miRNA-144-5p/ITGA3 Suppressed the Tumor-Promoting Behaviors of Thyroid Cancer Cells by Downregulating ITGA3

Objective To ascertain the mechanism of miRNA-144-5p and ITGA3 in thyroid cancer (TC). Methods From The Cancer Genome Atlas (TCGA), RNA expression profiles were obtained for the expression analysis of miRNAs and mRNAs in TC. qRT-PCR and western blot were utilized to measure the expression of miRNA-144-5p and ITGA3 at RNA and protein levels, respectively. The association between miRNA-144-5p and ITGA3 was validated by the dual-luciferase assay. CCK-8, scratch healing, transwell, and flow cytometry assays were employed to evaluate tumor-related cell behaviors. Results Low-expressed miRNA-144-5p and high-expressed ITGA3 were found in TC cells relative to normal cells. miRNA-144-5p expression was positively associated with suppressive effects on proliferative, invasive, and migratory ability of TC cells while negatively associated with cell apoptosis. miRNA-144-5p inhibited ITGA3 expression in TC, and its overexpression remarkably reversed the tumor-promoting effects of overexpressed ITGA3 on the biological functions of TC. Conclusion It is verified in our study that cell growth of TC is inhibited by the miRNA-144-5p/ITGA3 axis, which represents an underlying target for TC. This research proposed a new insight into the strategy of TC treatment.


Introduction
Thyroid cancer (TC) is considered endocrine cancer, with continuous increasing morbidity in the past decades [1]. TC could be classified into different subtypes, where papillary thyroid cancer (PTC) occupies the majority of the new TC cases [2]. Treated with specific methods such as surgery, radioiodine, and thyroid-stimulating hormone suppression, a great number of patients showed a relatively optimal improvement in their condition. However, there was still recurrence and metastasis of the tumor [3][4][5]. Also, due to the unmatured prognostic assessment for TC patients, many patients suffered the overtreatment [6]. Considering the challenges of TC therapy and prognosis, it is of significance to carry out an in-depth exploration into the molecular mechanisms of TC, which is able to provide valuable insights into the developments of therapy and prognostic assessment.
Dysregulation of miRNAs was indicated by many studies to be heavily associated with tumor process [7]. Also, miRNAs are regarded as novel cancer biomarkers [8][9][10]. Among many miRNAs dysregulated in many cancer, miRNA-145 was reported as the tumor suppressor of cervical cancer [11], lung adenocarcinoma [12], and gastric cancer [13]. By bioinformatics analysis, miRNA-144-5p is lowly expressed in TC and implicated with cell adhesion and cell migration [14]. But experimental data are lacking, within a need of experiments to validate the possible impact of miRNA-144-5p on TC progression.
Integrin α3 (ITGA3), a cell surface adhesion molecule, is mainly expressed in the cell membrane. It was reported that ITGA3 can interact with extracellular matrix protein, and its expression is associated with cancer metastasis [15]. Besides, research on the molecular mechanism, results of bioinformatics analysis showed that ITGA3 is regarded as a pivotal biomarker for pancreatic cancer and can facilitate cancer progression through P53/TGF-β-related pathways [16]. Upregulation of ITGA3 exerts a promotive role in the cell proliferation of intrahepatic cholangiocarcinoma and indicates a poor prognosis [17]. Although some mechanisms of ITGA3 in TC were reported [18], the detailed functions and potential mechanisms of ITGA3 in TC are still unknown. Therefore, the study of the regulatory effect of ITGA3 on TC can improve the theoretical construction of ITGA3 as a cancer regulator.
To sum up, the current study focused on the effects of miRNA-144-5p and ITGA3 on TC, and miRNA-144-5p/ ITGA3 regulatory axis was determined by bioinformatics methods and dual-luciferase assay. The findings in this study provided a new theoretical support for TC therapy.  2.3. Cell Transfection. miRNA-144-5p mimics (miRmimics), negative control mimics (miR-NC), pcDNA3.1-ITGA3 plasmid (oe-ITGA3) encoding ITGA3, and blank pcDNA3.1 plasmid (oe-NC) vectors were all procured from RiboBio (China). Complying with the operation instructions, Lipofectamine 2000 kit (Invitrogen, Carlsbad, USA) was in use to transfect mimics/plasmids into TC cell lines TPC-1 and FTC-133. Cells were used for further experiments 24 h after transfection.

