MicroRNA‐524‐5p suppresses the progression of papillary thyroid carcinoma cells via targeting on FOXE1 and ITGA3 in cell autophagy and cycling pathways

Abstract MicroRNAs are beneficial for cancer therapy as they can simultaneously downregulate multiple targets involved in diverse biological pathways related to tumor development. In papillary thyroid cancer, many microRNAs were identified as differentially expressed factors in tumor tissues. In another way, recent studies revealed cell proliferation, cell cycling, apoptosis, and autophagy are critical pathways controlling papillary thyroid cancer development and progression. As miR‐524‐5p was approved as a cancer suppressor targeting multiple genes in several types of cancer cells, this study aims to characterize the role of miR‐524‐5p in the thyroid cancer cell. The expression of miR‐524‐5p was decreased in the papillary thyroid cancer tissues and cell lines, while forkhead box E1 (FOXE1) and ITGA3 were increased. In the clinical case, expression of miR‐524‐5p, FOXE1, and ITGA3 were significantly correlated with papillary thyroid cancer development and progression. FOXE1 and ITGA3 were approved as direct targets of miR‐524‐5p. miR‐524‐5p could inhibit papillary thyroid cancer cell viability, migration, invasion, and apoptosis through targeting FOXE1 and ITGA3. Cell cycling and autophagy pathways were disturbed by downregulation of FOXE1 and ITGA3, respectively. Collectively, miR‐524‐5p targeting on FOXE1 and ITGA3 prevents thyroid cancer progression through different pathways including cell cycling and autophagy.

cell-cycle progression were also involved in the PTC progression.
After decades of studies in molecular biology, researchers have identified several oncogenes related proliferation and cell cycle of cancer cell, such as LCN2, CRABP1, C1QL1, PTEN, and BRAF (Celestino et al., 2018;Loo, Khalili, Beuhler, Siddiqi, & Vasef, 2018;Razavi, Modarressi, Yaghmaei, Tavangar, & Hedayati, 2017). In recent years, it was implicated that autophagy has a vital role in PTC development and progression. Without FAM129A induced autophagy in normal thyroid cells, carcinomas are developed under certain nutrient and growth condition (Nozima et al., 2019). miR-125b could enhance autophagy in PTC through repression of Foxp3 . miR-144 reduces the tumor growth by suppressing transforming growth factor-α signaling pathway to promote autophagy of PTC (J. Liu et al., 2018). Obviously, targets for PTC therapy exist in multiple biological pathways. Both EMT and autophagy attracted researchers' attention to survey underline mechanisms related to PTC development and progression.
In the complicated network of carcinoma development, micro-RNAs (miRNAs) have the critical role as they can simultaneously downregulate multiple targets (Dallaire, Frederick, & Simard, 2018;Fitzwalter et al., 2018;Kasper et al., 2017;Singh, Lee, Hartman, Ruiz-Whalen, & O'Reilly, 2018). Thus, it is possible to modify cell proliferation, cycling, and autophagy in carcinoma cells at the same time though controlling miRNAs expression. In PTC, there are several differentially expressed miRNAs were identified by miRNA sequencing (Graham et al., 2015). PTC cell proliferation is prevented by miR-7 targeting CDC28 protein kinase regulatory subunit 2 (CKS2) (Hua et al., 2016). This interaction also influences on the migration and invasion of PTC. Whereas, miR-150-5p targeting on BRAFV600E mutation could accelerate PTC development by enhancing EMT . During the processes, the PTC cell proliferation and apoptosis are also affected by miR-150-5p with regulation on BRAFV600E. The typical tumor pathway AKT/mTOR in PTC development and progression is also regulated by miR-451a (Minna et al., 2016). To turn down the activation of AKT/mTOR pathway, multiple targets including AKT1, MIF, and c-MYC are downregulated by miR-451a. Attenuated AKT/mTOR signaling pathway through ectopic expression of miR-451a could damage the processes of PTC cell proliferation and migration. Another typical miRNA function as a tumor suppressor in PTC is let-7 miRNA targeting on RAS. Recent studies suggested that the let-7 family from circulating miRNA could be served as a clinical marker to noninvasively diagnose PTC and prognose survival after treatment (Perdas, Stawski, Nowak, & Zubrzycka, 2016 Zhen et al., 2017). The growth of glioma is decreased by miR-524-5p targeting on EZH2, Jagged-1, and Hes-1 (L. Chen et al., 2012;Zhi et al., 2017).
Oncogene BRAF and ERK2 could be downregulated by miR-524-5p in melanoma to reduce tumor growth (S. M. Liu, Lu, Lee, Chung, & Ma, 2014). However, the role of miR-524-5p is still unclear in PTC where miR-524-5p was already detected with differential expression level.
In this study, we investigated the relationship between miR-524-5p and PTC development and uncovered the underlying mechanisms by identification of its major targeting genes and biological pathway.

| RNA extraction and isolation
TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA), NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and the Agilent RNA 6000 (Thermo Fisher Scientific) Nano assay was utilized to isolate the total RNA and assess the RNA quantity, purity, and integrity. The experiments performed following the manufacturer's instructions. Gel electrophoresis was used to detect genomic DNA contamination. RNA with A260/A280 ≥ 1.6 and A260/A230 ≥ 1 were used for the following experiments.

