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. 2011 May 24;19(8):1521–1528. doi: 10.1038/mt.2011.64

Tumor-specific Expression of MicroRNA-26a Suppresses Human Hepatocellular Carcinoma Growth via Cyclin-dependent and -independent Pathways

Lizao Chen 1, Jianming Zheng 2, Yan Zhang 1, Luxi Yang 1, Jiaqi Wang 1, Jian Ni 3, Daxiang Cui 3, Chaoqin Yu 4, Zailong Cai 1
PMCID: PMC3149175  PMID: 21610700

Abstract

MicroRNA-26a (miR-26a) is a tumor suppressor that is reduced in hepatocellular carcinoma (HCC). Increasing evidence indicates that the liver is a hormone-responsive organ like the breast. The purpose of this study was to investigate whether miR-26a, regulated by a human α-fetoprotein (hAFP) and human telomerase reverse transcriptase (hTERT) dual promoter, could be specifically expressed in liver tumor cells to suppress their growth and to clarify whether estrogen receptor-α (ERα) is regulated by miR-26a and involved in the HCC process. Our data show that miR-26a expression driven by a hAFP-TERT dual promoter was tumor-specific and decreased the viability of tumor cells by regulating ERα, progesterone receptor (PR) and P53 except for cyclin D2 or cyclin E2 in vitro and in vivo. Our data also show that estradiol (E2) promotes the growth of liver cancer cells similar to breast cancer cells partly via the E2-ERα pathway and that miR-26a significantly down regulates ERα and prevents the stimulation of hepatoma cell growth by E2. These data suggest that ERα, which is regulated by miR-26a, is important for liver tumor cell growth. Moreover, hAFP-TERT dual promoter-mediated miR-26a expression could specifically exert potential antitumor activity and provide a novel targeting approach for cancer therapy.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common human malignancies in the world, especially in eastern Asia and Africa,1 with an annual death rate exceeding 500,000. Unfortunately, it is not sensitive to chemotherapy or radiotherapy.2,3

In addition, increasing evidence indicates that, similar to breast tissue, the liver is a hormone-responsive organ. HCC could therefore be an estrogen-dependent cancer. The importance of sex hormone receptors, such as estrogen receptor (ER), androgen receptor, and progesterone receptor (PR), in normal liver physiology is not clear, but it has been shown that estrogens play an important role in the control of liver cell proliferation.4 Experimental models have shown that estrogens act as tumor promoters in the liver and may induce hepatocarcinogenesis in hamsters and mice.5 Further experiments suggest that in utero exposure to inorganic arsenic results in the alteration of estrogen signaling and the persistent and widespread overexpression of ERα mRNA and protein in the livers of adult male mice-bearing arsenic-induced HCC.6

A recent study clearly demonstrated that microRNA-26a (miR-26a) is a tumor suppressor that is reduced in HCC. Decreased levels of miR-26a have been associated with poor prognosis and are predictive of the therapeutic response of HCC patients to interferon-α.7 Furthermore, in an MYC-inducible model of liver cancer, animals treated systemically with miR-26a [or with control microRNA (miRNA)] using adeno-associated virus for delivery shown significant tumor regression, indicating that the reintroduction of miR-26a may be an effective strategy to treat cancer.8

Tumor-specific promoters have shown great potential for the delivery of exogenous genes to specifically targeted tissues. Human telomerase reverse transcriptase (hTERT)9 and human α-fetoprotein (hAFP) promoters have recently been reported to be effective in this capacity.10 However, the expression of genes regulated by a single tumor-specific promoter is much lower than that of genes regulated by other nontumor-specific strong promoters such as the cytomegalovirus (CMV) promoter and the SV40 promoter, and the previous research has shown that the combination of the regulatory regions of two tumor-specific promoters can enhance its transcriptional activities.11

Specific expression of a miRNA regulated by tumor-specific promoters and a combination of the regulatory regions of hTERT and hAFP promoters has not been reported. We explored the impact of miR-26a overexpression in normal liver tissues and whether the hTERT and AFP dual promoter was specific and able to efficiently control miR-26a expression and thereby suppress human cancer cell growth in vitro and in vivo. Additionally, we asked whether ERα is a target of miR-26a, and its contribution to liver cancer proliferation was investigated.

