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), which restored RHOB expression (59.22± 4.58 vs. 30.12± 3.25 in BCPAP transfected only by mocks,P=0.028; 47.71± 2.91 vs. 28.58± 2.21 in K1 cells transfected with mimic alone, P=0.048) Wediscovered that RHOB overexpression restored the inhibitoryresult of miR-223 on proliferation and invasion in miR-223-overexpression cells (Fig. 4B and 4C). A time-dependent increase in cell proliferation was observed in the BCPAP and K1 cells also overexpressing RHOB compared tothat in BCPAP and K1 cells overexpressing miR-223 (10.8± 0.7, 14.2± 2.1, 24.5± 3.7, and 39.4± 5.1% at 24, 48, 72, and 96 h in BCPAP cells, respectively, P =0.031; 4.5± 0.3, 17.4± 2.5, 22.2± 3.1, and 30.7± 4.3% at 24, 48, 72, and 96 h in BCPAP cells, respectively, P =0.044). Furthermore, RHOB overexpression obviously alleviated apoptosis (Fig. 4D and 4E). These data suggested that miR-223 inhibited PTC progression by inhibiting RHOB.