miR-32-5p Inhibits the Proliferation, Migration and Invasion of Thyroid Cancer Cells by Regulating Twist1

Background: Thyroid cancer is the most prevalent malignancy and one of the leading causes of cancer-related deaths. Recent studies have revealed that microRNAs (miRNAs) play an important role in tumorigenesis in various cancer types by affecting the expression of its targets. However, the role of miR-32-5p in thyroid cancer remains limited. Methods: In this study, we attempt to explore the role of miR-32-5p in thyroid cancer and elucidate the underlying mechanism. Expression of miR-32-5p was determined by quantitative reverse transcription PCR. Functional assays were performed by CCK-8 assay, cell colony assay, cell apoptosis assay, cell migration and invasion assays, cell cycle assay and luciferase assay. Protein expression was analyzed by Western blot. Results: In the present study, the role of miR-32-5p in thyroid cancer was rstly explored. It is found that miR-32-5p was downregulated in thyroid cancer tissues and cells. Overexpression of miR-32-5p inhibited thyroid cancer cells proliferation, migration, invasion and epithelial ‐ mesenchymal transition process; while suppression of miR-32-5p exhibited an opposite effect on thyroid cancer cells. In addition, In addition, a luciferase assay showed Twist1 was identied as a direct target of miR-32-5p in thyroid cancer, and further study showed that restoration of Twist1 attenuated the biological effect of miR-32-5p on thyroid cancer cells. Conclusion: In conclusion, our results demonstrated miR-32-5p functions as a tumor suppressor by targeting Twist1 in thyroid cancer, providing a novel insight into thyroid cancer therapy.


Introduction
Thyroid cancer is a common malignant tumor in the head and neck and the most common malignant tumor in the endocrine system [1]. The treatment of thyroid cancer mainly adopts comprehensive treatment based on surgery, but its limitations still exist. Due to the popularity of early cancer screening, the incidence of thyroid cancer is increasing every year globally. [2]. Although the patients with thyroid cancer present a good prognosis, some patients still exhibit recurrence or distant metastasis [3]. Therefore, exploring the potential molecular mechanisms of thyroid cancer is particularly important for treating patients.
MicroRNA (miRNA), an additional product produced during gene transcription, not only plays an important role in cell morphology, structure, and functional transformation by participating in epigenetic, transcriptional, and post-transcriptional regulation, but also plays an important role as an oncogene or tumor suppressor gene in the occurrence and development of malignant tumors, including thyroid cancer [4][5][6]. More and more studies have con rmed that miR-32-5p plays a very important role in the occurrence and development of different malignant tumors in the human body. For example, overexpression miR-32-5p was able to inhibit the triple negative breast cancer cells (TNBC), and further results elucidated that the LncRNA WEE2 antisense RNA 1 (WEE2-AS1) and transducer of ERBB2, 1(TOB1) were the upstream gene and downstream gene of miR-32-5p, respectively [7]. Additionally, the level of miR-32-5p was downregulated in osteosarcoma (OS) tissues and cells, and Lou et al. found that long noncoding RNA HNF1A antisense RNA 1 (HNF1A-AS1) bound to miR-32-5p to regulate the expression of high-mobility group protein B1 (HMGB1) in OS progression [8]. Moreover, miR-32-5p was found to signi cantly regulate the radiosensitivity, migration and invasion of colorectal cancer cells via targeting transducer of ERBB2, 1(TOB1) [9]. The application of miR-32-5p in prognosis evaluation of malignant tumors is of great signi cance. Nevertheless, the relationship between the progression of thyroid cancer and miR-32-5p remains largely unclear. Thus, the expression of miR-32-5p in thyroid cancer and its potential molecular mechanisms were investigated in our study.

Tissue samples
Tumor tissues and matched adjacent nontumor tissues were collected from 10 thyroid cancer patients in the Department of General Surgery, Department of pathology, Hubei Provincial Hospital of TCM, (Wuhan, China). All patients received no chemotherapy or radiotherapy before surgery. Tissue samples were frozen immediately in liquid nitrogen following resection and stored at -80°C until RNA extraction.