Results

Overexpression of miR-26a driven by the H1 promoter negatively regulates normal liver cell proliferation

To examine the potential influence of miR-26a overexpression on normal liver cells, we used a vector containing a strong, nonspecific mammalian promoter (h1 promoter) in this experiment. The expression of mature miR-26a in L02 cells stably transfected with the high-expression vector ph1MCG (h1-miR-26a-CMV-GFP) was twofolds higher than that of the control vector ph1CG (h1-CMV-GFP) (P < 0.05) (Figure 1a). The cell cycle assay indicated that h1MCG-transfected L02 cells significantly induced cell cycle arrest at the G1 phase (P < 0.05) (Figure 1b). Accordingly, the CCK-8 proliferation assay shown that cell growth was reduced in ph1MCG-transfected L02 cells compared with ph1CG-transfected L02 cells (Figure 1c). This result suggests that overexpression of miR-26a could negatively regulate normal liver cell proliferation.

Figure 1.

Figure 1

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MicroRNA-26a (mir-26a) negatively regulates normal liver cell proliferation, and specific expression of miR-26a specifically suppresses tumor cell proliferation. (a) Real-time PCR detected the expression of mature miR-26a in L02 cells transfected with ph1MCG or its control vector ph1CG (normalized to U6 levels). (b) Compared with the control vector ph1CG, overexpression of miR-26a negatively regulated cell proliferation. (c) In the ph1MCG group, the percentage of cells in the G1 phase of the cell cycle increased by ~20%. Accordingly, cell growth was reduced significantly. (d) Expression of mature miR-26a was detected by real-time PCR in Huh-7, SMMC-7721, and L02 cells transfected with pAT, pTM, pAM, and pATM. Relative expression was normalized to U6 levels. (e) Growth of Huh-7, SMMC-7721, and L02 cells after transfection with the indicated plasmids. The growth index was assessed at 0, 1, 2, 3, and 4 days. (f) Western blot analysis of cyclin D2 and cyclin E2 in Huh-7, SMMC-7721, and L02 cells transfected with pAT, pAM, pTM, and pATM. β-Actin served as an internal control. *P < 0.05, **P < 0.01.

Tumor-specific expression of miR-26a suppresses tumor cell proliferation significantly

To avoid the overexpression of miR-26a in normal liver cells, the hAFP and hTERT promoters were used to control miR-26a expression. Real-time PCR analysis shown that the transcription of mature miR-26a increased significantly in two tumor cell lines, Huh-7 and SMMC-7721, but not in L02 cells when they were transfected with pAT (AFP-hTERT), pTM (hTERT-miRNA26a), pAM (AFP-miRNA26a), or pATM (AFP-hTERT-miRNA26a). This suggests that the hAFP and hTERT dual promoter was effectively activated in tumor cells. Strikingly, the complex dual promoter increased transcriptional activity in both cancer cell lines (7.5-fold in Huh-7 and 2.3-fold in SMMC-7721) compared with the control vector (P < 0.05). However, the transcriptional activity of the hAFP promoter and the hTERT promoter alone or in combination in the normal liver cell line L02 was not significantly different (Figure 1d).

We next examined the effect of miR-26a expression driven by the hAFP and hTERT dual promoter on cell viability. As shown in Figure 1e, compared with control group, the cell viability of two tumor lines stably transfected with pTM, pAM, or pATM decreased in a time-dependent manner (no statistically significant difference between pTM, pAM, or pATM-transfection group, P > 0.05). No significant changes in the viability of the normal liver cell line L02 were observed up to 4 days. This result was confirmed by cell cycle analysis (Table 1) and cyclin-dependent protein analysis (known target genes of miR-26a: cyclin E2, cyclin D2) (Figure 1f).8 The basic expression of cyclin D2 and cyclin E2 differed in the two tumor lines (Supplementary Figure S1). In particular, the expression of cyclin E2 was undetectable in the Huh-7 cell line.

