Histone Demethylase Retinoblastoma Binding Protein 2 is Overexpressed in Hepatocellular Carcinoma and Negatively Regulated by hsa-miR-212

Background The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues. Methods We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay. Results The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR. Conclusions RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common tumors in China [1][2][3]. Its early diagnosis is difficult. HCC can be diagnosed effectively in some cases with serological assessment [4][5][6], but the molecular mechanism of cell proliferation and effective biological markers for diagnosis are unclear.
We found RBP2 overexpressed in gastric cancer, and its inhibition could trigger cancer-cell senescence [16]. We also found RBP2 overexpressed in HCC. RBP2 was significantly underexpressed in normal liver tissue but strongly expressed in cancerous liver tissues, so RBP2 may take part in the development of HCC. Furthermore, siRNA inhibition of RBP2 expression could decrease the proliferation and induce senescence of high-differentiated HepG-2 cells and low-differentiated SMMC-7721 cells, 2 representative HCC cell lines, but the mechanism of RBP2 overexpression has not been elucidated.
MicroRNAs (miRNAs), non-coding 20-to 23-nt RNAs, play a key role in gene expression in different kinds of cancer [17][18]. MiRNAs can bind to the seed sequence of the 39 UTR of target genes and negatively regulate their expression in cell proliferation, differentiation, senescence, and apoptosis, for example. MiRNAs also participate in cancer metastasis and cancerous angiogenesis by regulating specific genes. Homo sapiens miRNA-212 (hsa-miR-212) has 2 sites binding in the 39 UTR of RBP2. It is downregulated and suppresses methyl-CpG-binding protein MeCP2 in human gastric cancer [19]. Downregulation of hsa-miR-212 increasing the expression of HB-EGF may be involved in cetuximab resistance in head and neck squamous cell carcinoma [20].
In this research, we investigated the involvement of RBP2 in HCC and the miRNA implicated in its regulation in human HCC. RBP2 inhibition and hsa-miR-212 overexpression may lead to cell proliferation arrest and cellular senescence by depressing cyclindependent kinase inhibitors (CDKIs; p16 ink4a , p21 CIP2 and p27 kip1 ). RBP2 and hsa-miR-212 may be biomarkers for diagnosis of HCC and therapeutic targets in its treatment.

Materials and Methods
The ethics committee of Shandong University School of Medicine approved this study and all participants provided written informed consent.

Clinical Samples
We obtained 20 samples of HCC from patients at Bengbu Medical University, AnHui Province, China, immediately after surgery and stored them in formalin. The diagnosis of HCC was confirmed by histological examination; no patient had undergone chemotherapy or radiotherapy before surgery. The general characteristics of patients are in Table S1. We excluded patients with a history of alcohol abuse.

Other Materials and Methods
Immunohistochemistry, cell culture [21][22][23] and siRNA interference, RNA extraction, RT-PCR and real-time PCR (primers are in Table 1), plasmid transfection and luciferase reporter gene assay (primers and methods are in Table 2), protein extraction and western blot assay, clonal formation assay and senescence-associated b-galactosidase (SA-b-Gal) staining are described in Data S1.

Statistical Analysis
Quantitative data are expressed as mean6SD or SEM. Student's t test was used to analyze the differences between groups. P,0.05 was considered statistically significant.

Results
RBP2 is overexpressed and hsa-miR-212 underexpressed in human HCC.
Immunohistochemical staining revealed higher RBP2 protein expression in HCC than normal liver tissue ( Figure 1A), and the mRNA level of RBP2 was higher in HCC than normal tissue (P,0.001; Figure 1C) [24]. RBP2 was highly expressed in most HCC tissues (75%), whereas most normal tissues (70%) showed low RBP2 expression ( Figure 1B). Overexpression of RBP2 at the mRNA and protein levels in HCC tissue suggested that RBP2 might participate in the initiation and development of HCC.
Hsa-miR-212, the predicted miRNA that could bind to the 39 UTR of RBP2 ( Figure 1D), may regulate its expression in these clinical samples. Hsa-miR-212 level was significantly lower in HCC than normal tissue (P,0.001; Figure 1E), which suggested a negative regulation between RBP2 and hsa-miR-212 in HCC. RBP2 and miR212 levels were negatively correlated (R 2 = 0.7941, Figure 1F).

