MicroRNA-543 acts as a prognostic marker and promotes the cell proliferation in cervical cancer by BRCA1-interacting protein 1

Abstract

Human cervical cancer is one of the most common malignancies worldwide. Recent studies have focused on microRNAs that play crucial roles in cancer development and progression of cervical cancer. In this study, we aimed to analyse the biological function of microRNA-543 in cervical cancer. Samples of human cervical cancer and matched adjacent normal cervical tissues were collected, and expression level of microRNA-543 and the clinical characteristics of cervical cancer were investigated. We found that microRNA-543 expression was significantly elevated in cervical cancer and its aberrant expression levels were positively correlated with tumour size (p = 0.0315), differentiation (p = 0.0134), clinical stage (p = 0.0315) and overall (p = 0.0426) and disease-free survival (p = 0.0396) of cervical cancer. Overexpression of microRNA-543 in cancer-derived HeLa and SiHa facilitated cell growth and suppressed cell apoptosis, while down-regulation of microRNA-543 exerted a reverse effect on cell growth and apoptosis. In addition, we demonstrated that BRCA1-interacting protein 1 was directly regulated by microRNA-543 and the restoration of BRCA1-interacting protein 1 expression reversed the effects of microRNA-543 on cell proliferation. Taken together, these findings collectively demonstrate that microRNA-543 exerts its oncogene function by directly targeting BRCA1-interacting protein 1 in cervical cancer, indicating a potential novel potential prognostic biomarker and therapeutic target for cervical cancer.

Introduction

Cervical cancer is the second most common cause of cancer-related mortality in women worldwide. According to statistics, there are over 300,000 deaths annually, and more than 85% of these deaths occur in developing countries.^1,2 Cervical cancer is characterized by a multistep process that involves the transformation of the normal cervical epithelium into a preneoplastic cervical intraepithelial neoplasia (CIN) that is subsequently transformed into invasive cervical cancer. ³ As is well known, human papillomavirus (HPV) infection is associated with cervical cancer. ⁴ However, HPV infection is not sufficient to induce the malignant transformation of the cervix, and other factors are also critical in the process of malignant transformation of the cervix. ⁵ Despite great progress in cervical cancer treatments, including radiotherapy, chemotherapy and surgical intervention, the clinical outcomes of cervical cancer vary markedly and are difficult to predict. It is necessary to identify novel molecular markers that can predict and reverse malignant transformation of the cervix.

Recently, microRNAs (miRNAs), a class of small and non-coding RNAs that are 19- to 24-nucleotide in length, have emerged as potent regulators of gene expression by silencing messenger RNAs (mRNAs) that bind to the 3′-untranslated region (3′-UTR) of target genes. ⁶ MicroRNAs are considered to be master regulators of biological processes, including cell motility, cell differentiation, cell proliferation and apoptosis. ⁷ All of these studies suggest that miRNAs are involved in the pathogenesis and progression of carcinomas. ⁸ Moreover, miRNAs are deeply implicated in multiple steps of cervical cancer occurrence and development, including proliferation, adhesion, invasion and metastasis. ⁹

This study aimed to examine the expression of miR-543 in the cervical cancer and to investigate its possible correlation with certain clinicopathological features, such as tumour size, clinical stage and metastasis. In addition, we also identified the role of miR-543 on proliferation in cervical cancer cell lines and found the potential regulatory mechanism.

Materials and methods

Patients and tissue samples

A total of 56 human cervical cancer samples and matched adjacent normal tissues were obtained by cervical biopsy from the Department of Gynaecology and Obstetrics, the Second Affiliated Hospital, Xi’an Jiaotong University between May 2013 and October 2015. Collected samples were flash frozen in liquid nitrogen and stored at −80°C for later RNA extraction or formalin-fixed and paraffin-embedded for immunohistochemistry. The histopathologic diagnoses of these tissue samples were confirmed by two experienced pathologists. None of the patients received either radiotherapy or chemotherapy prior to biopsy. This study was approved by the Ethics Committee of Department of Gynaecology and Obstetrics, the Second Affiliated Hospital, and informed consents were obtained from all patients.