Real-Time PCR.
Trizol reagent (Life Technologies, USA) was used for extracting total RNA. The miScript II RT kit (Qiagen, USA) was used to revert miRNA and mRNA to cDNA. The miScript SYBR Green PCR Kit (Qiagen, Germany) was used to measure expression levels of miRNA and mRNA. Then, qRT-PCR was operated on Applied Bio-systems® 7500 Real-Time PCR Systems (Thermo Fisher Scientific, MA). GAPDH was taken as the internal parameter for ITGA3, and U6 for miRNA-144-5p. Primer sequences are exhibited in Table 1. The 2 -ΔΔCt value was employed to assess the relative expression of miRNA-144-5p and ITGA3.
2.5. Western Blot Assay. Radio immunoprecipitation assay lysis buffer (Beyotime, China) was used to extract cell lysates. Quantified by the BCA protein assay kit (Beyotime, China), the proteins were separated by 12% SDS-PAGE and transferred onto the PVDF membrane. 5% skimmed milk powder was harnessed to keep the membrane sealed for 2 h; then, it was incubated overnight with primary antibodies at 4°C. Next, phosphate-buffered saline with Tween® was harnessed to rinse the membrane and it was cultured at room temperature with the secondary antibody for 2 h. Eventually, an enhanced chemiluminescence (ECL) kit (GE Healthcare, USA) was in use to detect protein signals. The primary antibodies rabbit anti-ITGA3 (ab131055, 0.3 μg/ml) and rabbit anti-GAPDH (ab181602, 1 : 10,000) and secondary antibody goat anti-rabbit IgG (ab205718) were provided by Abcam (China).

Transwell Assay.
A 24-well transwell chamber (8 μm aperture, BD Biosciences) was utilized in this assay for migratory ability and invasiveness of cancer cell detection. Coated with Matrigel, the upper chamber was filled with nearly 2 × 10 4 cells. And a medium containing 10% FBS was added to the lower chamber. After 48 h, the invaded cells were fastened with methanol as well as stained with 0.1% crystal violet. Finally, they were photographed under an inverted microscope (DSX510i, Olympus, Japan).

Scratch
Healing Assay. Six-well plates were employed here for cell inoculation. Upon the overall coverage of cells reached 80%, the monolayer of cells was gently scraped with the tip of a 200 μl pipette. After 24 h, the wound areas of cells both in the transfection group and in the control group were measured. Those isolated cells were removed by twice washing with the culture solution. As for the regrowth of cells (24 h), a fresh medium was used. Finally, at 0 h and 24 h, the cell migration was observed and photographed.
2.9. Flow Cytometry. Transfected for 48 h, cells were collected and digested. Then, they were washed twice with PBS, resuspended in binding buffer, and then added with Annexin V-FITC and propidium iodide (PI). Finally, the cell apoptosis was tested by FACS Calibur flow cytometry (Becton Dickinson, CA, USA) and the data were analyzed. 2.11. Statistical Analysis. Analysis of data from 3 independent experiments was conducted on GraphPad Prism 6.0 (La Jolla, CA). The results are expressed as mean ± standard deviation. Comparison between the two groups was Student's t-test, and comparison between multiple groups was one-way analysis of variance used for pretest. Then, the Bonferroni method was used for posttest.

Results
3.1. The Expression of miRNA-144-5p Is Notably Decreased in TC Cells. miRNA-144-5p was pointed out by the previous study as a TC-related miRNA by bioinformatics analysis [14]. However, no practical supports determine its molecular mechanisms in TC. To determine its roles in TC, we intro-duced the experiments in the following. A total amount of 82 DEmiRNAs were screened via differential expression analysis, including miRNA-144-5p which was the focus of our research (Figure 1(a)). Bioinformatics analysis of TCGA TC data indicated that TC was implicated in low miRNA-144-5p expression (Figure 1(b)). miRNA-144-5p expression in TC cell lines was markedly low as manifested in the qRT-PCR result (Figure 1(c)). These findings indicated that miRNA-144-5p was significantly low-expressed in TC.  Reverse primer 5′-ACCACCCTGTTGCTGTAGCCAA-3′

Computational and Mathematical Methods in Medicine
Remarkable overexpression of miRNA-144-5p was observed in these two thyroid cancer cells compared with stimulated transfected cancer cells (Figure 2(a)), indicating these cells could be used in the subsequent experiments. Proliferation of TC cells was dramatically suppressed by miRNA-144-5p overexpression (Figure 2(b)). The invasiveness and mobility    (Figure 2(e)). Collectively, proliferative, migratory, and invasive TC cells could be weakened by overexpression of miRNA-144-5p, while activating apoptosis.

