MicroRNA-92a Promotes Cell Proliferation in Cervical Cancer via Inhibiting p21 Expression and Promoting Cell Cycle Progression.

MicroRNA-92a (miR-92a) generally plays a promoting role in human cancers, but the underlying mechanism in cervical cancer remains unclear. Here we studied the expression and clinical significance of miR-92a in cervical cancer, as well as the regulatory mechanism in the proliferation of cervical cancer cells. Our data indicated that miR-92a was significantly upregulated in cervical cancer tissues compared to their matched adjacent nontumor tissues (ANTs), and the increased miR-92a levels were significantly associated with a higher grade, lymph node metastasis, and advanced clinical stage in cervical cancer. In vitro study revealed that inhibition of miR-92a led to a significant reduction in the proliferation of HeLa cells via induction of cell cycle arrest at the G1 stage. In contrast, overexpression of miR-92a markedly promoted the proliferation of HeLa cells by promoting cell cycle progression. Further investigation revealed that miR-92a has a negative effect on protein levels, but not the mRNA levels, of p21 in HeLa cells, suggesting that p21 is a direct target of miR-92a. Overexpression of p21 eliminated the promoting effects of miR-92a on the proliferation and cell cycle progression of HeLa cells. However, knockdown of p21 reversed the suppressive effects of miR-92a downregulation on HeLa cell proliferation and cell cycle progression. Moreover, p21 was significantly downregulated in cervical cancer tissues compared to ANTs, suggesting that the increased expression of miR-92a may contribute to the decreased expression of p21, which further promotes cervical cancer growth. In conclusion, our study demonstrates that miR-92a promotes the proliferation of cervical cancer cells via inhibiting p21 expression and promoting cell cycle progression, highlighting the clinical significance of miR-92a in cervical cancer.

INTRODUCTION

Cervical cancer is the third most common cancer in women worldwide, especially in developing countries like China (1). Moreover, it is the fourth most frequent cause of cancer-related deaths in females (1). Despite improvements in the diagnosis and treatment of cervical cancer, the overall survival time of patients is still not satisfactory (2). Therefore, revealing the underlying mechanism of cervical cancer progression may contribute to developing effective therapeutic strategies for this disease.

MicroRNAs (miRs), a kind of short noncoding RNA, can cause mRNA degradation or translation inhibition via directly binding to the 3′-untranslational region (UTR) of their target genes (3). Various miRs have been reported to participate in various biological processes, including cell survival, proliferation, differentiation, cell cycle progression, as well as tumorigenesis (4–6). Moreover, deregulation of miRs has been implicated in the development and progression of a variety of malignancies including cervical cancer (7,8). Some miRs play promoting or suppressive roles in cervical cancer by regulating the expression of tumor suppressors or oncogenes, which make them become potential diagnostic and therapeutic targets for this disease (9–11). For instance, miR-101 was found to inhibit the proliferation of cervical cancer cells by targeting Fos, leading to cell cycle arrest at the G1 stage (12). miR-143, which is significantly downregulated in cervical cancer, was reported to promote cervical cancer cell apoptosis and inhibit tumor formation by targeting Bcl-2 (13). However, the underlying mechanism of miRs in the development and progression of cervical cancer still needs to be fully uncovered.

miR-92a, a member of the miR-17-92 family, has been reported to play an oncogenic role in some common human cancers (14–16). For instance, He et al. reported that miR-92a could promote the proliferation of pancreatic cancer cells by directly targeting DUSP10 and activating the JNK signaling pathway (17). Ke et al. found that miR-92a had a promoting effect on colorectal cancer metastasis by inhibiting the protein expression of PTEN and promoting activity of the PI3K/AKT pathway (14). Recently, miR-92a was found to promote cervical cancer cell proliferation and invasion by targeting FBXW7 (18). However, at the same time, miR-92a may function by targeting other tumor suppressors in cervical cancer cells.

Therefore, we aimed to study the regulatory mechanism of miR-92a in cervical cancer progression.

