Introduction

MicroRNAs (miRNAs or mirs) are 20–25-nt small non-coding RNAs, conserved across evolution, which regulate gene expression post-transcriptionally. miRNAs are usually transcribed by RNA polymerase II as long primary miRNA transcripts (pri-miRNAs) that are subsequently cleaved by drosha/pasha to form approximately 70-nt stem-loop pre-miRNAs in the nucleus [1, 2]. The pre-miRNAs are exported into the cytoplasm by exportin 5 and then processed by dicer to generate mature miRNAs [3, 4]. Finally, the mature miRNAs interact with argonaute proteins to form the RNA-induced silencing complex, which results in the decay of the target mRNAs or the inhibition of translation [5, 6].

By regulating target gene expression, miRNAs play important roles in many processes, such as cell proliferation, apoptosis, differentiation, invasion and metastasis [7–11]. Recently, the mir-200 family has been reported as a powerful marker and determining factor of the epithelial phenotype of cancer cells. By targeting the zinc finger E-box-binding homeobox (ZEB) proteins 1 and 2, the mir-200 family could regulate the epithelial to mesenchymal transition (which is known to promote tumor invasion and metastasis) and protect tumor cells from apoptosis [12, 13]. The mir-200 family can be divided into two clusters according to their chromosomal location: the mir-200a/mir-200b/mir-429 cluster on chromosome 1 and the mir-200c/mir-141 cluster on chromosome 12. They also can be grouped into two subfamilies according to their function: mir-200b, mir-200c and mir-429 have the same seed region while those of mir-200a and mir-141 are different.

In our previous study, we demonstrated that the mir-200 family is overexpressed in endometrial adenocarcinomas, and that mir-200b showed the most significant change [14]. Other groups have demonstrated that the mir-200 family is abnormally expressed in tumors of many other cancers, such as hepatocellular, ovarian and gastric cancers [15–17]. To investigate the function of mir-200b further, we transfected a mir-200b mimic into HeLa cells and the human hepatocellular liver carcinoma cell line HepG2. HeLa cells transfected with the mir-200b mimic showed a high percentage of S-phase entry. Using luciferase assays, quantitative (q) PCR and western blotting, we demonstrated that mir-200b could directly reduce the expression of RND3 in HeLa cells and promote expression of the downstream protein cyclin D1 (CCND1) and S-phase entry.

Materials and methods

Plasmid construction

The wild-type 3′ untranslated region (UTR) of the RND3 gene, containing predicted miRNA target sites, was amplified by PCR from HeLa cell genomic DNA, then cloned into a modified pGL3-control plasmid (Promega, USA) downstream of the firefly luciferase coding region between the PstI and EcoRI sites, as described [18]. Mutant constructs containing deletions of the predicted target sites were generated using a MutanBest Kit (Takara Bio, Japan).

Cell culture, miRNA and small interfering (si) RNA transfection

HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10% FBS and 100 μg/ml penicillin/streptomycin. The miRNA mimics, miRNA inhibitors, siRNA and negative control were synthesized by GenePharma (Shanghai, China). miRNA and siRNA transfections were performed using Lipofectamine 2000 (Invitrogen, USA). Cells were plated in 6-well plates at a density of to 2 × 105 cells per well. For each well, 5 μl siRNA or miRNA (100 pmol) was added to 250 μl DMEM, and 5 μl of Lipofectamine 2000 was added to 250 μl DMEM. The siRNA/miRNA and Lipofectamine dilutions were then mixed together and incubated for 20 min. The mixture was added to the cells and incubated for 4 h before replacing the medium with fresh DMEM. Total RNA and protein, for use in qRT-PCR or western blotting, respectively, were prepared 48 h after transfection.

The mimic and siRNA sequences were: mir-200b sense: 5′-UAAUACUGCCUGGUAAUGAUGA-3′; mir-200b anti-sense: 5′-AUCAUUACCAGGCAGUAUAAAU-3′; RND3 siRNA sense: 5′-GUAGAGCUCUCCAAUCACAdTdT-3′ and RND3 siRNA anti-sense: 5′-GUAGAGCUCUCCAAUCACAdTdT-3′.

Luciferase assays

HepG2 cells were transfected in 24-well plates using Lipofectamine 2000 (Invitrogen). The transfection mixtures contained 100 ng firefly luciferase reporter plasmid, 5 ng pRL-TK plasmid (Promega) for a normalization control and 1 μl (20 pmol) mir-200b mimic or negative control. Cells were harvested 48 h after transfection, and luciferase activity was measured using a dual-luciferase reporter assay system (Promega).