Table 1. Percentage of Huh-7, SMMC-7721, and L02 cells in G1 phase.

graphic file with name mt201164t1.jpg

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miR-26a directly inhibits the expression of ERα through its 3′-UTR

To further explore the underlying mechanisms by which miR-26a executes its function based on bioinformatics and cell signal nets, we tested the expression of several proteins involved in cell proliferation by western blot analysis, among them, ER-a and Bcl-6 mRNA have binding sites for miR-26a in their 3′-UTRs according to TargetScan analysis, and P53 and P21 are closely associated with cell proliferation. Our results show that the expression of ERα, P53, and P21 remarkably changed in miR-26a-transfected cells compared with control vector-transfected cells (Figure 2a), and a potential binding site of miR-26a was found in the 3′-UTR of ERα mRNA using bioinformatic algorithms (TargetScan). Moreover, perfect base pairing exists between the “seed sequence” of mature miR-26a and ERα, and the seed sequence was exactly conserved across species (Figure 2c). To further investigate whether ERα is a direct target of miR-26a, Huh-7 cells were co-transfected with a reporter plasmid (ERα_WT, Figure 2c) and miR-26a mimics or negative control. miR-26a mimic-transfected cells shown a marked reduction (34.5%) in luciferase activity compared with negative control-transfected cells, and this effect was strengthened with increasing concentrations of miR-26a mimics (P < 0.01) (Figure 2b). The same assay was then performed for another reporter plasmid, ERα_MUT (Figure 2c). As expected, the inhibition of luciferase activity by miR-26a mimics was almost abolished in the ERα_MUT-transfected cells, suggesting that the conserved region was fully responsible for miR-26a function (Figure 2b). This result suggests that miR-26a directly inhibits the level of ERα mRNA through its 3′-UTR.

Figure 2.

Figure 2

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MicroRNA-26a (miR-26a) targets ERS1, and expression of estrogen receptor-α (ERα) and miR-26a in patient hepatocellular carcinoma (HCC) tissues (T) and pair-matched adjacent tissues (N) n = 12. (a) Western blot analysis demonstrates an abundance of ERα in pATM-transfected Huh-7 cells. The band intensities were normalized to β-actin levels. (b) Dual luciferase assay of Huh-7 cells co-transfected with the firefly luciferase constructs containing the ESR1 WT or Mut 3′-UTR and miR-26a mimics or scrambled oligonucleotides as the negative control. (c) Sequence and evolutionary conservation of the miR-26a binding site in the 3′-UTRs of transcripts encoding ERα (ESR1). The interaction sites between WT and Mut 3′-UTRs of ESR1 and miR-26a are indicated. (d) Western blot analysis of ERα protein in human HCC. β-Actin serves as an internal control (gray scale is represented as the mean ± SD). Expression of (e) mature miR-26a and (f) ESR1 was detected by real-time PCR. Relative expression was normalized to U6 and β-actin levels, respectively. *P < 0.05, **P < 0.01.

A relationship between the expression of ERα and miR-26a in HCC patient samples (n = 12) was observed. Surprisingly, the expression of the ERα protein was ~31.37% lower in tumor tissues than in adjacent tissues (Figure 2d), whereas the expression of miR-26a was lower in all tumor tissues (n = 12) (Figure 2e). To clarify this phenomenon, we further analyzed the ERα mRNA level in human HCC (n = 12). Our results revealed a marked decrease of 56.61% in the HCC tissues (Figure 2f). Notably, the decrease in mRNA level was far greater than that in protein level. The data for each of the 12 samples is in Supplementary Figure S3.

miR-26a prevented hepatoma cell growth stimulated by E2 through significantly downregulating ERα

It is clear that E2 promotes breast cancer cell growth.12,13 As shown in Figure 3a, the same physiological function was found in the liver cancer cell line, in which 10−7 mol/l of E2 was more effective than 10−8 mol/l of E2. Cell growth was induced in E2-treated Huh-7 cells compared with dimethyl sulfoxide-treated cells. However, the degree of stimulation was markedly reduced in E2-treated pATM-transfected Huh-7 cells compared with dimethyl sulfoxide-treated pATM-transfected cells (P < 0.05), and ERα overexpression (pcDNA3.1-ERα) relieved the miR-26a-mediated growth inhibition of HCC cell lines. Alternatively, Fulvestrant, which blocks the physiological response of tissues to estrogen,14 was introduced in this experiment (5 × 10−6 mol/l). As expected, miR-26a blocked ERα in a similar manner to that of Fulvestrant (Figure 3b).