Inhibition of RBP2 Expression in HCC Cells Repressed Cell Proliferation and Induced Cell Senescence by Upregulating CDKIs
Next, we investigated the mechanism by which RBP2 participates in hepatic oncogenesis in 2 hepatic cancer cell lines, HepG-2 and SMMC-7721 cells. The mRNA level of RBP2 was decreased with RBP2 siRNA knockdown, by about 80% and 72% in HepG-2 and SMMC-7721 cells, respectively (Figure 2A; Figure S1A), and RBP2 protein level was decreased ( Figure 2A). Table 1. PCR primer sequences used in the study. At the same time, H3K4me2 and H3K4me3 expression was significantly increased with RBP2 depletion ( Figure S2C). The expression of known targets of RBP2, namely, the CDKIs p16 ink4a , p21 CIP2 and p27 kip1 , was markedly increased with RBP2 siRNA knockdown ( Figure 2B; Figure S2A). As a result, the proliferation of HCC cells was sharply restricted ( Figure 2C).

Genes Primers
Cell senescence was induced with RBP2 siRNA knockdown for 72 hr in cancer cells ( Figure 2D). Therefore, CDKI expression could be regulated by RBP2, and knocking down RBP2 expression could repress cell proliferation and induce cell senescence.

Overexpression of RBP2 Blocked CDKI Expression in vitro
RBP2 mRNA and protein expression was induced with an overexpression plasmid ( Figure 3A; Figure S1B) and the expression of the CDKIs p16 ink4a , p21 CIP2 and p27 kip1 was downregulated ( Figure 3B and C; Figure S2B).
Expression of the CDKIs p21 CIP2 and p27 kip1 was Directly Regulated by RBP2 To determine whether p21 CIP2 and p27 kip1 were directly regulated by RBP2, siRNA knockdown of RBP2 expression greatly increased luciferase activity of p21 CIP2 and slightly but significantly increased activity of p27 kip1 in HepG-2 cells ( Figure 3D); ChIP assay confirmed the results ( Figure 3E). Therefore, RBP2 could directly bind to p21 CIP2 and p27 kip1 promoters and suppress their expression.
HepG-2 cells were injected into nude mice and tumors were formed ( Figure S1F). P21 CIP2 and p27 kip1 co-located with RBP2 on immunohistochemical staining of serial tissue sections of tumors: with RBP2 overexpression, the expression of p21 CIP2 and p27 kip1 was relatively low ( Figure 3F). Thus, RBP2 regulated the expression of CDKIs and affected cell proliferation, thereby contributing to the development of HCC in humans.

RBP2 was Negatively Regulated by hsa-miR-212
We confirmed that RBP2 was overexpressed in HCC and investigated the mechanism of the increased RBP2 expression. We found that RBP2 is a target of hsa-miR-212, and hsa-miR-212 could bind with 2 regions of the 39 UTR of RBP2 ( Figure 1D). With hsa-miR-212 overexpression in HepG-2 and SMMC-7721 cells, hsa-miR-212 expression was significantly increased, which confirmed the success of hsa-miR-212 overexpression ( Figure 4A). RBP2 expression was markedly repressed with hsa-miR-212 overexpression ( Figure 4B and C; Figure S1C). The expression of p16 ink4a and p27 kip1 , targets of RBP2, was increased in the 2 cell lines ( Figure 4D), which agreed with previous findings. Accordingly, cell proliferation was repressed and cell senescence induced with hsa-miR-212 overexpression ( Figure 4E and F). To further confirm that RBP2 was negatively regulated by hsa-miR-212, transfection of hsa-miR-212 inhibitor plasmid in the 2 cell lines decreased the expression of hsa-miR-212 ( Figure 5A) and increased that of RBP2 ( Figure 5A and B; Figure S1D). Accordingly, the expression of p16 ink4a and p27 kip1 was decreased with hsa-miR-212 inhibition ( Figure 5C).
To ascertain whether hsa-miR-212 directly regulated the expression of RBP2, use of an RBP2 39-UTR reporter vector significantly decreased luciferase activity with hsa-miR-212 overexpression in SMMC-7721 cells. However, co-transfection of hsa-miR-212 with the 2 mutants of the RBP2 39 UTR reporter vector (mutation of the 2 binding sites of RBP2, Table 2) reversed the luciferase activity ( Figure 5D). Thus, RBP2 was a direct target of hsa-miR-212.

Discussion
RBP2 is involved in the pathogenesis of gastric cancer, but its role and regulation in HCC was unknown. We determined the function of RBP2 and its regulation in HCC in vivo and in vitro. The expression of RBP2 was stronger in cancerous than noncancerous human tissues, but that of its binding miRNA, hsa-miR-212, was reversed. SiRNA knockdown of RBP2 significantly upregulated CDKIs, with suppression of HCC cell proliferation and induction of cell senescence. Overexpression of hsa-miR-212 had a similar effect. Conversely, inhibition of hsa-miR-212 expression upregulated RBP2 expression. We found that RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212-RBP2-CDKI pathway may be important in the pathogenesis of HCC.
Epigenetic modulation participates in various biological processes by affecting chromatin stability and transcriptional repression or activation of target genes. Epigenetic molecules may play Table 2. Primer sequences for construction of wild type (WT) and mutants (MUT1 and 2) of the 39 UTR of retinoblastoma binding protein 2 (RBP2) by the overlap extension PCR method.