MATERIALS AND METHODS

Tissue Collection

This study was approved by the Ethics Committee of First Affiliated Hospital of Xiamen University, Xiamen, P.R. China. We collected 74 cervical cancer tissues and their matched adjacent nontumor tissues (ANTs) at our hospital. Written informed consent was obtained from all studied patients, and their clinical information is summarized in Table 1. No patients received radiation therapy or chemotherapy before surgical resection. The tissue samples were immediately snap frozen in liquid nitrogen after surgical resection and stored at −80°C until use.

Table 1

Association Between miR-92a Expression and Clinicopathological Characteristics of Patients With Cervical Cancer

Variables	No.	Low miR-92a	High miR-92a	p Value
Age				0.45
<55	30	13	17
≥55	44	23	21
Tumor size				0.082
≤4 cm	46	26	20
>4 cm	28	10	18
Differentiation				0.013
Well moderate	54	31	23
Poor	20	5	15
Clinical stage				0.019
I–II	39	24	15
III–IV	35	12	23
Lymph node metastasis				0.024
No	48	28	20
Yes	26	8	18
Distant metastasis				0.073
No	62	33	29
Yes	12	3	9

Real-Time qPCR

Total RNA was extracted using TRIzol reagent (Thermo Fisher, USA) according to the manufacturer’s instruction. The total RNA (1 μg) was then converted into cDNA using PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Tokyo, Japan). For detection of miR-92a expression, qPCR was performed using miRNA Q-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) on an ABI 7500 thermocycler, according to the manufacturer’s instruction. U6 gene was used as an internal control. For examining the mRNA expression, SYBR Green I Real-Time PCR kit (Biomics, Nantong, P.R. China) was used to perform qPCR on an ABI 7500 thermocycler (Thermo Fisher), according to the manufacturer’s instruction. GAPDH was used as an internal control. The PCR steps were 95°C for 10 min, and 40 cycles of denaturation at 95°C for 15 s and annealing/elongation step at 60°C for 60 s. The relative expression was analyzed by the 2−ΔΔCt method (19).

Cell Culture and Transfection

Human cervical cancer HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, P.R. China. Cells were cultured in DMEM (Thermo Fisher) supplemented with 10% fetal bovine serum (Thermo Fisher) in a 37°C humidified atmosphere of 5% CO2. Cells were transfected with scramble miR mimic (miR-NC), miR-92a mimic, NC inhibitor, and miR-92a inhibitor, or cotransfected with miR-92a mimic and pcDNA3.1-p21 expression plasmid, using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer’s instruction.

Western Blot

Cells were lysed with ice-cold lysis buffer [50 mM Tris-HCl, pH 6.8, 100 mM 2-ME, 2% (w/v) SDS, 10% glycerol]. After centrifugation at 20,000 × g for 10 min at 4°C, proteins in the supernatants were quantified and separated with 10% SDS-PAGE. Proteins (50 μg) were then transferred onto a polyvinylidene difluoride membrane (Amersham Bioscience, Buckinghamshire, UK), which was then incubated with PBS containing 5% milk overnight at 4°C. Afterward, the membrane was incubated with primary antibodies including p21 and GAPDH (all from Abcam, Cambridge, MA, USA) at room temperature for 3 h, respectively. After washing with PBS three times, the membrane was incubated with secondary antibody (Abcam) at room temperature for 1 h. After being washed with PBS three times, Super Signal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, USA) was used to detect signals, according to the manufacturer’s instruction. The relative protein expression was represented as the density ratio versus GAPDH.

MTT Assay

HeLa cell suspension (5 × 104 cells/well) was plated in a 96-well plate and cultured for 0, 24, 48, or 72 h. MTT (10 μl, 5 mg/ml) was added into each well and then incubated at 37°C for 4 h. The supernatant was removed, and 100 μl of DMSO was added into each well. The absorbance at 570 nm was determined using the Model 680 Microplate Reader (Bio-Rad, USA).

Cell Cycle Distribution Analysis

Cells (1 × 106) were washed twice with DPBS, resuspended in 70% ethanol, and fixed overnight at −20°C. Afterward, cells were washed twice in PBS with 3% BSA and incubated for 30 min at room temperature in propidium iodide (PI) staining buffer containing 3% BSA, 40 μg/ml PI, and 0.2 mg/ml RNase in PBS. DNA content analyses were carried out using flow cytometry (FACSCalibur; Beckman Coulter).