RNA extraction and miRNA expression assay

Total RNA from tissues or cell was isolated using a miRCut miRNA Isolation Kit (Tiangen, Shanghai, China) according to the manufacturer’s instructions. Reverse transcription was then performed with primers for miR-543 (5′-GTCGTATCCAGTGCGTGTVGTGGA-3′); U6 (CGCTTCACGAATTTGCG TGTCAT). The expression of miR-543 was quantified by Taqman microRNA assay kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed using ABI7500 (Life Technologies, Carlsbad, CA, USA). The primers for qPCR were as follows: miR-543 forward, 5′-GGGGAAACATTCGCGGTGCA-3′, reverse, 5′-TGCGTGTCGTGGAGTC-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. The relative quantification of miR-543 was calculated using the 2^−ΔΔCt method. For data analysis, the data were normalized using U6 small nuclear RNA as an internal control.

Cell culture

Human cervical cancer cell lines (HeLa and SiHa) were obtained from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS, GIBCO) and 100 U/mL penicillin/streptomycin in a cell culture incubator at 37°C in a humidified incubator with 5% CO₂.

RNA oligonucleotide and cell transfection

Mature miR-543 mimics/inhibitor and miRNA negative control (non-special sequence) were synthesized by GenePharma (Shanghai, China).The cells were plated in six-well plates and cultured in RPMI 1640 medium for 24 h. The cells were transfected with the miRNA mimics/inhibitor at a final concentration of 50 nM using Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. At 48 h post-transfection, the cells were harvested for analysis.

³H thymidine incorporation assay

The cells were plated onto six-well plates a density of 3 × 10⁵ cells/well and grew to 70% confluent. Cells were serum-starved overnight followed by addition of serum and ³H thymidine (1 Ci/mM) for 4 h. The reaction was terminated by aspirating the culture medium off, washed three times with cold phosphate-buffered saline (PBS) followed by the addition of 2 mL/well of ice-cold 10% trichloroacetic acid (TCA) for 20 min at 4°C. Then, the cells were washed by ice-cold water and solubilized with 1 mL of 0.5 M NaOH/well at 37°C for 30 min. The ³H thymidine incorporation was quantitated by liquid scintillation counting system (Beckman Coulter, Inc., Fullerton, CA, USA).

Cell cycle analysis

Transfected cells were collected and washed with cold PBS followed by fixation in 70% cold ethanol at 4°C overnight. The fixed cells were washed with PBS, and treated with RNaseA (50 µg/mL) and stained with propidium iodide (PI, 25 µg/mL) for 30 min at 37°C. The DNA content was determined using a Becton Dickinson FACS/Calibur cytometer (Becton Dickinson Biosciences, Inc., NJ, USA). Cell cycle profile was evaluated using the ModFit 3.0 software packages.

Apoptosis assay

Cell apoptosis was assessed using the Annexin-V fluorescein isothiocyanate (FITC) Apoptosis Kit (Calbiochem, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, transfected cells were washed with cold PBS and re-suspended in staining buffer containing 1 µg/mL PI and 0.025 µg/mL annexin V-FITC, followed by 10 min incubation at room temperature in the dark. Then, the cells were immediately analysed using FACScan and the Cellquest program (Becton Dickinson Biosciences).

Western blot assays

Cells were homogenized and extracted in NP-40 buffer, followed by 5 min boiling and centrifugation to obtain the supernatant. A total of 50 µg protein was separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (NC) membranes (Bio-Rad Laboratories, Hercules, CA, USA). After saturation with 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) with 0.1% (w/v) Tween 20. The blots were then probed with antibodies against the following primary antibodies: BRIP1 (Sigma, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, Boston, USA), at 4°C overnight. The bands were visualized with the Odyssey Infrared Imaging System (LI-COR Biotechnology).

