27a-5p (Fig. 4c), while the cell proliferation ability was significantly enhanced after inhibition of miR-27a-5p expression (Fig. 4d).

3.6. Effect of miR-27a-5p on DTC cell cycle and apoptosis

To further investigate the role of miR-27a-5p in influencing the proliferative capacity of DTC cells, we used flow cytometry for cell cycle detection. The results showed that compared with the control group, the cell cycle of BCPAP cells was blocked in S phase and the cells in G2/M phase were significantly reduced after overexpression of miR-27a-5p, whereas the cell cycle of WRO cells was blocked in G0/G1 phase, and the cells in both S phase and G2/M phase were significantly reduced (Fig. 5a). The opposite was true after inhibitor treatment, as both BCPAP and WRO cells had fewer cells in the S phase of the cell cycle, indicating that the cell cycle was promoted (Fig. 5b). This also suggests that miR-27a-5p can affect the proliferation of differentiated thyroid cancer cells by regulating cell cycle transitions and thus affecting the proliferation of differentiated thyroid cancer cells. It is well known that apoptosis also plays an important role in the proliferation process. To further investigate the effect of miR-27a-5p on apoptosis in differentiated thyroid cancer cells. In this study, flow cytometry was utilized for the detection of apoptosis. The experimental results showed that apoptosis was significantly increased after overexpression of miR-27a-5p (Fig. 5c) and vice versa after inhibition of miR-27a-5p expression (Fig. 5d). This suggests that miR-27a-5p can also regulate apoptosis, which in turn affects the progression of thyroid cancer.

3.7. MiR-27a-5p inhibits invasive migration of DTC cells

To explore the potential role of miR-27a-5p in DTC metastasis. We used scratch and Transwell assays to detect the invasive and migratory abilities of cells. The results of the scratch assay showed that the invasion and migration abilities of both BCPAP and WRO cell lines were significantly reduced after overexpression of miR-27a-5p compared with the control group, and the difference was statistically significant (Fig. 6a); the opposite was true for inhibitor-treated BCPAP and WRO cells (Fig. 6b). It indicated that low expression of miR-27a-5p could enhance the migration ability of cells, and thus promote tumor metastasis.Transwell assay results showed that low expression of miR-27a-5p could enhance the invasion and migration ability of thyroid cancer cells, and thus promote tumor metastasis (Fig. 6c, d).

Thyroid cancer can be classified into different types based on histological features and biological behavior, among which differentiated thyroid cancer (DTC) is the most common type, including papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) [20, 21]. Currently, treatments for thyroid cancer include surgical resection, radiation therapy, and thyroid hormone replacement therapy. Surgical resection is the main treatment modality, and cure can be achieved for early stage thyroid cancer. Radiation therapy is mainly used for high-risk or advanced patients to reduce the risk of recurrence and metastasis [3]. For some refractory or metastatic thyroid cancers, targeted therapies and immunotherapy are also being researched and tested to provide more treatment options for patients. A variety of genes and signaling factors regulate the occurrence and development of thyroid cancer by influencing the biological behaviors of tumor cells such as proliferation, metastasis, and drug resistance, and the search for small molecule markers for the diagnosis and treatment of thyroid cancer can help to understand the pathogenesis and biological characteristics of thyroid cancer.MiRNA expression is either up-regulated or down-regulated in cancers playing the role of either a proto-oncogene or an oncogene suppressor. It has been found that in cancers such as ovarian, colorectal, breast and gastric cancers, miR-27a-5p expression levels are upregulated, promoting cell proliferation and metastasis, and correlating with malignant progression of cancer [22–26]. In tumors such as cervical cancer, esophageal squamous carcinoma and prostate cancer, the expression level of miR-27a-5p was significantly reduced, and it functioned as an oncogene to inhibit tumor growth and metastasis [27–29]. miR-27a-5p's regulatory role for cancer may depend on the cancer type and cellular microenvironment [30]. In thyroid cancer, the expression status of miR-27a-5p is unknown. To investigate the expression of miR-27a-5p in thyroid cancer, 75 pairs of cancer and paired paracancerous tissue specimens from patients diagnosed with DTC by pathology were collected in the present study, and it was found that the expression level of miR-27a-5p was reduced in thyroid cancer tissues compared with the adjacent normal thyroid tissues. A statistically negative correlation between miR-27a-5p expression and SREBP1 expression was also observed in thyroid cancer tissues.