Category Primers
Sequences      an important role in many types of tumorigenesis [25]. An important histone demethylase family, the JARID1 protein family, has been investigated widely in the biological process of higher organisms [18]. JARID1B/RBP2-H1/KDM5B was found to be a transcriptional regulator with tumor-suppressive potential in melamoma cells [26] and was used as a biomarker for a small subpopulation of slow-cycling melanoma cells [27]. The von Hippel-Lindau tumor-suppressor protein regulates gene expression and tumor growth via the histone demethylase JARID1C/ KDM5C/SMCX in clear-cell renal cell carcinoma [28]. The expression pattern of JARID1D/KDM5D/SMCY significantly differed in meningiomas of male and female patients and contributes to the female predominance of this tumor [29]. RBP2, a member of the JARID family protein, also named JARID1A or KDM5A, has histone demethylase activity that could demethylate H3K4me3 and H3K4me2 significantly [8,12]. Thus, RBP2 may affect the stability of chromatin through histone modulation. We first investigated the expression of RBP2 in human HCC and found the same expression as in gastric cancer. Recently, upregulation of RBP2/KDM5A expression was seen as characteristic of a drug-tolerant cancer cell subpopulation, cancer stem cells [15], which highlights the role of RBP2 in tumors and suggests its role in the initiation and development of HCC, but its mechanism in cancer was not eluciated.
MiRNAs affect tumorigenesis [30][31][32]. They can negatively regulate oncogenes and play a role as tumour suppressor genes but can also target tumour-suppressor genes and play a role as oncogenes. MiR-21 is overexpressed in most types of cancer [33][34] and Let-7 has an exclusively strong effect in lung cancer development [35]. Here, we found that RBP2 is a direct target of hsa-miR-212, and overexpressed RBP2 in HCC tissues may result from downregulated hsa-miR-212 expression. Hsa-miR-212 has been widely investigated [36][37][38][39]. We found that Hsa-miR-212 directly binds to the 39 UTR of RBP2 and suppressed its expression. The suppressed RBP2 expression led to inhibited proliferation and induced senescence of hepatocellular cells by upregulating CDKIs. So, the low expression of hsa-miR-212 in HCC may result in overexpression of RBP2 and downregulation of RBP2 targets such as CDKIs, accompanied by cell proliferation and blocking cellular senescence in HCC, which contributes to its pathogenesis. Here, we provide more evidence of epigenetic molecule regulation by miRNAs.
However, the mechanism by which hsa-miR-212 is downregulated in HCC is not known. Infection with hepatitis B virus (HBV) may play a role in this process because HCC can result from HBV infection. As well, downregulation of hsa-miR-212 may be due to hypermethylation of DNA in the hsa-miR-212 promoter because downregulation of hsa-miR-212 expression is through DNA hypermethylation in human gastric cancer [40]. Recently, miR-212 expression was found to be deregulated in lung cancer [41]. However, other mechanisms may lead to the suppression of hsa-miR-212 expression in HCC. Also, downregulated hsa-miR-212 expression may not be the only mechanism leading to the overexpression of RBP2. We found that depletion of one miRNA, hsa-miR-212, antagonized the suppression of RBP2, which led to RBP2 overexpression in HCC, but further investigation is needed to elucidate the reason for the overexpressed RBP2 in HCC. Induced epigenetic molecules could also regulate the expression of miRNAs [42][43][44], but we found no evidence of induced RBP2 affecting hsa-miR-212 expression because siRNA knockdown of RBP2 did not change the expression of hsa-miR-212 ( Figure  S1E).
In conclusion, our findings suggest that overexpressed RBP2 in HCC resulted from downregulated hsa-miR-212 expression and that overexpressed RBP2 could promote cell proliferation and suppress cell senescence by downregulating p16 ink4a , p21 CIP2 and p27 kip1 . The hsa-miR-212-RBP2-CDKI pathway in HCC may contribute to its initiation and development. Low expression of hsa-miR-212 and overexpression of RBP2 may be prognostic markers for HCC. As well, RBP2 and hsa-miR-212 expression may be a therapeutic target for HCC.

Supporting Information
Data S1 Supporting Materials and Methods.