Cell Culture
The human thyroid cancer cell lines B-CPAP, TPC-1, KTC-1, HTh-7, C643 and the normal human thyroid cell line HTORI-3 were purchased from Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, China). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Gibco) at 37˚C in a humidi ed chamber with 5% CO 2 atmosphere.

Transfection
The mimics-miR-32-5p, inhibitor-miR-32-5p, Twist1 and Twist1 siRNA(si-Twist1), and their respective negative controls were obtained from Shanghai GenePharma Co., Ltd., and separately transfected into thyroid cancer cell lines using Lipofectamine 2000 according to the manufacturer's protocols. After 48 h of transfection, the cells were harvested and used in experiments.

Cell Counting Kit-8 (CCK-8) assay
The CCK-8 assay was performed to measure cell proliferation according to the manufacturer's instructions. In brief, after effectively transfection, cultured cells were plated and incubated in 96-well plates at 5×10 4 cells/well. Then, 10 µL CCK-8 solution was added to each well. Proliferation rates were measured with a microplate reader at an absorbance of 450 nm at 24, 48, 72 and 96 h.

Cell colony assay
Cell samples were seeded in 6-well culture dishes at 200 cells/well for incubation of 2 weeks. After xed in 4% paraformaldehyde, samples were processed with 0.1% crystal violet, and colony numbers were then counted 2.7 Wound healing assay Cell samples were seeded in 6-well culture dishes at 5×10 5 cells/well for incubation of 24 h. The cell monolayer was scratched with a 10 µL pipette tip to in a straight line and then washed twice with cold phosphate-buffered saline (PBS). Cells were then cultured with serum-free medium at 37°C for 48 h. Finally, the wounds were observed under a microscope (Carl Zeiss, Germany) and the relative migration ability was calculated.

Cell apoptosis assay
Cell samples were seeded in 6-well culture dishes at 1×10 6 cells/well for incubation of 24 h. Cells were collected by the pancreatin without EDTA, and cells were stained with AnnexinV-APC and 7-AAD for 5-15 min at room temperature avoiding light, and immediately analyzed on FACSCalibur ow cytometer.

Cell migration and invasion assays
Cell samples were seeded in 6-well culture dishes at 1×10 6 cells/well for incubation of 24 h. A Transwell chamber with 8-µm pores was used for the migration assay. Complete RPMI-1640 medium containing 10% FBS, was added in the lower layer, and the cell suspension in serum-free media was added in the upper chamber. After 48 h incubation, the cells on the lower surface of the chamber were xed with 70% ice ethanol solution for 60 min and stained with 0.5% crystal violet for 20 min at room temperature. A total of 10 elds from each chamber were selected randomly for counting and the relative migration ability was calculated. Cell samples were seeded in 6-well culture dishes at 1×10 6 cells/well for incubation of 24 h. Then the cells were plated into the upper layer of the chamber covered with Matrigel, and the same culture method was used to perform cell invasion assays. After staining with 0.5% crystal violet, at least 10 elds from each chamber were selected and the invasive cells were counted and quanti ed.

Cell cycle assay
Cell samples were seeded in 6-well culture dishes at 1×10 6 cells/well for incubation of 24 h. All cells were harvested by digesting with trypsin and washed with PBS and then xed with ice-cold 70% ethanol in PBS for 30 min at -20°C. Fixed cells were washed with PBS, treated with 10 µlL RNase A (1 mg/mL) and resuspended in 10 µl of L 400 µg/mL propidium iodide (PI) for staining. Cell cycle distribution was performed with FACSCalibur ow cytometer.

Luciferase activity assay
The wild-type (WT) 3′-UTR of Twist1 containing miR-32-5p binding site or the mutant (mut) Twist1 3′-UTR was ampli ed and then inserted into pUC57 reporter vector. For luciferase activity assay, two thyroid cancer cell lines were cotransfected with mimics-miR-32-5p or the scramble with the WT or mut 3′-UTR of Twist1 reporter vector using Lipofectamine 2000 according to the manufacturer's protocol. Following 48 hours of transfection, cells were harvested and lysed. The luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega) and normalized against renilla luciferase activity.

