MicroRNA-367 directly targets PIK3R3 to inhibit proliferation and invasion of oral carcinoma cells

Recently, microRNA-367 (miR-367) has been reported to function as both tumor suppressor and oncogene in several cancer types, including gastric cancer, hepatocellular cancer and lung cancer. However, the biological function of miR-367 and its precise mechanisms in oral squamous cell carcinoma (OSCC) have not been well clarified. The aim of the present study was to study the roles of miR-367/PIK3R3 axis in OSCC. The levels of PIK3R3 and miR-367 were detected by quantitative PCR assay in OSCC tissues and cell lines. Moreover, the biological roles of miR-367 and PIK3R3 in OSCC cells were assessed by cell proliferation and invasion. The mRNA and protein levels of PIK3R3 were determined by using quantitative PCR and Western blotting assays. Luciferase assays were used to confirm that PIK3R3 was one target of miR-367. In the present study, the miR-367 level was dramatically reduced in OSCC tissues and cell lines, and the PIK3R3 expression was significantly enhanced. What’s more, the PIK3R3 expression was negatively related to the miR-367 level in OSCC tissues. Furthermore, up-regulation of miR-367 obviously restrained OSCC cells proliferation and invasion. We confirmed that miR-367 could directly target PIK3R3 by luciferase reporter assay. Besides, knockdown of PIK3R3 also could markedly inhibit the proliferation and invasion of OSCC cells. Finally, overexpression of miR-367 in OSCC cells partially reversed the promoted effects of PIK3R3 up-regulation. Overexpression of miR-367 restrained OSCC cells proliferation and invasion via regulation of PIK3R3.


Introduction
Oral squamous cell carcinoma (OSCC) has high incidence rate, and is a malignant tumor of oral maxillofacial region [1,2]. In recent years, advanced technologies have been developed in both experimental and clinical fields. Due to invasive characteristics and highly malignancy, the prognosis of OSCC is still unfavorable. Moreover, the 5-year survival rates are still less than 50%, and have not been improved in the last 3 decades [3][4][5]. Up to now, it is unable to meet the needs of the patients for the traditional treatment method, and then it needs new therapeutic strategies to improve the prognosis of OSCC patients. Therefore, in order to identify useful biomarkers and novel therapeutic targets, it is critical to find out the biological mechanisms of OSCC.
MicroRNAs (miRNAs) are a family of endogenous, small noncoding RNAs. They regulate the translation or induce degradation of specific protein coding genes through binding to the 3 -untranslated regions of the mRNA [6]. According to the bioinformatic analysis, it predicted that miRNAs targeted more than 60% of human genes [7]. Previous reports demonstrated that altered miRNAs expressions were participated in tumorigenesis and the development of various cancers [8][9][10]. Thus, miRNAs are thought to be markers of cancers diagnosis, progression and prognosis [11]. Many human miRNAs have been confirmed to be dysregulated in OSCC, including miR-488, miR-199a, miR-211, miR-106a and miR-16 [12][13][14][15][16]. Up to now, miR-367 has been reported to function as an oncogene in melanoma, non-small cell lung cancer, osteosarcoma [17][18][19], or function as a tumor suppressor in gastric cancer [20], the functions of miR-367 in OSCC were rare unexplored previously. Therefore, we investigated the functional roles and its mechanisms of miR-367 in OSCC.
Here, in order to investigate the functional role of miR-367 in OSCC, we detect the miR-367 level in OSCC tissues and cell lines. Next, we predicted that miR-367 directly targeted PIK3R3 according to the online database TargetScan 7.2. For further study, we explored the relationship between miR-367 and PIK3R3 in OSCC tissues. At last, the effects of miR-367 overexpression or PIK3R3 silencing on proliferation and invasion of OSCC cells were determined.

Human tissue samples
Thirty pairs of human OSCC tissues and their adjacent non-cancer tissues were collected from patients at the Beijing Tongren Hospital Affiliated to Capital Medical University between Feb 2017 and June 2018. All samples were immediately frozen in liquid nitrogen for subsequent quantitative RT-PCR and Western blot analysis. All participants signed written informed consent. The present study was approved by the Ethical Committee of Beijing Tongren Hospital Affiliated to Capital Medical University (BJTRH2017012212) and complied with the guidelines and principles of the Declaration of Helsinki.