Plasmid construction

The 3′-UTRs of potential RNAs targeted by miR-543 were synthesized by PCR. The PCR product placed in the pMiR-Report vector (Invitrogen). A QuikChange^® II XL Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to generate a mutant-type 3′-UTR of BRIP1, according to the manufacturer’s protocols. The BRIP1 plasmid (lacking 3′-UTR) was amplified by PCR with the following primers: forward, 5′-CTGCCATCATGCCGATGTTCAT-3′ and reverse, 5′-CGGCTCTTAGGCGAAG GTG-3′. The PCR product was cloned into the T vector (Promega, Madison, WI, USA).

Luciferase assays

For the luciferase reporter assay, the cells were seeded into a 24-well plate and cultured to 70% confluence. Then, cells were transfected with the wild/mutant recombinant plasmid (200 ng), with 50 nM miRNAs mimics using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Following incubation for 48 h, luciferase assays were performed using dual-luciferase system kit (Promega) on the LD400 Luminometer (Beckman Coulter, Inc.). Firefly luciferase activity was normalized against Renilla luciferase gene activity.

Statistical analysis

All data are presented as the mean ± standard deviation (SD). SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analyses. The statistical significance between groups were analysed using Student’s t-test or one-way analysis of variance (ANOVA). Count data were analysed by Fisher’s exact tests. Univariate survival analysis was performed using Kaplan–Meier method and the Log-rank test. p < 0.05 was considered to indicate a statistically significant difference.

Results

Association between miR-543 expression and clinicopathological characteristics in cervical cancer

The miR-543 expression level of cervical cancer sample and matched adjacent normal tissues was examined by qPCR. We also found that the expression of miR-543 was significantly up-regulated in cervical cancer tissues than that in normal tissues (Figure 1(a)). Then, miR-543 expression level was classified either low or high according to the median of the cohort. As shown in Table 1, high expression of miR-543 was found to significantly correlate with unfavourable variables, including tumour size (p = 0.0315), differentiation (p = 0.0134), Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.0315). However, no significant difference was observed between the expression of miR-543 and other clinicopathological characteristics such as gender, age, histologic type and lymph node metastasis. Furthermore, Kaplan–Meier analysis with log-rank showed that high expression of miR-543 indeed associated with worse overall survival (p = 0.0426) (Figure 1(b)) and disease-free survival in patients (p = 0.0396) (Figure 1(c)).

Figure 1.

The relationship between miR-543 expression clinical characteristics. (a) The relative expression of miR-543 in cervical cancer and paraneoplastic tissues was detected by qPCR. Data are expressed as the mean ± SD. *p < 0.05, n = 20. Kaplan–Meier curves of survival time in patients with in patients divided according to miR-543 expression. (b) Overall survival rate. (c) Disease-free survival rate.

Table 1.

Correlations between miR-543 expression in cervical cancer and clinical characteristics.

Group	No.	Relative miR-543 expression		p value
		Low	High
Age (years)
>50	30	17	13	1.0000
⩽50	26	14	12
Menopausal status
Yes	24	14	10	0.4303
No	32	15	17
HPV status
Positive	24	13	11	0.4184
Negative	32	13	19
Size of diameter (cm)
>4	29	10	19	0.0315*
⩽4	27	18	9
Histologic type
Squamous cell cancer	36	21	15	0.4076
Adenocarcinoma	20	9	11
Differentiation
Well and mediate	22	15	7	0.0134*
Poor	34	11	23
FIGO stage
I–II stage	28	19	9	0.0315*
III–IV stage	28	10	18
Lymph node metastasis
Negative	31	16	15	0.7925
Positive	25	14	11

miR-543: microRNA-543; HPV: human papillomavirus; FIGO: Federation of Gynecology and Obstetrics.

*

p < 0.05.

MiR-543 regulates proliferation and apoptosis in cervical cancer cell

In order to elucidate whether miR-543 influences cell proliferation, miR-543 was overexpressed in cervical cancer cells (HeLa and SiHa) by transfection with miR-543 mimics. The expression of miR-543 was measured by qPCR after transfection with miR-543 mimics (Figure 2(a)). ³H thymidine incorporation assay showed that cell proliferation was significantly promoted by the overexpression of miR-543 compared with control group (Figure 2(b)). Moreover, cell cycle revealed that miR-543 overexpression significantly increased the percentage of cells in the S phase compared with the control group (Figure 2(c) and (d)). Apoptosis assay suggested that miR-543 overexpression reduced cell apoptosis rates (Figure 2(e) and (f)).