RNA extraction and qRT-PCR

Total RNA was extracted from the cultured cells using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. RNA was reverse transcribed using M-MLV reverse transcriptase (Promega), according to the manufacturer’s protocol. qPCR was performed according to the protocol of the SYBR Green I kit (Toyobo Life Science, Japan) with Mx3000p (Stratagene, USA). Beta-actin mRNA levels were used for normalization. The primer sequences were: β-actin forward: 5′-TGAAGTGTGACGTGGACATCCGC-3′; β-actin reverse: 5′-GCCAATCTCATCTTGTTTTCTGCGC-3′; RND3 forward: 5′-GTGCTTGCATTTTTGGGTTT-3′; RND3 reverse: 5′-ATCCCATGGGTCCTGATACA-3′; mir-200b forward: 5′-TAATACTGCCTGGTAATGATGA-3′; mir-200b reverse: 5′-GCGAGCACAGAATTAATACGAC-3′; U6 forward: 5′-CGCTTCGGCAGCACATATACTA-3′; U6 reverse: 5′-CGCTTCACGAATTTGCGTGTCA-3′.

Western blotting

Cells were washed with PBS, harvested in ice-cold PBS and centrifuged at 2,000 rpm at 4°C. Then, cells were lyzed for 30 min on ice in RIPA buffer in the presence of a cocktail proteinase inhibitor (Sigma-Aldrich, USA). The protein was harvested, subjected to PAGE and then transferred to Hybond™-ECL membrane (GE Healthcare, USA). Immunodetection was performed using standard techniques. Antibodies against β-tubulin (Santa Cruz Biotechnology, USA), RND3 (Proteintech Group, USA) and CCND1 (Bioworld Technology, USA) were used in western blot analysis. Signals were visualized with a SuperSignal® West Femto Trial Kit (Thermo Fisher scientific, USA) and exposure to film.

Flow cytometric analysis

Cells were transfected with mir-200b or siRNA against RND3 for 24 h, starved for 24 h in 0.5% FBS-containing medium and then stimulated for 8 h in medium containing 10% FBS. The cells were then trypsinized and collected by centrifugation, washed in PBS and fixed overnight at 4°C in 70% ethanol. After being washed twice with PBS, DNA was stained with propidium iodide (50 μg/ml) in the presence of 1 mg/ml RNase A for 30 min. Analysis was performed on a FACScalibur flow cytometer (Becton Dickinson, USA).

MicroRNA target prediction software

TargetScan Release 5.1: http://www.targetscan.org/

Results

miRNA-200b promotes S-phase entry in HeLa cells

The mir-200b mimic and negative control were transfected into HeLa cells for 24, 48 or 72 h, and we examined the level of mir-200b 24–72 h after transfected by qRT-PCR which was described in [19] (Fig. 1a). The cells transfected by mir-200b showed a higher percentage of S-phase entry compared with the negative controls, which suggested that mir-200b could promote S-phase entry in HeLa cells (Fig. 1b).

Fig. 1
figure 1

A microRNA (mir)-200b mimic or negative control was transfected into HeLa cells for 24, 48 or 72 h. Cells transfected with mir-200b showed a higher level of mir-200b (a) and a higher percentage of S-phase entry (b) than cells transfected with the negative control

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Target validation of miRNA-200b

The 3′ UTR of RND3 mRNA contains two elements complementary to mir-200b seed regions, and both are conserved among human, mouse, rat, dog and chicken (Fig. 2a, b). To investigate whether RND3 mRNA is regulated by mir-200b, the 3′ UTR of RND3 was cloned into a modified pGL-3 control vector, placing it downstream of the luciferase coding sequence. The construct was cotransfected into HepG2 cells with a mir-200b mimic or negative control siRNA. Luciferase assays revealed that overexpression of mir-200b could significantly reduce luciferase activity from the reporter vector containing the 3′ UTR of RND3.

Fig. 2
figure 2

mir-200b downregulates RND3 mRNA and protein. a, b Human RND3 3′ UTR and target sites predicted by TargetScan. c Luciferase reporter assays indicated that mir-200b regulates RND3 expression by targeting both putative target sites. The transfection mixtures contained 100 ng of firefly luciferase reporter construct, 5 ng pRL-TK plasmid for a normalization control and 1 μl (20 pmol) mir-200b mimic or negative control (NC). Cells were harvested 48 h after transfection. d, e Overexpression of mir-200b downregulated RND3 mRNA (d) and protein (e) in vivo. f mir-200b mimics, mir-200b inhibitors and negative control were transfected into HeLa cells and the RND3 mRNA was up-regulated in the presence of mir-200b inhibitors

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To identify which putative target site is regulated by mir-200b, two deletion constructs were generated in the modified pGL-3 control vector: the first contained putative target site 2 and the second contained no putative target site. Luciferase assays indicated that overexpression of mir-200b reduced the luciferase activity from the reporter vector containing the wild-type RND3 3′ UTR, but it had less effect on activity from the first deletion construct and no effect on activity from the second. Our results suggest that mir-200b regulates RND3 expression by targeting both putative target sites 1 and 2 (Fig. 2c).