Isolation of RNA and quantitative polymerase chain reaction analysis
Total RNA from OSCC cells were extracted using TRIzol (Invitrogen, U.S.A.) following the manufacturer's protocols. MiRNA-specific RT primers (RiboBio, Guangzhou, China) for miR-367 and random primers (TaKaRa, Dalian, China) for mRNAs were synthesized. Quantitative polymerase chain reaction (qPCR) was used to measure Reverse-transcribed cDNA with SYBR Green PCR Kit (QIAGEN, Shanghai, China) under the following conditions: predenaturation at 95 • C for 5 min, denaturation at 95 • C for 10 s, annealing and extension at 60 • C for 30 s, the followed steps were running for 40 cycles. The relative miRNA and mRNA expression levels were normalized by U6 and GAPDH, respectively. The relative expression levels of miR-367, PIK3R3, CDK2, CDK4, cyclin D1, cyclin E1, p21, p27, MMP-2, MMP-9 and TIMP-1 were normalized to those of internal control U6 or GAPDH using the comparative delta CT (2 − Ct ) method. Prime sequences are shown in Table 1.

Luciferase reporter assay
The luciferase reporter vectors (pGL3-PIK3R3-3 UTR WT and pGL3-PIK3R3-3 UTR MUT) were synthesized by GenePharma. SCC1 cells were seeded into 24-well plates and transfected with pGL3-PIK3R3-3 UTR WT or pGL3-PIK3R3-3 UTR MUT, along with miR-367 mimics or miR-NC using Lipofectamine 3000 per the manufacturer's instructions. After transfection for 48 h, luciferase reporter assays were performed with the Promega Dual-Luciferase Reporter Assay System. The relative firefly luciferase activities were measured by normalizing to renilla luciferase activities.

Statistical analysis
The data were expressed as the means + − standard error of the mean (S.E.M.). The number of independent experiments was represented by "n". Correlations between miR-367 and PIK3R3 levels were analyzed using Pearson's correlation coefficient. Two-tailed Student's t-test was used for other comparisons. The associations between miR-367 level and clinicopathological features were analyzed using χ 2 test. P<0.05 was considered statistically significant.  Statistical analyses were performed by the χ 2 test. *P<0.05 was considered significant.

High level of miR-367 in OSCC tissues and cells
In the present study, the miR-367 level in OSCC tissues and cells were detected by using qRT-PCR. Our findings demonstrated that the miR-367 level in the OSCC tissues was lower than that in the adjacent tissues ( Figure 1A). In addition, as we saw in Table 2, miR-367 was highly related to stage and tumor size. The results above indicated that miR-367 was correlated with OSCC progression. Next, the data further confirmed that the miR-367 level was lower in SCC1 cells than that in the other five OSCC cell lines ( Figure 1B). Therefore, SCC1 cells were used in the following experiments.

The effect of miR-367 on proliferation of OSCC cells
After transfection with miR-367 mimic or inhibitor, the results showed that the miR-367 level was significantly up-regulated or down-regulated in a miR-367 mimic or inhibitor group compared to a miR-NC group (Figure 2A), respectively. To study the role of miR-367 in regulating SCC1 cells proliferation, our results suggested that knockdown of miR-367 significantly promoted SCC1 cells proliferation ( Figure 2B). However, transfection with miR-367 mimic suppressed the cell proliferation of SCC1 cells compared with the miR-NC group ( Figure 2B). Next, overexpression of miR-367 reduced expressions of cyclin D1, cyclin E1, CDK2, CDK4 and enhanced the expressions of p21, p27 at the protein and mRNA levels ( Figure 2C). In addition, down-regulation of miR-367 had opposite effects on regulating expressions of these cell cycle genes ( Figure 2C).

