MiR-223 down-regulates RHOB to inhibit progression of papillary thyroid cancer

Backgrounds The incidence of thyroid carcinoma continues to increase every year. The incidence of papillary thyroid carcinoma (PTC) is the largest of all thyroid cancers, and is the most widely seen. Thus, Do some identification and testing of biomarkers related to tumorigenesis and development, which will help understand and treat PTC. Methods The expression of miR-223 and RHOB needs to be detected by polymerase chain reaction and western blot, respectively. Thyroid cell-line BCPAP and K1 cells were cultured, transfected for overexpression of miR-223or RHOB, and evaluated using MTT, Transwell, and apoptosis assays. In order to better understand the relationship between miR-223 and RHOB, dual luciferase reporter gene detection and RHOB rescue experiments were performed. In addition, clinical pathological parameters and correlation analysis of patients with miR-223 were also performed. Results Aberrant expression of miR-223 was detected in PTC samples. We also determined that miR-223 expression was associated with TNM staging and cervical metastasis. Moreover, miR-223 inhibits the increase in cell number and position, as well as defense against cancer cells and accelerates cell death. Furthermore, miR-223 excessive expression was associated with a significant inhibition of RHOB levels. According to the luciferase test report, it can be known miR-223 was able to bind RHOB 3′-UTR, it is possible to change its stability through these, and finally achieve the purpose of reducing RHOB. Finally,excessive expression of RHOB has undergone earth-shaking changes in the effect of miR-223 overexpression on cell number growth, invasion and disappearance. Conclusion miR-223/RHOB expression took part in PTC make headway and

miR-223 inhibition caused RHOB overexpression and cancer progression. Therefore, miR-223 may be a potential therapeutic target for PTC.

Background
The incidence of thyroid cancer has also become higher and higher, but the incidence of thyroid papillary cancer (PTC) has been ranked first [1]. In addition, the fifth most common tumor in women is PTC [2,3]. The biological behavior of different PTCs varies widely. Some exhibit few clinical symptoms while others are aggressive and may be fatal. This phenomenon is associated with different genetic abnormalities [4]. Therefore, Do some identification and testing of biomarkers related to tumorigenesis and development, which will help understand and treat PTC.
Microrna (miRNAs) are a class of non-coding rna that can be directly contacted with the 3'-untranslation region (3'-UTRs) of a gene to affect the expression of a proteincoding gene [5,6]. Coding for this gene the miRNA miR-223 in humans is on the X chromosome to q12 this site [7]. Recently, miR-223 phenotypes have been reported in both hematological malignancies [8][9][10][11][12] and in solid tumors, such as breast cancer [13,14], gastric cancer [15,16], hepatic cancer [17,18], and colon cancer 4 cancer [27][28][29]. Whereas, it is still not very clear about the impact of RHOB on PTC in humans.. In the current study, we attempted to identify any inhibiting act of miR-223 in human PTCs and to clear the potential mechanisms involved. The results may help define the process of tumorigenesis, identify a potential biomarker for PTC progression, and determine if miR-223 may be Potential therapeutic targets for PTC a treatment site that is not obvious to PTC.  According to the manufacturer's instructions, we can use the dual luciferase reporter assay to speculate on luciferase activity.Implanted miR-223 mimics or BCPAP and K1 cells used for reverse control were placed in 96-well plates.When 12 h have passed, the cells were furtherinfection 50 ng pGL3-WT-3′-UTR-Rhob or pGL3-Mut-3′-UTR-Rhob using Lipofectamine 2000 reagent. When it has been cultivated for 24 hours, the luminescence was discovered using a Dual-Luciferase

Study subjects and patientorganized system specimens
Reporter assay system (Promega, USA).

Apoptosis assays
The apoptosisof BCPAP or K1 cells were detection by flow cell flow meter. Briefly, for Annexin V dyeing, 5 µL phycoerythrin-Annexin V, 5 µL propidium iodization(BD Pharmingen), and 300 µL 1 × binding cushion were incubatedwith the cells allow to stand for 15 minutes at room temperature. The samples were then analysed within 1 h of staining flow cytometry (FACS Canto II, BD Biosciences) was used. Data processing is performed by FlowJo software.The test were repeated three times.
Colorimetric caspase-3 assays BCPAP and K1 splitting cells with RIPA bufferand then their protein chromas were resoluted by Bradford assay. Every 100 µg of protein was dealed with 10 µL Ac-DEVD-pNA (Abcam, USA) and incubate for 2 hours at 37 degrees Celsius. The absorbance at 405 mm was measured by a microplate reader (Bio-Tek instrument).

Statistical Analysis
The above experiments were performed three times. SPSS 17.0 software (SPSS, Inc., Chicago, USA) was employed to perform the statistical analyses. The representation of the data is: mean ± standard deviation. Statistical differences can be analyzed using Student's t test or one-way variance. P < 0.05 is a statistically significant difference.