Figure 3.

Figure 3

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MicroRNA-26a (miR-26a) downregulates the expression of estrogen receptor-α (ERα) and partly blocks the E2-ERα pathway. (a) Growth curve of Huh-7 cells treated with different concentrations of E2 at 0, 1, 2, and 3 days. (b) Proliferation index of Huh-7 cells stably transfected with or without pATM as well as transient transfected with pcDNA3.1-ERα/pcDNA3.1 and treated with E2 or Fulvestrant on day 2. (c) Immunofluorescence assay of Huh-7 cells stably transfected with or without pATM and treated with E2 or Fulvestrant at 8 hours to detect ERα expression (red) in the cell nucleus. DAPI (blue) was used to stain the nuclei. *P < 0.05, **P < 0.01.

To further confirm that miR-26a prevents hepatoma cell growth stimulated by E2, an immunofluorescence assay was used to detect ERα expression in the cell nucleus. Interestingly, E2 caused more nuclear accumulation of ERα at 8 hours compared with control cells. However, overexpression of miR-26a driven by pATM led to a reduction in ERα in the nucleus, as did Fulvestrant (Figure 3c). This result suggests that miR-26a suppresses the nuclear accumulation of ERα and prevents hepatoma cell growth stimulated by E2 through significantly downregulating ERα.

Tumor-specific expression of miR-26a inhibits tumor cell growth in vivo

An in vivo tumorigenesis assay revealed that tumor growth was significantly slower in nude mice inoculated with pATM-transfected Huh-7 cells than in nude mice inoculated with pAT-transfected Huh-7 cells (Figure 4a). This result was also confirmed by tumor volume measurements (Figure 4b). Moreover, the level of ERα was much lower in tumor tissues in which miR-26a was overexpressed (Figure 4c).

Figure 4.

Figure 4

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MicroRNA-26a (miR-26a) driven by the human α-fetoprotein (hAFP) and human telomerase reverse transcriptase (hTERT) dual promoter inhibits tumor cell growth in vivo. (a) Nude mice were inoculated with pATM- or pAT-transfected Huh-7 cells in their flanks. Two of the five mice are shown here. (b) Representative tumor growth 5 weeks after inoculation. Tumor volume was calculated. Data are represented as the mean ± SD. n = 5. (c) Expression of estrogen receptor-α (ERα) was measured by immunohistochemistry in the tissues of mice inoculated with pATM- or pAT-transfected Huh-7 cells. (d) Hematoxylin-eosin (HE) staining of tumor sections demonstrates the character of the hepatocellular carcinoma (HCC)-bearing nude mouse model generated by inoculation with HCC cell lines (×200).

To further estimate the therapeutic effects of miR-26a, we prepared a human HCC-bearing mouse model, SMMC-LTNM (generated by inoculation with focus of HCC patient), which more closely models the clinical progression of HCC. Hematoxylin-eosin staining shown that the HCCs in the mouse model SMMC-LTNM were arranged in cords separated by blood sinusoids, which had enlarged and polymorphic nuclei, prominent nucleoli, and hyperchromatism and might contain vacuoles in affluent cytoplasm (Figures 4d and 5b).

Figure 5.

Figure 5

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Specific expression of microRNA-26a (miR-26a) for therapeutic experiments. (a) Tumor growth was observed in nude mice whose tumors were treated with the pATM or pAT mixture. (b) Hematoxylin-eosin (HE) staining of the hepatocellular carcinoma (HCC) mouse model SMMC-LTNM, which more closely models the clinical character of HCC (×200). (c) Representative tumor suppression 4 weeks after treatment. Tumor volume was calculated. Data are represented as the mean ± SD. n = 5.