Western blot assay
Cell samples were seeded in 6-well culture dishes at 1×10 6 cells/well for incubation of 24 h. The cultured cells were collected, washed twice with cold PBS and lysed with radioimmune precipitation assay buffer containing Protease Inhibitor Cocktail. Total protein concentration was measured using a bicinchoninic acid (BCA) protein kit. Protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene uoride membranes. Membranes were blocked in 5% nonfat milk and then incubated with primary antibodies at 4°C overnight. The following day, membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. Finally, the signals were detected using an enhanced chemiluminescent (ECL) Western blot analysis kit.

Statistical analysis
All experimental data were presented in the form of mean ± standard deviation from three independent experiments. GraphPad Prism 8 (GraphPad Software, Inc.) was utilized for statistical analysis. Student's t-test was used to compare the statistical difference between two groups, while one-way ANOVA with Dunnett's or Tukey's multiple comparisons test was employed to compare the statistical differences among multiple groups. P < 0.05 was considered to indicate a statistically signi cant difference.

Results
3.1 miR-32-5p was down-regulated in thyroid cancer Firstly, the relative expression of miR-32-5p in thyroid cancer tissues and normal tissues was measured by qRT-PCR. It was found that the expression of miR-32-5p in thyroid cancer tissues is signi cantly lower than normal tissues (Fig. 1A). Moreover, the miR-32-5p expression in ve thyroid cancer cell lines and normal thyroid cell lines was also examined. Compared to normal thyroid cell lines Htori-3, miR-32-5p was also downregulated in ver thyroid cancer cell lines, which is consistent with the results of tumor tissue (Fig. 1B). More importantly, among these thyroid cancer cell lines, the TPC-1 and KTC-1 cell lines were contained the most signi cant upregulation and downregulation of miR-32-5p, respectively, thus they were used for following assays (Fig. 1B). In conclusion, the data indicated that miR-32-5p is downregulated in thyroid cancer tissues and cells lines.
Compared with the inhibitor NC, the proliferation of TPC-1 cell was increased in the miR-32-5p inhibitortreated group (Fig. 3B). As exhibited in Fig. 3C, results showed that the numbers of colonies are increased, while the cell apoptosis was decreased when miR-32-5p inhibition in TPC-1 cells (Fig. 3D). Moreover, our results showed that inhibition of miR-32-5p is able to enhance the migration and invasion ability of TPC-1 cells (Fig. 3E-3G). Furthermore, the S phase was arrest when TPC-1 cells treat with miR-32-5p inhibitor (Fig. 3H). Besides, results indicated that miR-32-5p suppression can remarkably promote EMT process (Fig. 3I). These ndings suggested that the increased proliferation, migration, invasion and cell cycle of TPC-1 cells are associated with miR-32-5p deletion.

miR-32-5p directly targeted Twist1
Based on miRanda data, we found that Twist1 has miR-32-5p binding sequences in its 3 '-UTR, suggesting that Twist1 may the target of miR-32-5p (Fig. 4A). To con rm the Twist1 as the target of miR-32-5p in our study, the luciferase assay was further studied. As shown in Fig. 4B, the results showed that luciferase activity of WT was repressed by overexpression of miR-32-5p, but not the mut 3 '-UTR of miR-32-5p (Fig. 4B). More importantly, results also revealed that the level of Twist1 is elevated or reduced when inhibition of miR-32-5p in TPC-1 cells or overexpression of miR-32-5p in KTC-1 cells, respectively ( Fig. 4C-4D). Together, these results demonstrated that Twist1 is a target of miR-32-5p.