Figure 2.

Up-regulation of miR-543 expression enhances cell proliferation. 48 h after transient transfection with miR-543 mimics (or control mimics) in HeLa and SiHa cells. (a) qPCR analysis was used to detect the transfection efficiency of miR-543 mimics in cells. (b) ³H thymidine incorporation assay was performed to examine cells proliferation 96 h after transfection. (c and d) Frequencies of cells at different stages of the cell cycle. (e and f) The percentage of apoptotic cells (Annexin V-FITC positive, PI negative).

In contrast, we down-regulated the expression of miR-543 by transfection with miR-543 inhibitor in cervical cancer cells (Figure 3(a)). ³H thymidine incorporation assay suggested that cell proliferation was inhibited after transfection with miR-543 inhibitor (Figure 3(b)). Flow cytometry assay showed that knockdown of miR-543 significantly decreased the percentage of S phase and induced the cells apoptosis (Figure 3(c)–(f)).

Figure 3.

Down-regulation of miR-543 expression inhibits cell proliferation. 48 h after transient transfection with miR-543 inhibitor (or control inhibitor) in HeLa and SiHa cells. (a) qPCR analysis was used to detect the transfection efficiency of miR-543 inhibitor in cells. (b) ³H thymidine incorporation assay was performed to examine cells proliferation 96 h after transfection. (c and d) Frequencies of cells at different stages of the cell cycle. (e and f) The percentage of apoptotic cells.

MiR-543 directly inhibits BRIP1 gene expression via targeting its 3′-UTR

miRNAs play important roles in biological processes by targeting the 3′-UTR site of multiple genes. To identify candidate targets of miR-543, we employed miRnada (http://www.microrna.org) to search for putative protein coding gene targets of miR-543. The gene for BRIP1 was identified as a potential target. We first examined the correlation between BRIP1 and miR-543 expression in cervical cancer specimens. Immunohistochemistry analysis showed that there was a strong negative correlation between the BRIP1 expression and miR-543 levels in cervical cancer samples (Figure 4(a)). Western blot analysis demonstrated that BRIP1 expression was significantly down-regulated after transfection with miR-543 mimics, while down-regulation of miR-543 significantly elevated BRIP1 protein level in cervical cancer cells (Figure 4(b)). To further confirm whether BRIP1 was a direct target of miR-543, BRIP1 3′-UTR was cloned into a luciferase reporter vector and the putative miR-543 binding site in the BRIP1 3′-UTR was mutated (Figure 4(c)). Luciferase reporter assay showed that up-regulation of miR-543 significantly reduced the luciferase activity of pMir-BRIP1 3′-UTR. Mutation of the miR-543-binding site in the BRIP1 3′-UTR abolished the effect of miR-543 (Figure 4(d)).

Figure 4.

miR-543 inhibits BRIP1 expression by directly targeted its 3′-UTR. (a) Immunohistochemistry showed an inverse relationship between the expression of miR-543 and BRIP1 in cervical cancer specimens (amplification 200×). Spearman’s correlation analysis was used to determine the correlation between the expression levels of BRIP1 and miR-543; Spearman’s correlation, r = −0.7805 (n = 20). (b) Western blot analysis of BRIP1 protein expression in cells after transfection. (c) Schematic construction of wild-type (WT) and mutant (Mut) 3′-UTR of BRIP1 were shown. (d) A dual-luciferase reporter system analysis was performed in cervical cancer cells co-transfected with wild-type 3′-UTR BRIP1 reporter plasmid or mutant type 3′-UTR BRIP1, along with miR-543 mimics or control mimics.