The effects of miR-miR-367 on invasion in OSCC cells
Compared with the miR-NC group, the results from Transwell assays showed that increased miR-367 level significantly reduced the number of invading OSCC cells ( Figure 3A). However, miR-367 inhibitor could promote cell invasion ability compared with anti-miR-NC group ( Figure 3A). In addition, we detected MMP-2, MMP-9 and TIMP-1 expressions by qRT-PCR and Western blot. The results indicated that MMP-2 and MMP-9 expressions were markedly decreased by enhancing miR-367 level in SCC1 cells ( Figure 3B,C), while expression of TIMP-1 was dramatically increased ( Figure 3B,C). However, reduced miR-367 level up-regulated the expressions of MMP-2, MMP-9 and down-regulated TIMP-1 expression ( Figure 3B,C).

miR-367 directly targeted PIK3R3 3 UTR
Actually, we found a miR-367-binding site in the 3 UTR of PIK3R3 by using TargetScan 7.2 online database ( Figure  4A). Then, we validated PIK3R3, a critical oncogene, is a direct target of miR-367 by luciferase reporter assay. Introduction of miR-367 significantly suppressed WT PIK3R3 reporter activity but not the activity of the mutated reporter construct in SCC1 cells, suggesting that miR-367 could specifically target the PIK3R3 3 UTR by binding to the seed sequence ( Figure 4B). Up-regulation of miR-367 could obviously reduce PIK3R3 expression, whereas down-regulation of miR-367 enhanced PIK3R3 expression ( Figure 4C). These results suggested that miR-367 directly targeted PIK3R3 through 3 UTR sequence binding. Next, the PIK3R3 expression was detected by qRT-PCR in OSCC tissues. Our results demonstrated that the PIK3R3 expression was obviously up-regulated in OSCC tissues compared with the adjacent tissues ( Figure 4D). Next, we also determined the PIK3R3 expression in five OSCC cell lines (such as SCC1, SCC4, H157, Cal-27, HSC-2) and a human normal oral keratinocyte cell culture (NHOK). The PIK3R3 expression in SCC1 cells was higher than that in other five cell lines ( Figure 4E). Finally, the Pearson's correlation analysis revealed a significant inverse correlation between PIK3R3 and miR-367 in OSCC tissues ( Figure 4F). From the above data, we predicted that miR-367 might negatively regulate PIK3R3 expression.

Knockdown of PIK3R3 restrained OSCC cells proliferation and invasion
To investigate the functional roles of PIK3R3 in OSCC cells, the proliferation and invasion of OSCC cells were detected after transfection with si-NC or si-PIK3R3. Transfection with si-PIK3R3 significantly decreased the PIK3R3 expression in SCC1 cells compared with the si-NC group ( Figure 5A). Then, we found that silencing PIK3R3 evidently suppressed the proliferation of SCC1 cells ( Figure 5B). Moreover, down-regulation of PIK3R3 reduced the expressions of cyclin D1, cyclin E1, CDK2, CDK4, and enhanced the expressions of p21, p27 ( Figure 5C). Next, Transwell assay revealed that decreased PIK3R3 expression suppressed invasion of SCC1 cells ( Figure 5D). For further study, knockdown of PIK3R3 markedly down-regulated expressions of MMP-2, MMP-9 and up-regulated expression of TIMP-1 in SCC1 cells ( Figure 5E). Consequently, silencing PIK3R3 remarkably restrained OSCC cells proliferation and invasion.

Up-regulation of miR-367 markedly reversed the effects of PIK3R3 overexpression on the proliferation and invasion of OSCC cells
To determine whether miR-367 regulated the proliferation and invasion of OSCC cells by directly targeting PIK3R3, we cotransfected pcDNA3.1 or pcDNA-PIK3R3 with or without miR-367 mimic into SCC1 cells ( Figure 6A). In-     troduction of miR-367 abrogated the promoted effect of PIK3R3 up-regulation on cell proliferation ( Figure 6B). At the same time, expressions of cyclin D1, cyclin E1, CDK2, CDK4 were decreased and expressions of p21, p27 were increased in PIK3R3-overexpressing SCC1 cells after exogenous up-regulation of miR-367 ( Figure 6C). Next, the data showed that introduction of miR-367 partially reversed the invasion of SCC1 cells promoted by PIK3R3 overexpression (Figure 6D), through decreasing MMP-2, MMP-9 expressions and increasing TIMP-1 expression ( Figure 6E). Hence, the promoted effects of PIK3R3 overexpression were reversed by miR-367 mimic. Altogether, all above results suggested that overexpression of miR-367 restrained OSCC cells proliferation and invasion via directly down-regulating PIK3R3 expression.