Restoration of Twist1 attenuated the biological effect of miR-32-5p on thyroid cancer cells
In order to further study the role of Twist1 in thyroid cancer, the KTC-1 and TPC-1 cells were transfected with pcDNA3.1-Twist1 plasmid or siRNA-Twist1, respectively. The results found that Twist1 expression was signi cantly increased in KTC-1 cells, while there was opposite phenomenon in TPC-1 cells. (Fig. 5A and 5B). Upregulated Twist1 was found to increase cells proliferation, but downregulation of Twist1 partly abolished the effect of inhibitor miR-32-5p on cells proliferation regression ( Fig. 5C and 5D). Additionally, the numbers of colonies were increased when overexpression Twist1, while knockdown of Twist1 attenuated the inhibitor miR-32-5p-induced thyroid cancer cell colony (Fig. 5E). Moreover, it is found that restoration of Twist1 increased the proapoptotic effect of mimics-miR-32-5p on thyroid cancer cells (Fig. 5F). it indicated that overexpression of miR-32-5p signi cantly decreases the migrated and invaded of cell numbers, respectively, while restoration of Twist1 increased the number of migrated and invaded cells on the basis of the up-regulation of miR-32-5p. However, decreased Twist1 shows the opposite effect, implying that Twist1 reverses the ability of miR-32-5p to promote thyroid cancer cell migration and invasion (Fig. 5H-5I). Furthermore, restoration of Twist1 signi cantly increased the proportion of S phase of mimics-miR-32-5p on thyroid cancer cells, while si-Twist1 decreased t the proportion of S phase on the base of knockdown of miR-32-5p (Fig. 5J). In addition, the results showed that the recovery or knockout of Twist1 could effectively weaken the effect of mimic-miR-32-5p or inhibitor miR-32-5p on the EMT process of thyroid cancer cells (Fig. 5K). These ndings suggest that miR-32-5p promotes thyroid cancer cell proliferation by the regulation of Twist1.

Discussion And Conclusion
Abnormal regulation of miRNA plays an important role in tumorigenesis and development [5,6]. During the occurrence and development of different types of human cancers, miRNAs consider an oncogene or tumor suppressor gene in the occurrence and development, respectively [5,6]. The application of miRNAs in the diagnostic classi cation and prognostic evaluation of thyroid cancer has been a research hotpot in recent years. For example, Hou et al. found that the proliferation and migration of dedifferentiated thyroid cancer cells are inhibited when miR-146b-3p deletion, and the results of further study showed that regulation the expression and localization of sodium/iodine cotransporter related with targeting MUC20 [10]. Moreover, miR-153-3p was found to inhibit the cell proliferation and glycolysis by inhibiting the expression of E3F3, and it may consider as a potential biomarker for thyroid cancer diagnosis [11].
Additionally, the results from Wu et al. demonstrated that the The miR-199a-3p/ DNMT3A pathway is involved in the aggression of papillary thyroid carcinoma and directly targets RAP2A [12]. MiR-32-5p has been extensively studied in different human diseases, including cancer, metabolic syndrome, and neuropathic pain [13][14][15]. However, the relationship between miR-32-5p and thyroid cancer has not been studied. Therefore, it is of great signi cance to study the occurrence and development of miR-32-5p mediated thyroid cancer. In our study, miR-32-5p was down-regulated in both thyroid cancer tissues and cancer cells, and further study showed that it is positively correlated with cell proliferation, migration and invasion. Each miRNA can regulate its downstream target, and then control to promote or inhibit the proliferation of tumor cells [16]. In previous study, Twist1 is a pro-malignant transcription factor, which plays an important role in the invasion and metastasis of various malignant tumors [17]. It was reported that the induction of Twist1can can regulate tumor metastasis in hepatocellular carcinoma [18]. Furthermore, Twist1 was regulated by several miRNAs in cancers. Yin et al. found that miR-361-5p can inhibit tumorigenesis and EMT in hepatocellular carcinoma by targeting Twist1 [19]. In addition, miR-186 also targets Twist1 to mediate proliferation, migration, and EMT inhibition in breast cancer cells [20]. In our research, we found that Twist1 is a direct target of miR-32-5p. Moreover, rescue experiments showed that overexpression or knockdown of Twist1 can decrease the role of mimics or inhibitor miR-32-5p on thyroid cancer cells proliferation and EMT process. All results indicate that miR-32-5p may play an antithyroid cancer effect by targeting Twist1.
In conclusion, this study is the rst to show that miR-32-5p is downregulated in thyroid cancer tissues and cell lines and miR-32-5p inhibited tumorigenesis and the EMT of thyroid cancer by targeting Twist1. These ndings suggest that miR-32-5p may serve as a novel biomarker in thyroid cancer diagnosis or have potential clinical values in thyroid cancer treatment.

Declarations
He and Silei Li revised it critically for important intellectual content. All the authors read and approved the nal manuscript.

Funding
Not applicable.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study protocol has been approved by the Department of General Surgery, Department of pathology, Hubei Provincial Hospital of TCM, (Wuhan, China)