Therapeutic experiments shown a marked suppression of tumor growth in nude mice whose tumors were injected with the pATM mixture compared with those injected with the pAT mixture. This result was also confirmed by tumor volume measurements (Figure 5a,c). Similarly, immunohistochemical analysis shown that the expression of AFP, PCNA, Ki-67, PR, CEA, cyclin D2, cyclin E2, and ERα decreased (Figure 6) and that of P53 and PTEN increased (Figure 6) in tumor tissues from nude mice injected with the pATM mixture. Among them, cyclin D2, cyclin E2, and ERα are direct target genes of miR-26a; AFP, PCNA, Ki-67, and CEA are important diagnostic and prognostic biomarkers for human HCC,15,16 whereas P53 and PTEN are important antioncogenes. The change in these proteins suggests that tumor-specific expression of miR-26a could inhibit tumor growth in vivo.

Figure 6.

Figure 6

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Immunohistochemistry assay of tumor tissues treated with the pATM or pAT mixture, respectively. Proteins were divided into five groups: (a) tumor marker [α-fetoprotein (AFP) and CEA] (b) cyclin-dependent pathway (cyclin D2 and cyclin E2); (c) cyclin-independent pathway [estrogen receptor-α (ERα), PR, and P53] (d) proliferation index (PCNA and Ki-67) and (e) antioncogene (P53 and PTEN). The brown color seen in the cells denotes positive staining for the aforementioned proteins. The white frame indicates a representative ×400 magnification field. The original magnification is ×200. (f) Quantitative analysis of PCNA- and Ki-67-positive cells in each group. *P < 0.05.

Discussion

One miRNA may target several mRNAs, while one mRNA may be targeted by a number of miRNAs. miRNAs are predicted to take part in the regulation of ~30% of all proteins and to play an important role in almost every biological process in cells.17 Therefore, miRNAs represent a promising new family of targets in the current era of molecular therapies in oncology.18,19 Our results show that the nonspecific overexpression of miR-26a contributes to the negative regulation of normal liver cell progression. To obtain high suppression of tumor proliferation and avoid this negative impact on normal liver cells, we designed and constructed a hAFP and hTERT dual promoter to specifically drive the expression of miR-26a in tumor tissue. The results show that the specific expression of miR-26a in tumor cells could specifically suppress tumor cell proliferation in vitro and in vivo. In addition, as shown in the Supplementary Figure S5, there was no significant difference of expression of mature miR-26a in cells transfected with pAT and scrambled sequence (purchased from Genechemat, Shanghai, China) as well as untreated cells.

Nonviral delivery methods present certain advantages over viral methods, including simple, large-scale production, and low host immunogenicity.20,21 Previously, low levels of transfection and expression of the gene held nonviral methods at a disadvantage. However, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses. Plasmid delivery by nanoparticles whose diameters are <100 nm shows a high level of transfection efficiency in the liver by systemic administration.22 We are now developing a biocompatible, nonimmunogenic, cell- and tissue-specific miR-26a systemic delivery system for liver cancer therapy.

In humans, the chronic use of estrogens is associated with an increased risk of developing liver neoplasms, such as benign nodular hyperplasia and hepatic adenoma.23 Current research shows that estrogens promote breast cell proliferation through the estrogen-ERα pathway.24,25 ERα likely plays an important role in the control of liver cell proliferation. Our results suggest that E2 promotes the growth of liver cancer cells growth similar to breast cancer cells via the E2-ERα pathway and that miR-26a significantly downregulates ERα and prevents hepatoma cell growth stimulated by E2 by reducing the level of ERα in the nucleus. This function of miR-26a is similar to the selective ER down regulator Fulvestrant. Moreover, the Dual Glo Luciferase Assay demonstrated the direct regulation of ESR1 (ERα mRNA) by miR-26a. Based on previous studies,26,27 we predicted that the expression of miR-26a is downregulated in most HCC tissues, whereas the level of ERα protein is upregulated. However, our data shown that the expression of the ERα protein and mRNA were lower in tumor tissues than that in adjacent tissues. Furthermore, the decrease in the mRNA level was far greater than that of the protein level. Based on data from a combination of in vitro experiments, we believe that the expression of ERα is regulated in the transcriptional level as well as the post-transcriptional level by miR-26a in liver cancer progression. The mechanism of transcriptional regulation is not fully understood at this time. Dhasarathy et al. found that the transcription factor snail directly downregulated the expression of ERα by binding to the ESR1 promoter sequence in breast cancer.28 Alternatively, our results and previous studies show that the expression of snail was higher in tumor tissues than that in adjacent tissues.29,30 Moreover, snail is upregulated but ERα is downregulated in human clinical specimens (Supplementary Figure S3). Thus, we speculated that ERα might be regulated at the transcriptional level by snail in human HCC. In this work, our group only focused on the mechanism of post-transcriptional regulation in which the expression of ERα is regulated by miR-26a in human HCC progression.