| Real-time quantitative PCR (RT-qPCR)
The complementary DNA was produced by the Prime Script™ RT-qPCR Kit (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. The quantification PCR were carried out in 7900HT fast RT-qPCR instrument (Applied Biosystems, Life Technologies, Cologne, Germany) with SYBR ® Premix Ex Taq™ II (Takara Bio Inc., Tokyo, Shiga, Japan). The endogenous control was the expression of glyceraldehyde-3-phosphate dehydrogenase was used as the control. The level of genes were calculated by the −ΔΔ 2 Ct method. All qPCR was performed in triplicate. 2.8 | 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) migration and invasion assays Cell Counting Kit-8 Reagent (Beyotime Institute of Biotechnology) was used to cell proliferation assay. In colony formation assay, cell was grown in six-well transwell plates for 2 weeks. Crystal violet solution (0.5%) was used to stained the positive cells after fixation.
For scratch test, A 10 μl pipet tip was used to make lines and after certain hours, cells were imaged and determine the distance of gap.
All assays were carried out biological independently in triplicate. To determine the miR-524-5p expression in cancer and papa-cancer tissues, RT-qPCR was carried out with tissues from 57 PTC patients.

| Cell cycle and apoptosis analysis
Results displayed that expression of miR-524-5p in PTC was lower than adjacent normal tissues with a significant fold change < 0.3 and highest in TPC-1. Increased expression of FOXE1 and ITGA3 was found in TPC-1, K1, and NPA compared with Nthyori 3-1 (Figure 1b).
Western blot analysis was carried out to investigate the protein level of FOXE1 and ITGA3. As results displayed, FOXE1 and ITGA3 have more higher protein level in the PTC cell lines than the normal cell line (Figure 1c).
3.3 | The progression of PTC is associated with the level of miR-524-5p The clinical characteristics of PTC patients were collected for further analysis of the relationship between the PTC progression and miR- UTR to knockdown the expression (Figure 2a).
To confirm the downregulation of miR-524-5p on FOXE1 and   3.7 | PTC cell-cycle arrest was enhanced by miR-524-5p or siRNA-ITGA3 To investigate the role of miR-524-5p, FOXE1, and ITGA3 in PTC cell cycle, TPC-1 and NPA transfected with NC, miR-524-5p mimic, inhibitor, siRNA-FOXE1 or siRNA-ITGA3 were measured by flow cytometry. Percentage of G0/G1 phase TPC-1 and NPA cells was significantly increased in miR-524-5p mimic groups whereas significantly decreased in miR-524-5p inhibitor groups compared with the NC and blank groups (p < 0.05; Figure 5a,b). The proportion of the S phase TPC-1 and NPA cells were reversely changed in miR-524-5p mimic and inhibitor groups (p < 0.05; Figure 5a,b). Proportion of G0/ G1 phase and the S phase TPC-1 and NPA cells in siRNA-ITGA3 group were increased and decreased, respectively (p < 0.05; Figure   5a,b). Other groups had no significant differences in the pattern of cell cycle (Figure 5a,b).
Protein level of CDK2, CDK4, and CDK6 were compared by western blot analysis in TPC-1 and NPA under different transfection.
Result demonstrated that the protein level of CDK2, CDK4, and CDK6 were fall off in miR-524-5p mimic group and raised in miR-524-5p inhibitor group (Figure 6c,d). siRNA-ITGA3 group has similar pattern to miR-524-5p mimic group (Figure 6c,d).  (Minna et al., 2016). In glioma, miR-524-5p could also directly knockdown expression of EZH2, Jagged-1, and Hes-1 (L. Chen et al., 2012;Zhi et al., 2017 (Table 1). ITGA3 has been identified as a factor to enhance cell proliferation and cell cycle in intrahepatic cholangiocarcinoma (Huang et al., 2018). ITGA3 could also play in bladder cancer to regulate cell apoptosis (Sakaguchi et al., 2017). In contrast, previous Genome-Wide Association Study suggested that SNP in FOXE1 is strongly associate with the risk of PTC (He et al., 2015). Together, these results reveal a potential mechanisms underlying suppression of PTC progression through miR-524-5p, ITGA3, and FOXE1 pathway.
Third, ITGA3 or FOXE1 might play in a different role during PTC development. In MTT assay, miR-524-5p mimic has more effect than RNAi-ITGA3 or RNAi-FOXE1 alone (Figure 3). The protein level of autophagy-related protein LC3-II, Bcl-2, and BECLIN1 are changed under the regulation of miR-524-5p or FOXE1 but not ITGA3 (Figure 6). The effect of FOXE1 on cell cycle might be attenuated that ITGA3 (Figure 6). Recent studies reveal that the increase of FOXE1 is associated with autophagy markers such as LC3B, ATG5, ATG12, and BECLIN1 (Ji, Cui, Yu, & Cui, 2016). Taken together, ITGA3, and FOXE1 might effect on cell cycle and autophagy, respectively.

ACKNOWLEDGMENTS
We would like to acknowledge the helpful comments on this paper received from our reviewers. This study was supported by the Natural Science Foundation of Fujian Province (Grant No.

2017Y0036) and Fujian Provincial Department of Science and
Technology (Grant No. 2017J01530)