Aberrant expression of miR-223 in human PTC organizations
To evaluate the utterance of miR-223 in papillary thyroid carcinoma (PTCs), 47 paired PTC and adjacent non-tumor tissue samples were examinedusingqRT-PCR.
Thirty-eightof the PTC specimens were found to have lower miR-223 expression than the adjacent non-tumor tissue specimens ( Figure 1A).We alsoobserved that PTC specimens exhibited lower levels of miR-223 expressionthan the paired adjacent non-tumor tissues ( Figure 1B).

MiR-223 may have inhibited PTC progression and metastasis in patients
After determining the utterance of miR-233 in PTC organizations, we then analyzed the correlation between miR-223 and the clinicopathological parameterscan be obtained from clinical experience. We can get the results from Table 1. The expression of miR-233 is not affected by age, sex, and tumor size., multicentricity, or extrathyroidal invasion. However, lower miR-223 levels were detected in patients with advanced TNM stages and with cervical lymph node metastasis. All the above results reflect that the evolution and transfer of PTC may be affected by miR-233.

Overexpression of miR-223 in PTC cells restrained proliferation and migration and promoted apoptosis PTC cells in vitro
MTT assays were used to evaluate the effect of overexpression of miR-223 on the proliferation of BCPAP and K1 cells. We transientlytransfer infection BCPAP and K1 cells with miR-223 mimic or negative control (Figure 2A). The forced overexpression of miR-223 inhibited the development of BCPAP and K1 cells emulated to that observed in negative control cells in a time-dependent manner ( Figure 2B). The migration potential of BCPAP and K1 cells overexpressing miR-223 can be identified by transwell migration assay.Forced overexpression of miR-223lead todecreased cell invasion (BCPAP cells, P = 0.035; K1 cells, P = 0.022; Figure 2C). Tofix the effect of miR-223 overexpression on cell apoptosis, Annexin-V staining was performed.
Overexpression of miR-223 in PTC cells significantly promoted cell apoptosis (Fig.   2D). Similar results were observed for the colorimetric caspase 3 assays (Fig.   2E).Collectively, these dataindicated that the overexpression of miR-223 inhibits proliferation and invasion of PTC, accelerates cell deathin vitro.

MiR-223 directly inhibited RHOB expression in vitro
Based on the bioinformatics analyses (miRWalk,TargetScan, and PicTar), RHOB was predictedto bea potential mark of miR-223. MiR-223 overexpression in PTC cellswas related to a significantreduce in RHOB expression beside that in the negative control cells ( Figure 3A). Moreover,The relationship between miR and RHOB can be judged by measuring the luciferase reporter gene. Construct a cell that can transfer the infection, which contains the RHOB wild type or the 3'-UTR luciferase reporter plasmid (as shown in Figure 3B). When the wild-type RHOB 3'-UTR plasmid was transfected with the miR-223 mimetic, the activity was significantly inhibited.
However, the amount of miR-223 contained did not affect the luciferase activity of the reporter gene containing the mutant 3'-UTRs (as shown in Figure 3C).From the above results can be derived that RHOB was a first-hand mark of miR-223 in PTC.

RHOB overexpression reversed the result of miR-223 overexpression
To promotedetermine the RHOB-mediated result of miR-223 in PTC, a RHO-specific overexpression vector was transfected into BCPAP and K1 cells overexpressing miR-223 (Fig. 4A) Fig. 4D and 4E). These data suggested that miR-223 inhibited PTC progression by inhibiting RHOB.

Discussion
PTC is common in all diagnosed thyroid cancers, accounting for 80% of the rate and RHOB promotes colon cancer progression [29][30][31]. In the latest research, we also used the BCPAP and K1 cells to investigate the relationship between miR-223 and RHOB. MiR-223 overexpression is associated with significant inhibition of RHOB content. Furthermore, We can get from the luciferase test report that miR-223 was able to bind RHOB 3′-UTR. It has a certain influence on its stability, which eventually leads to a decrease in RHOB content. Furthermore, we proved that the overexpression of RHOB deteriorate the acts of miR-223 overexpression on cell proliferation, invasion, and apoptosis.
Overall, our results showed that the aberrant miR-223/RHOB expression was related to PTC progression. The miR-223 inhibition caused RHOB overexpression and cancer progression. Thence, miR-223 is perhaps a potential therapeutic mark for PTC.

Ethics approval and consent to participate
The research had been supported by the Ethics Committee of Shengjing Hospital.
Relevant clinical information was gathered from patients' records and informed written consent had been gained. The papillary thyroid carcinoma cell lines BCPAP and K1 were obtained from Cell bank of typical culture preservation Committee of Chinese Academy of Sciences, and did not require ethics approval for their use in this study.

Consent to publishcation
Not applicable

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Authors' Contributions
XZ designed the study and drafted the manuscript. WJ reviewed the article. WZ condected the experiments. All authors have read and approved the final manuscript.

Competing interests
The authors declare there isn't any conflict of interest.

Funding
The National Natural Science Foundation of China gave a lot of support to the paper (grant No.81302364).