The paired tissue samples from primary tumor and adjacent non-tumor sites were obtained from 14 RCC patients during surgery prior to any therapeutic intervention at the Shanghai Tenth People’s Hospital, Tongji University. All of the samples were subsequently verified by histology. Informed written consent was provided by all of the patients. The study protocol was approved by the Ethics Committee of Shanghai Tenth People’s Hospital. To identify the differential expression of miR-34a, YY1 and C/EBPα in tumor specimens versus adjacent non-tumor renal tissue, we pooled these samples in two subsets. Pool 1 contained 14 primary tumor samples. Pool 2 was generated from the matched 14 normal renal tissue samples.

RCC cell lines, 786–0 and ACHN, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. All cells were maintained in 5% CO₂ atmosphere at 37°C.

Cells were transfected with 50 nM miR-34a mimics and inhibitors (GenePharma Co., Ltd., Shanghai China), YY1-siRNA (Sigma-Aldrich Co., LLC, St. Louis, MO, USA), YY1 and C/EBPα plasmids (Genechem Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA), respectively. RNA and proteins were harvested 48 h after transfection.

Total RNA was extracted from the tissues or cultured cells following the indicated treatment using TRIzol (Invitrogen Corp.) method. qRT-PCR or Stem-loop PCR (24) was performed using the SYBR Green PCR kit (Takara Biotechnology Co., Ltd., Dalian, China), Real-time PCR data for mRNA (YY1, C/EBPα) and miRNA (miR34a) are expressed relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6, respectively. The primers used were described as follows: YY1 forward, 5′-CCT GGC ATT GAC CTC TCA GAT CCA-3′ and reverse, 5′-GGG CAA GCT ATT GTT CTT GGA GCA-3′; C/EBPα forward, 5′-AAC ATC GCG GTG CGC AAG AG-3′ and reverse, 5′-TTC GCG GCT CAG CTG TTC CA-3′. The stem-loop miR-34a RT primer was 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG ACA ACC AG-3′; miR-34a forward, 5′-ACA CTC CAG CTG GGT GGC AGT GTC TTA GCT GG-3′ and reverse, 5′-TGG TGT CGT GGA GTC G-3′. All reactions were performed in triplicate. The relative expression levels of target genes or miRNA were calculated using the 2^−ΔΔCt method (25).

The wild-type YY1–3′UTR region, generated by PCR amplification, was cloned into the pcheck-2 luciferase reporter plasmid (Promega, Madison, WI, USA). Specific primers were designed for this system. The forward primer (5′-CCA CTC GAG GCA TCT TCC AGA AGT GTG AT-3′) was Xhol-site-linked and the reverse primer Notl-site-linked (5′-CCA GCG GCC GCC ATT CTA CAA CTG AGC ACC AC-3′). Mutation of the miR-34a binding site was generated by a PCR-based site-directed mutagenesis method, using wild-type YY1–3′UTR reporter plasmid as the template. For the reporter assay, ACHN cells were plated onto 24-well plates and transfected with wild-type or mutant YY1–3′UTR reporter plasmid and miR-34a mimics or inhibitors using Lipofectamine 2000. After transfection for 48 h, cells were harvested and assayed with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The tests were repeated in triplicate. Firefly luciferase activity was normalized to Renilla luciferase activity.

Proteins were resolved on an SDS/PAGE gel (10% gel) and subjected to immunoblot analysis using monoclonal antibodies against YY1, C/EBPα or GAPDH (Epitomics, Burlingame, CA, USA). All of the antibodies were used at working concentration (1:1000) in PBS-T with 5% non-fat milk. The membrane was further probed with HRP (horseradish peroxidase)-conjugated rabbit anti-(mouse IgG) (1:2000 dilution; Santa Cruz Biotechnology), and the protein bands were visualized using enhanced chemiluminescence (Amersham Pharmacia).

To measure the cell migration activity, Transwell assays were performed using Corning 8.0-μm Transwell^® cell culture inserts (Corning Inc., Corning, NY, USA). Membranes were coated with purified fibronectin (Sigma) at a concentration of 10 μg/ml. For assessment of invasion, 1:10 diluted Matrigel-coated Transwell inserts (BD Biosciences, San Jose, CA, USA) were used. After transfection with either siRNA or expression vectors in opti-MEM medium for 5 h, cells were collected and resuspended in serum-free DMEM medium containing 0.1% bovine serum albumin (BSA). Subsequently, cells (4×10⁵/ml) were seeded in Transwell chambers. After 24 h of incubation, the cells on the upper surface of the filter were completely wiped away with a cotton swab. The cells on the lower surfaces of the membrane were fixed with 100% methanol, and counted under a microscope.