Statistical Methods

Data in this study are expressed as the mean ± SD of three independent experiments. The difference between two groups was analyzed using Student’s t-test. SPSS.18.0 was used to perform statistical analysis. A value of p < 0.05 was considered statistically significant.

RESULTS

miR-92a Is Upregulated in Cervical Cancer

In the present study, we first performed real-time RT-PCR to examine the miR-92a expression in 74 cases of cervical cancer tissues and their matched ANTs. Our data showed that the miR-92a levels were significantly higher in cervical cancer tissues when compared with those in matched ANTs (Fig. 1). Accordingly, we speculated that miR-92a might play a role in cervical cancer.

Figure 1

Real-time RT-PCR was performed to examine the miR-92a expression in cervical cancer tissues and their matched adjacent nontumor tissues. **p < 0.01 versus Adjacent.

Increased Expression of miR-92a Is Associated With Cervical Cancer Progression

To further confirm our speculation, we studied the clinical significance of miR-92a in cervical cancer. On the basis of the mean value of the miR-92a level, all patients involved in this study were divided into two groups: the high miR-92a expression group and the low miR-92a expression group. Statistical analysis indicated that high expression of miR-92a was significantly associated with high-grade lymph node metastasis as well as advanced clinical stage (Table 1). In addition, the expression of miR-92a was not associated with age, tumor size, or distant metastasis (Table 1). According to these findings, we demonstrated that increased expression of miR-92a is associated with malignant progression of cervical cancer.

miR-92a Promotes the Proliferation of Cervical Cancer Cells via Enhancing Cell Cycle Progression

To further investigate the potential role of miR-92a in cervical cancer, HeLa cells were transfected with the miR-92a inhibitor to knock down its expression. Real-time RT-PCR data indicated that transfection with the miR-92a inhibitor significantly decreased the miR-92a levels when compared to the NC inhibitor group (Fig. 2A). MTT assay was used to further examine cell proliferation. Our data showed that inhibition of miR-92a caused a significant decrease in HeLa cell proliferation (Fig. 2B). As cell cycle regulation is essential for cell proliferation, we further examined the cell cycle distribution. As shown in Figure 2C, downregulation of miR-92a caused a significant cell cycle arrest at the G1 stage when compared to the NC inhibitor group. To confirm these findings, HeLa cells were then transfected with miR-92a or miR-NC, respectively. As shown in Figure 2D, transfection with the miR-92a mimic significantly increased the miR-92a levels in HeLa cells when compared to the miR-NC group. MTT assay further indicated that overexpression of miR-92a increased the proliferation of HeLa cells (Fig. 2E). Moreover, miR-92a upregulation significantly promoted the cell cycle progression of HeLa cells when compared to the miR-NC group (Fig. 2F). Therefore, our data demonstrate that miR-92a may play a role in promoting HeLa cell proliferation via enhancing cell cycle progression.

Figure 2

(A) Real-time RT-PCR was performed to determine the relative expression of miR-92a in HeLa cells transfected with miR-92a inhibitor or negative control (NC) inhibitor as control. (B) MTT assay and (C) flow cytometry were used to examine cell proliferation and cell cycle distribution. (D) Real-time RT-PCR was performed to determine the relative expression of miR-92a in HeLa cells transfected with miR-92a mimic or scramble miR (miR-NC) as control. (E) MTT assay and (F) flow cytometry were used to examine cell proliferation and cell cycle distribution. **p < 0.01 versus NC inhibitor (A–C). **p < 0.01 versus miR-NC (D–F).

p21 Is a Target Gene of miR-92a in Cervical Cancer Cells

Recently, Su et al. used an miRNA in vivo precipitation (miRIP) method to find that miR-92a could interact robustly with p21 mRNA both in hepatocellular carcinoma HepG2 cells and in prostate cancer PC-3 cells (20). However, the relationship between miR-92a and p21 in cervical cancer cells has never been studied previously. Therefore, we detected the protein levels of p21 in HeLa cells after overexpression or downregulation of miR-92a. Western blotting data showed that overexpression of miR-92a significantly decreased the p21 protein expression (Fig. 3A), while knockdown of miR-92a markedly increased the p21 protein expression in HeLa cells (Fig. 3B). However, neither miR-92a upregulation nor knockdown affected the mRNA levels of p21 in HeLa cells (Fig. 3C and D). Therefore, miR-92a negatively regulates the expression of p21 at the posttranscriptional levels, suggesting that p21 is a target of miR-92a in HeLa cells.