miR-27a is a key miRNA that plays an important role in the regulation of lipid metabolism. Several studies have confirmed that miR-27a is able to play a regulatory role in lipid metabolism by targeting binding to multiple key genes and proteins. It affects the transcriptional regulation of lipid metabolic pathways by inhibiting the expression of RXRα and influencing the activity of nuclear receptors [15]. In addition, miR-27a regulates cholesterol metabolism by targeting and inhibiting the expression of ABCA1, which affects the transport of cholesterol and phospholipids [31]. It is also involved in regulating the gene expression of fatty acid synthase, which affects fatty acid synthesis and metabolic processes [32, 33]. In addition, the regulation of SREBP-1 and SREBP-2 by miR-27a affects the regulation of the lipid de novo synthesis pathway [15]. In this study, we demonstrated that miR-27a-5p mimics could effectively reduce SREBP1 expression by dual luciferase assay in human thyroid cancer cell lines BCPAP and WRO. miR-27a-5p targeted binding to the SREBP1 3'-UTR region thereby repressing the SREBP1 gene. miR-27a-5p has been shown to play a role in the regulation of lipid synthesis in a multiple cancers has been reported, but plays different roles in multiple cancers [34].

In this study, we found that the expression level of miR-27a-5p in human thyroid cancer tissues was significantly lower than that in normal tissues of the human thyroid gland, saying that miR-27a-5p is lowly expressed in thyroid cancer tissues and plays the role of an oncogene. miR-27a-5p expression was significantly correlated with the tumor size and TNM stage of the patients. the expression of SREBP1 and miR-27a-5p was significantly correlated with patient The correlation between the efficacy of postoperative TSH inhibitory therapy and the occurrence of adverse effects caused by the In in vitro cellular experiments, thyroid cancer BCPAP cells and WRO cells transfected with miR-27a mimics, mimics control were detected by MTT assay. miR-27a mimics significantly inhibited the proliferation of thyroid cancer BCPAP cells and WRO cells, which verified the oncogenic effect of miR-124. miR-27a-5p Significantly inhibited the malignant progression of thyroid cancer cell proliferation, invasion and metastasis as well as apoptosis. miR-27a-5p inhibited the transcription and expression of the rate-limiting enzyme genes in the lipid follower synthesis pathway by regulating the expression of SREBP1, thereby inhibiting the proliferation, migration and invasion of thyroid cancer cells. In-depth investigation of the regulatory role of miR-27a-5p in thyroid cancer and its potential role in the SREBP1-regulated lipid metabolism pathway is of great significance for understanding the pathogenesis of thyroid cancer and finding new therapeutic targets. Based on the results of this study, we conclude that in DTC, miR-27a-5p regulates SREBP1 expression and participates in the malignant progression of the cancer, providing new clues for individualized treatment of TSH inhibition.

Acknowledgments

Zilan Xie and Jianqiu Liu are the co-first authors of this article.

Funding

This work was supported by the Natural Science Foundation of of Hunan, China Grant[ No. 2020JJ4888].

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Jianqiu Liu], [Jiating Zhou], [Xuan Zhang], and [Zhi Li]. The first draft of the manuscript was written by [Zilan Xie] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known financial interests or personal relationships that might influence the work reported here.

Data availability

All data in this study are available from the corresponding author upon request.

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MiR-27a-5p inhibits malignant progression of differentiated thyroid cancer through SREBP1 and is associated with thyrotropin suppressive therapy

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Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1. Clinical samples and ethics statement.

2.2. Cell culture.

2.3. Transfection.

2.4. Reverse transcriptase quantitative PCR.(qRT-PCR).

2.5. Western blot.

2.6. Dual luciferase reporter gene

2.7. MTS assay.

2.8. Plate cloning experiment.

2.9. Wound healing experiment

2.10. Transwell assay.

2.11. Cell cycle assay.

2.12. Cell apoptosis assay.

2.13. Statistical analysis.

3. Results

3.1. SREBP1 expression in DTC and its clinical significance

3.2. Targeting relationship between SREBP1 and miR-27a-5p in DTC

3.3. MiR-27a-5p expression in DTC and its clinical significance

3.4. Correlation of SREBP1 and miR-27a-5p expression with patients' postoperative TSH suppression therapy.

3.5. MiR-27a-5p inhibits DTC cell proliferation

3.6. Effect of miR-27a-5p on DTC cell cycle and apoptosis

3.7. MiR-27a-5p inhibits invasive migration of DTC cells

4. Discussion

Declarations

Funding

Author Contributions

Declaration of competing interest

Data availability

References

Additional Declarations

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