Cells were seeded into 96-well plates at an initial density of 1×10⁵ cells/well. MTT solution (200 μl) (5 mg/ml, Alfa Aesar) was added to each well and incubated for 5 h at 37°C. The supernatant was then discarded, and 100 μl of dimethyl sulfoxide (DMSO, Sigma) was added to each well to dissolve the precipitate. After 30 min at room temperature, the plates were scanned spectrophotometrically with a microplate reader (Beckman Coulter) set at 595 nm to measure the absorbance.

Statistical evaluations were conducted using the t-test. P-values <0.05 were considered to be statistically significant.

To confirm the tumor promotion effect of YY1 in RCC, we evaluated the impact of YY1 on the growth of RCC cell lines, 786-0 and ACHN. The growth curve of the RCC cell lines transfected with si-YY control siRNA is shown in Fig. 1A. The cell growth was significantly decreased in the si-YY1-transfected 786-0 and ACHN cells as compared with the control siRNA-transfected cells (P<0.05). To evaluated the impact of YY1 on cell migration and invasion, Transwell and Matrigel invasion assays were employed. We found that YY1 knockdown inhibited 786-0 and ACHN cell migration (Fig. 1B). Consistent with this finding, the Matrigel invasion assay showed that YY1 knockdown significantly inhibited the invasive capacity of RCC cells (Fig. 1C). These observations suggest that YY1 plays an important role in promoting cell growth, migration and invasive potential of RCC cells.

To associate miRNAs with the regulation of YY1 expression, a bioinformatics search was performed for potential miRNAs targeting the mRNA of YY1 by using the public database Targetscan (www.targetscan.org). Noteworthy, there were several predicated miRNAs to target YY1, including miR-34a. miR-34a was reported to play a tumor-suppressive role in a number of tumor types (15–19). We then performed a luciferase reporter assay to verify that miR-34a directly targets YY1. We found that co-transfection of miR-34a mimics and wild-type YY1–3′UTR significantly decreased the luciferase activity in ACHN cells as compared with the control. However, miR-34a mimics had no effect on the luciferase activity when co-transfected with mutant YY1–3′UTR (Fig. 2B). These data demonstrate a specific inhibitory effect of miR-34a on the 3′UTR of YY1 through direct interaction. Furthermore, to examine the potential negative regulatory effect of miR-34a on endogenous YY1, we transfected miR-34a mimics, miR-34a inhibitors or control mimics into ACHN cells. In comparison with the miR control, we found the miR-34a exerted a discernible inhibitory effect on the YY1 protein level, while an increased protein level of YY1 was found in ACHN cells following transfection with miR-34a inhibitors (Fig. 2C). These data showed that YY1 is one of the direct targets of miR-34a.

We next determined whether or not miR-34a overexpression impairs the oncogenic effect of YY1 on cell growth, migration or invasion of RCC cells. We transfected YY1 overexpression plasmids alone, or co-transfected YY1 plasmids and miR-34a mimics into ACHN cells. Notably, exogenous YY1 expression in ACHN cells promoted the migratory, invasive or proliferative potential of ACHN cells (Fig. 2D–F). Furthermore, constitutively expressed miR-34a rescued the previously aggressive phenotype in ACHN cells initiated by YY1 (Fig. 2D–F), suggesting that YY1 is indeed a functional target of miR-34a.

Since YY1 downregulates C/EBPα expression through a promoter-dependent manner in HCC cells (26) and C/EBPα leads to upregulation of miR-34a expression during granulocytic differentiation (27), we aimed to ascertain whether or not YY1 regulates miR-34a expression via C/EBPα in human RCC. We observed that miR-34a was downregulated in the pools of cancer tissues when compared to that of the normal tissues. Moreover, miR-34a displayed an inverse correlation with YY1, but a positive correlation with C/EBPα (Fig. 3A and B), suggesting that a low level of miR-34a may contribute to the upregulation of YY1 or the elevated expression of YY1 in turn inhibits miR-34a via C/EBPα suppression. We then revealed that YY1 siRNA was able to induce the expression of C/EBPα both at the mRNA and protein levels in ACHN cells (Fig. 3C and D). We also found that either C/EBPα overexpression or YY1 knockdown alone in ACHN cells increased miR-34a expression. However, when C/EBPα siRNA was transfected into ACHN cells, the upregulation of miR-34a by si-YY1 was abrogated (Fig. 3E). These data indicate that YY1, C/EBPα and miR-34a collaborate to form feedforward loops, which contributes to RCC progression (Fig. 4).