Discussion
miRNAs serve as important regulatory factors, which affects OSCC progression. Overexpression of miR-488 suppressed OSCC cell invasion and EMT via regulating ATF3 expression [12]. Wei et al. indicated that up-regulation of miR-199a inhibited OSCC cell proliferation and induced OSCC cell apoptosis through regulating the IKKβ/NF-κB signaling pathway [13]. Numerous studies have found that miR-367 not only affected cell proliferation, but also was closely associated with prognosis in cancers including osteosarcoma, melanoma, gastric cancer and non-small cell lung cancer [17][18][19][20].
In the present study, our results showed that the miR-367 level was down-regulated in OSCC tissues compared with the adjacent normal tissues. And miR-367 level was also down-regulated in OSCC cell lines compared with NHOK cells. Moreover, we explored the functional roles of miR-367 in OSCC cells. First, we found that overexpression of miR-367 significantly inhibited OSCC cell proliferation than the cells transfected with anti-miR-NC, whereas down-regulation of miR-367 promoted OSCC cell proliferation. Cell cycle regulation involved complex events. Such events revealed that cell cycle related proteins provided a promising mechanism for the inhibition of growth [21,22]. An earlier study suggested that the up-regulation of cyclin-dependent kinases (CDKs) and cyclin (D1 and E1) were participated in cell cycle progression and arrested cells at the G0/G1 phase [22]. The p21 and p27 has been reported as an inhibitor of CDK protein with an anti-proliferative effect on mesangial cells [23]. Here, we also found that introduction of miR-367 reduced expressions of CDK2, CDK4, cyclin D1 and cyclin E1 while it enhanced p21 and p27 expressions.
Invasion is one process of metastasis. Here, the data indicated that increased or decreased miR-367 level significantly inhibited or enhanced the invasive ability of SCC1 cells compared with the control group. Furthermore, one of very important steps for cancer cell invasion is that proteolytic enzymes degrade the extracellular matrix (ECM) components [24]. And the MMPs degrading ECM process contributes to cancer cells angiogenesis, invasion and metastasis [25][26][27]. Both MMP-2 and MMP-9 degrade components of the basement membrane to promote cancer invasion [26,28,29]. Moreover, it is reported that the imbalance between TIMPs and MMPs is important in early events of tumor progression [30]. Next, up-regulation of miR-367 significantly decreased expressions of MMP-2, MMP-9, and increased the TIMP-1 expression in SCC1 cells, whereas down-regulation of miR-367 enhanced both MMP-2 and MMP-9 expressions and reduced TIMP-1 expression. All above results indicated that miR-367 restrained cell proliferation and EMT of OSCC.
It is well known that miRNAs perform their function by regulating the expression of its target gene. Thus, we explore the functional target gene for miR-367 that was involved in OSCC progression. Targetscan and luciferase reporter assay suggested that PIK3R3 might be the functional target gene of miR-367. Furthermore, increased expression or knockdown of miR-367 significantly inhibited or promoted PIK3R3 expression, respectively. PIK3R3 is an oncogene and participated in development and progression of multiple cancers [31][32][33][34][35][36]. In our study, we also observed that PIK3R3 protein and mRNA expressions were frequently at high expression in tumor tissue when compared with its paired non-tumor tissues, which agreed with previous studies in ovarian cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer and glioma malignancy [31][32][33][34][35][36]. Hence, our result indicated that PIK3R3 expression may play a cardinal role in tumorigenesis of OSCC. Besides, miR-367 level was inversely correlated with PIK3R3 expression in OSCC. Moreover, the data showed that Silencing PIK3R3 could restrain the proliferation and invasion of OSCC cells. Next, our results showed that introduction of miR-367 inhibited the proliferation and invasion of OSCC cells promoted by overexpression of PIK3R3. Our findings demonstrated that miR-367 might act as a tumor suppressor in OSCC by directly targeting PIK3R3.