ITGA3 Was Negatively Correlated with miRNA-144-5p
Expression in TC Cells. To explore the downstream targets   (Figure 3(a)). The target genes of miRNA-144-5p were predicted using miRTarBase and Tar-getScan databases. The 3 DEmRNAs sharing binding sites with miRNA-144-5p were obtained by intersecting the predicted targets and the upregulated DEmRNAs (Figure 3(b)). It was revealed by the Pearson correlation analysis that miRNA-144-5p was negatively correlated with ITGA3 with the highest correlation coefficient (Figure 3(c)). Data from TCGA database indicated that ITGA3 was markedly overexpressed in TC tissue (Figure 3(d)). Combined with clinical information, the expression of ITGA3 was markedly different in patients with different T stages (Figure 3(e)) and N stages (Figure 3(f)) of the tumor. mRNA expression of ITGA3 in TC cell lines was significantly higher compared with that in normal cell line (Figure 3(g)). Similarly, the result of the western blot assay demonstrated that the protein level of ITGA3 in TC cell lines was considerably higher in contrast with that in normal cell line (Figure 3(h)). Based on the above results, ITGA3 could be one target of miRNA-144-5p.

miRNA-144-5p Suppresses ITGA3 Expression by
Targeting It. We bioinformatically predicted that on the 3 ′ UTR of ITGA3 existed the binding site of miRNA-144-5p (Figure 4(a)). Afterward, the targeted interaction was determined by dual-luciferase assay, where the luciferase activity was reduced by introducing miR-mimics in the ITGA3-WT group, indicating that the miRNA-144-5p binding site predicted by bioinformatics analysis was correct (Figure 4(b)). It was revealed by the qRT-PCR that overexpressed miRNA-144-5p markedly downregulated ITGA3 mRNA level in TC cells (Figure 4(c)). The ITGA3 protein expression was also notably reduced upon the overexpressing of miRNA-144-5p (Figure 4(d)). It was shown that miRNA-144-5p both reduced the translation and accelerated the mRNA degradation of ITGA3. In a word, these results suggested that miRNA-144-5p downregulated ITGA3 by targeting its 3 ′ UTR region in TC cells.
3.5. miRNA-144-5p Suppresses TC Progression by Targeting ITGA3. When both miRNA-144-5p and ITGA3 were overexpressed, ITGA3 was strikingly upregulated in the miR-NC +oe-ITGA3 group while it was subsequently restored, or even downregulated (Figures 5(a) and 5(b)). CCK-8 assay results  Figure 5(c)). It was uncovered that the invasiveness as well as mobility of TC cells was markedly promoted after ITGA3 was overexpressed. But they were hugely decreased resulting from the simultaneous overexpression of miRNA-144-5p and ITGA3, in contrast with that when ITGA3 was overexpressed alone (Figures 5(d) and 5(e)). The overexpression of ITGA3 could prominently inhibit the cell apoptosis of TC, while the simultaneous overexpression of miRNA-144-5p and ITGA3 could reduce this inhibitory effect ( Figure 5(f)). Thus, miRNA-144-5p partially rescued the phenotype induced by ITGA3 in TPC-1 cells. Overall, ITGA3 is suggested by these results to be a functional target of miRNA-144-5p.

Discussion
The expression status and potential roles of miRNA-144-5p were analyzed previously. For example, Pan et al. [14] pointed out that low expression of 4 miRNAs in TC, including miRNA-144-5p, may affect tumorigenesis through key pathways. Furtherly, it was verified by our study that expression of miRNA-144-5p was markedly downregulated in TC tissue and cells. The tumor-suppressing role of miRNA-144-5p in cell proliferation, migration, and invasion in TC was for the first time revealed by the results, presenting a novelty of our study. According to Rosignolo et al.'s research, miRNA-144-5p could be applied to classify PTC combined with other miRNAs [19], showing a promising potential; however; in our study, we failed to verify miRNA-144-5p expression status and effects among TC subtypes. In conclusion, we expanded the understandings on miRNA-144-5p to some extent based on the previous studies.
This study demonstrated that ITGA3 was possible to be the downstream target of miRNA-144-5p. It was reported that ITGA3 is highly expressed in different cancer types [16,17]. Moreover, highly expressed ITGA3 was proved to stimulate the proliferation and metastasis of tumor cells in former studies [20]. Liu et al. [18] revealed that the growth of TPC cells was able to be reduced by miRNA-524-5p targeting FOXE1 and ITGA3. However, the detailed functions of ITGA3 in TC are still incomplete. Through cell biology experiments, the expression of ITGA3 was verified to be upregulated in TC cells, and the overexpression of ITGA3 enhances the proliferative, migratory, and invasive ability while weakening cell apoptosis of TC cells. Additionally, dual-luciferase assay results further verified the binding relationship between ITGA3 and miRNA-144-5p. And it was also uncovered that miRNA-144-5p attenuated the proliferative, migratory, and invasive ability and promoted cell apoptosis by targeting ITGA3. These conclusions provided a molecular mechanism for the migration and invasion of TC, helping to develop new therapeutic methods for TC.
In conclusion, we demonstrated in this study that miRNA-144-5p inhibited the progression of TC cells. And that this effect was mediated, at least in part, by ITGA3. Although we have initially explored the role of miRNA-144-5p/ITGA3 in the TC cell phenotype, further elucidation of its downstream pathways in thyroid cancer is indispensable, which is crucial for the development of effective diagnosis and treatment of thyroid cancer.

Data Availability
The [DATA TYPE] data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no potential conflicts of interest.