Re-expression of BRIP1 reverses miR-543-mediated effects in cervical cancer

To determine whether up-regulation of miR-543 promotes the growth of cervical cancer cells in a BRIP1-dependent manner, we restored the BRIP1 expression in cervical cancer cells by transfecting BRIP1 recombinant plasmid. Western blot analysis determinate increased expression of BRIP1 protein after BRIP1 recombinant plasmid transfection (Figure 5(a)). Functionally, restoration of BRIP1 expression abrogated the enhanced effect of exogenous miR-543, resulting in significant decrease in cell proliferation (Figure 5(b)). Similarly, re-expression of BRIP1 exhibited an apparent decrease in the percentage of cells in the S phase and restored apoptosis rate in cervical cancer cells (Figure 5(c) and (d)).

Figure 5.

Re-expression of BRIP1 reverses the effect of miR-543 on proliferation. HeLa and SiHa cells were co-transfected with miR-543 mimics (or control mimics) and BRIP1 overexpressing plasmid (or empty vector) for 48 h. (a) Expression of BRIP1 protein was analysed by Western blot. (b) Cell proliferation ability was examined by ³H thymidine incorporation assay. (c and d) The cell apoptosis and cycle progression was analysed by flow cytometry.

Discussion

In recent decades, cervical cancer has been remained as one of the most frequent malignancies for women in world. ¹⁰ Although increasing evidence indicated that miRNAs plays an integral role in cervical tumourigenesis, their underlying functions and mechanisms in cervical cancer development have not been fully studied. In this work, our results indicated that miR-543 expression was raised in cervical cancer tissue relative to matched normal tissue, and patients with higher levels of miR-543 tended to exhibit larger tumour size, poor differentiation, advanced stages of disease. Moreover, patients with high expression of miR-543 had poor overall and disease-free survival rate. Our data identified that miR-543 was an independent prognostic marker for predicting overall and disease-free survival of cervical cancer patients. In addition, transfection with miR-543 mimics in cervical cancer cells remarkably promoted proliferation and suppressed apoptosis.

The miR-543 gene, located in a region that is named imprinted DLK1-DIO3 on human chromosome 14, encodes a large cluster of noncoding RNAs. ¹¹ The biological role of miR-543 in tumour is controversial. Previous study identified that miR-543 cooperates with other miRNAs in repressing the proliferation and epithelial-to-mesenchymal transition in breast ductal carcinoma. ¹² In endometrial cancer and ovarian cancer, miR-543 was also shown to suppress tumourigenesis and metastasis by inhibiting various targets.^12,13 Recent studies reported that miR-543 acted as an oncogene by promoting the proliferative and invasive potential of hepatocellular carcinoma ¹⁴ and lung cancer. ¹⁵ Our data were consistent with previous studies demonstrating that miR-543 improved the proliferation ability of cervical cancer cells in vitro. This study revealed miR-543 acts as an oncogene in cervical cancer pathogenesis.

We also identified BRIP1 as a direct target gene for miR-543, and we demonstrated that miR-543 negatively regulated BRIP1 expression by directly targeting the 3′-UTR of BRIP1 mRNA in cervical cancer cells. BRIP1, belonging to a DEAH helicase family, directly interacts with the BRCT domain of BRCA1 and plays a role in DNA damage repair.^16,17 Accumulating evidence suggest that BRIP1 regulates normal cell cycle progression and plays an anti-oncogenic role in multiple cancers.^18,19 Recently, a study reveals that BRIP1 is down-regulated in cervical carcinoma tissue, and overexpression of BRIP1 in cervical cancer cells inhibited the proliferation of cervical cancer, which suggests that the BRIP1 gene may play the role of a tumour suppressor in cervical cancer development. ²⁰ Furthermore, our previous study indicates that BRIP1 may represent a potential anti-cervical cancer agent and sensitizer of cisplatin and may also be a promising candidate for cervical cancer therapy. ²¹ This work strongly supported the results of previous studies and found that miR-543 promoted the cell proliferation in cervical cancer by suppressing BRIP1 expression.

In conclusion, the level of miR-543 is significantly increased in patients with cervical cancer, and this increase is significantly associated with larger tumour size, higher FIGO stage and poor survival. Our data reveal the important molecular mechanism by which miR-543 promotes proliferation and inhibits apoptosis in cervical cancer via targeting BRIP1. This newly identified target of miR-543 may be employed as a prognostic marker and therapeutic target for cervical cancer patients in future.