miRNA-200b directly regulates the expression of endogenous RND3

Having confirmed the interaction of mir-200b and the RND3 3′ UTR by luciferase assays, we next analyzed the capability of mir-200b to regulate endogenous RND3 expression. The mir-200b mimic, siRNA against RND3 or the negative control RNA duplex was transfected into HeLa cells, and qRT-PCR and western blotting were used to detect the expression levels of RND3 mRNA and protein, respectively. The qRT-PCR assay showed that the mRNA level of RND3 was reduced in cells transfected by the mir-200b mimic and RND3 siRNA compared with the negative control, and western blotting gave the same results. We conclude that mir-200b can directly regulate the expression of RND3 in vivo (Fig. 2d, e). We also measured the RND3 mRNA in presence or absence of mir-200b mimics, mir-200b inhibitors and corresponding negative control, and found the reversion of RND3 mRNA in the presence of mir-200b inhibitors (Fig. 2f).

miRNA-200b down-regulates RND3 and activates the expression of CCND1

Because RND3 has been reported to regulate CCND1 expression at the post-transcriptional level, we used western blotting to investigate whether mir-200b can regulate CCND1 by targeting RND3, hence promoting cell cycle progression. In HeLa cells transfected with mir-200b or siRNA against RND3, CCND1 expression levels were higher than those in cells transfected with the negative control, corresponding to the lower levels of RND3 expression (Fig. 3a). Cell cycle analysis confirmed that mir-200b and siRNA against RND3 led to less G0/G1-cell accumulation and promoted cell cycle progression. Our results suggest that mir-200b regulates CCND1 expression and promotes cell cycle progression by targeting RND3 (Fig. 3b).

Fig. 3
figure 3

mir-200b promotes cell cycle progression by regulating cyclin D1 (CCND1). a Western blotting demonstrated that both mir-200b and siRNA against RND3 downregulated RND3, thereby upregulating CCND1. b Cell cycle analysis confirmed that mir-200b and siRNA against RND3 led to less G0/G1-cell accumulation and promoted S-phase entry

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Discussion

Since miRNA was first identified in 1993 [20], over 700 miRNAs have been discovered in humans, and many of them are involved in tumor generation and development [21]. Recently, the mir-200 family has received much attention as it has been reported to inhibit the epithelial-mesenchymal transition [12, 13]. Members of the mir-200 family are abnormally expressed in hepatocellular tumors, ovarian cancer, gastric cancer and endometrial adenocarcinomas [15–17], and numerous studies have demonstrated that the mir-200 family inhibits migration and invasion by targeting ZEB1, ZEB2 and WAS protein family, member 3 (WASF3) in many kinds of cancer cells [22]. In embryonic stem cells, the mir-200 family is regulated by Myc and attenuates differentiation [23]. In bladder cancer cells, stable expression of mir-200 increases sensitivity to epidermal growth factor receptor (EGFR)-blocking agents by targeting ERBB receptor feedback inhibitor 1 (ERRFI1) [24]. The latest study has revealed that mir-200 could activate phosphoinositide-3-kinase by inhibiting feminization of germline 2 (FOG2) which suppresses cell growth; mir-200 and its target FOG2 are conserved components of the insulin pathway [25].

In this study, we investigated a novel role for mir-200b in cell cycle progression and identified a novel mir-200b target, RND3. We overexpressed mir-200b in HeLa cells, and found that the transfected cells showed a higher percentage of S-phase entry compared with the negative control cells. Luciferase assays, qPCR and western blotting suggested that mir-200b could directly regulate the expression of RND3 at the mRNA and protein levels. By comparing the data with that generated using an siRNA against RND3, we demonstrated that mir-200b could regulate the downstream protein CCND1 and promote S-phase entry by targeting RND3. CCND1 plays important role in cell cycle progression; overexpression of CCND1 may be connected with tumorigenesis. It is reported that RND3 could block CCND1 expression at the posttranscriptional level and induced cell cycle arrest [26]; as the downstream protein of RND3, CCND1 maybe up-regulated by mir-200b and promotes S-phase entry in HeLa cells.

Members of the Rho GTPase family are crucial regulators of cell shape and motility. RND3 is a member of the RND subfamily that is present in fish, birds and mammals, but not in invertebrates [27]. RND3 has two functions: one is to regulate the organization of the actin cytoskeleton [28] and the other is to inhibit cell cycle progression [26]. RND3 is regulated at multiple levels, from transcription to phosphorylation, protein stability and localization [29]. Here, we have demonstrated a novel miRNA-based mechanism for RND3 regulation.

In conclusion, this study demonstrated that mir-200b could promote CCND1 expression and S-phase entry by targeting RND3 in HeLa cells. Further research will explore the function of mir-200b in tumor generation and development.