Figure 3

(A, B) Western blot and (C, D) real-time qPCR were conducted to detect the protein and mRNA expression of p21 in HeLa cells transfected with scramble miR (miR-NC), miR-92a mimic, NC inhibitor, and miR-92a inhibitor, respectively. **p < 0.01 versus NC inhibitor (A). **p < 0.01 versus miR-NC (B).

p21 Acts as a Downstream Effecter in the miR-92a-Mediated Proliferation and Cell Cycle Progression in HeLa Cells

As p21 is a key regulator in cell cycle progression (21), it may be involved in the miR-92a-mediated HeLa cell proliferation. Therefore, we further transfected miR-92a-overexpressing HeLa cells with pcDNA3.1-p21 ORF plasmid in order to eliminate the suppressive effect of miR-92a on p21 protein levels. Western blotting assay indicated that the p21 protein levels were significantly higher in the miR-92a + p21 group than in the miR-92a group (Fig. 4A). MTT assay was then used to examine cell proliferation. As indicated in Figure 4B, the proliferation of HeLa cells was significantly decreased in the miR-92a + p21 group compared to the miR-92a group, indicating that overexpression of p21 eliminates the promoting effects of miR-92a on HeLa cell proliferation. Further investigation showed that p21 upregulation also reversed the promoting effects of miR-92a on cell cycle progression in HeLa cells (Fig. 4C). To further confirm these findings, miR-92a inhibitor-transfected HeLa cells were further transfected with p21 siRNA. After transfection, the p21 protein levels were significantly reduced in the miR-92a inhibitor + p21 siRNA group compared with the miR-92a inhibitor group (Fig. 4D). MTT assay and flow cytometry were then used to examine cell proliferation and cell cycle distribution. As indicated in Figure 4E, the proliferation of HeLa cells was significantly decreased in the miR-92a inhibitor + p21 siRNA group compared with the miR-92a inhibitor group. Moreover, knockdown of p21 also eliminated the promoting effects of miR-92a downregulation on cell cycle progression in HeLa cells (Fig. 4F). On the basis of these findings, our data suggest that miR-92a promotes HeLa cell proliferation via inhibiting p21 expression and promoting cell cycle progression.

Figure 4

(A) Western blot was used to examine the protein levels of p21 in cervical cancer cells transfected with miR-92a mimic or cotransfected with miR-92a mimic and p21 plasmid, respectively. (B) MTT assay and (C) flow cytometry were used to examine cell proliferation and cell cycle distribution. **p < 0.01 versus miR-92a (A–C). (D) Western blot was used to examine the protein levels of p21 in cervical cancer cells transfected with miR-92a inhibitor or cotransfected with miR-92a inhibitor and p21 siRNA, respectively. (E) MTT assay and (F) flow cytometry were used to examine cell proliferation and cell cycle distribution. **p < 0.01 versus miR-92a inhibitor (D–F).

p21 Is Downregulated in Cervical Cancer

To further reveal the relationship between miR-92a and p21 in cervical cancer, we examined the expression of p21 in cervical cancer tissues and matched ANTs. Real-time PCR data and Western blot data showed that the mRNA and protein levels of p21 were significantly lower in cervical cancer tissues than in ANTs (Fig. 5A and B). These findings suggest that the increased expression of miR-92a may contribute to the decreased expression of p21, which further promotes cervical cancer growth.

Figure 5

(A) Real-time RT-PCR and (B) Western blot were performed to examine the mRNA and protein expression of p21 in cervical cancer tissues and their matched adjacent nontumor tissues. **p < 0.01 versus Adjacent.

DISCUSSION

The underlying mechanism of miR-92a in regulating the proliferation of cervical cancer cells is largely unclear. In the present study, we found that miR-92a was significantly upregulated in cervical cancer tissues compared to their matched ANTs, and the increased miR-92a levels were significantly associated with higher grade, lymph node metastasis, and advanced clinical stage in cervical cancer. Knockdown of miR-92a led to a significant reduction in the proliferation of HeLa cells via induction of cell cycle arrest at the G1 stage, while overexpression of miR-92a markedly promoted the proliferation of HeLa cells by promoting the cell cycle progression. p21 was further suggested to be a direct target of miR-92a in HeLa cells, since the expression of p21 was negatively regulated by miR-92a at the posttranscriptional levels. Moreover, p21 overexpression eliminated the promoting effects of miR-92a on HeLa cell proliferation and cell cycle progression, while knockdown of p21 reversed the suppressive effects of miR-92a downregulation on HeLa cell proliferation and cell cycle progression. In addition, p21 was significantly downregulated in cervical cancer tissues compared to ANTs.

miR-92a, an important member of the miR-17-92 family, has been demonstrated to be deregulated and play a key role in some cancers (14,22,23). For instance, Sharifi and Salehi found that knockdown of miR-92a could prevent cell proliferation while inducing cell apoptosis in acute megakaryoblastic leukemia, suggesting that miR-92a may become a potential therapeutic target for this disease (24). Ren et al. reported that miR-92a was significantly upregulated in gastric cancer tissues compared with ANTs, and higher miR-92a levels were significantly associated with shorter survival time of patients with gastric cancer at stage II and stage III (25). Moreover, deregulation of miR-92a has also been implicated in cervical cancer (18). Zhou et al. reported that miR-92a was significantly upregulated in cervical cancer and could promote the proliferation and invasion of cervical cancer cells (18). Besides, miR-92a was suggested to be used as an important biomarker for oncogenic papillomavirus infections (26). Here we also showed that miR-92a was significantly upregulated in cervical cancer tissues compared to their matched ANTs. Afterward, we investigated the clinical significance of miR-92a in cervical cancer and found that the increased expression of miR-92a was significantly associated with higher grade, lymph node metastasis, and advanced clinical stage, suggesting that upregulation of miR-92a contributes to cervical cancer progression. In vitro study further showed that knockdown of miR-92a significantly inhibited the proliferation of HeLa cells through induction of cell cycle arrest at the G1 stage. On the contrary, miR-92a overexpression significantly promoted enhanced proliferation and cell cycle progression of HeLa cells.

p21, also known as cyclin-dependent kinase inhibitor 1A, is a potent cyclin-dependent kinase inhibitor, which can bind to and inhibit the activity of cyclin–CDK2 or cyclin–CDK4 complexes, acting as a negative regulator of cell cycle progression at G1 (27,28). Moreover, p21 can also interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and thus plays a key role in S phase DNA replication and DNA damage repair (29). Upregulation of p21 could effectively suppress the cell cycle progression and thus cell proliferation in various human cancers including cervical cancer (30–32). For instance, Slug inhibits the proliferation and tumor formation of human cervical cancer cells by upregulating the p21 and p27 proteins (30). In the present study, we found that the expression of p21 was negatively mediated by miR-92a at the posttranscriptional levels in HeLa cells, suggesting that p21 is a direct target gene of miR-92a. In fact, the targeting relationship between miR-92a and p21 was recently reported in hepatocellular carcinoma and prostate cancer cell lines (20), consistent with our data. Further investigation showed that overexpression of p21 eliminated the suppressive effects of miR-93 on cell proliferation and cell cycle progression in HeLa cells, while knockdown of p21 reversed the suppressive effects of miR-92a downregulation on HeLa cell proliferation and cell cycle progression. Accordingly, our findings suggest that miR-92a promotes the proliferation of cervical cancer cells via inhibition of p21 and thus promotion of cell cycle progression. In addition, we found that the mRNA and protein expression of p21 was significantly downregulated in cervical cancer tissues compared to their matched ANTs. As we have found that the expression of p21 was negatively regulated by miR-92a, we suggest that the decreased p21 expression may be due to the upregulation of miR-92a in cervical cancer.

In conclusion, our study indicates that miR-92a, which is upregulated in cervical cancer, plays an oncogenic role in cervical cancer cell proliferation by directly targeting p21 and thus promoting cell cycle progression. Therefore, we suggest that miR-92a may become a useful therapeutic target for cervical cancer.