In addition, immunohistochemical analysis shown a significant decrease in AFP, PCNA, PR, and CEA and an increase in P53 and PTEN in pATM-transfected cell tissues, indicating that miR-26a directly or indirectly regulates the expression of these genes in HCC. The “seed sequences” of mature miR-26a and the 3′-UTR of PR mRNA exhibit perfect base pairing, and the miR-26a seed sequence is exactly conserved across species (Supplementary Figure S4). In other words, PR is likely a direct target of miR-26a and plays a potential role in estrogen-dependent cancer.

Our study shown that the expression of cyclin D2, cyclin E2, ERα, and PR was altered in pATM-transfected tissues. However, the expression of other proteins that shown no binding sites for miR-26a in their 3′-UTRs by TargetScan analysis, including AFP, CEA, P53, and PTEN, was also altered in pATM-transfected cell tissues. We presume that miR-26a inhibits tumor growth via more complex regulatory networks.

In summary, hAFP and hTERT dual promoter-driven miR-26a expression was tumor-specific and decreased the viability of tumor cells, but not of normal cells, in vitro and in vivo. In addition, miR-26a significantly downregulated ERα and prevented the stimulation of hepatoma cell growth by E2. HAFP and hTERT promoter-mediated miR-26a expression could exert potential antitumor activity and provide a novel targeting approach for the clinical therapy of a variety of cancers.

Materials and Methods

Patient samples. The use of human samples was approved by the local ethical committee. HCC tissues were collected from patients undergoing liver resection for HCC at the Eastern Hepatobiliary Surgery Hospital (Second Military Medical University, Shanghai, China). Written informed consent was obtained from each patient. All tissues were stored at −80 °C until the time of use. The characteristics of the patients are described in Supplementary Table S1.

Construction of vectors. Recombinant plasmids for tumor-specific expression were constructed using the pEGFP-N1 expression vector, which contains a CMV promoter, as the backbone. The hTERT and hAFP promoters and the pri-miRNA26a sequence were amplified by PCR using HEK293 cell genomic DNA as a template. The promoter sequence was inserted into pEGFP-N1 in place of the CMV promoter, and then the pri-miRNA26a sequence was inserted into vectors controlled by either the hTERT or hAFP promoter or both. These vectors were named as follows: hTERT-miRNA26a (pTM), AFP-miRNA26a (pAM), AFP-hTERT-miRNA26a (pATM), and control vector AFP-hTERT (pAT). A vector for upregulated, nonspecific expression of miR-26a (h1-miR-26a-CMV-GFP, ph1MCG) and its control vector h1-CMV-GFP (ph1CG) were purchased from Genechemat. The 3′-UTR of human ERα mRNA with or without a mutation that was generated with the GeneTailor site-directed mutagenesis system (Invitrogen, Carlsbad, CA,) was cloned into PGL3 (Promega, Madison, WI) using a PCR-generated fragment. The ERα expression plasmid (pcDNA3.1-ERα) was constructed using pcDNA3.1 vector and PCR-generated fragment from a human cDNA library (Supplementary Figure S2). All constructs were confirmed by DNA sequencing analysis. Sequences of primers are shown in Supplementary Table S2.

Cell culture and transfection. Huh-7, SMMC-7721, and L02 cells were obtained from the China Center for Type Culture Collection (Wuhan, China). Cells were grown in Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum and maintained in an atmosphere of 5% CO2 in a humidified 37 °C incubator.

To generate stable cell lines, 4 × 105 cells in each well of a 6-well plate were transfected with 4 µg of the desired plasmids and then selected with 1,000 µg/ml G418 for 2 weeks. For transient transfection, cells in a 6-well plate (confluency of 80–90%) were transfected with the aforementioned plasmids (4 µg) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Reverse transcription reaction and quantitative real-time PCR. Total RNA was extracted with TRIzol reagent (Invitrogen). First-strand complementary DNA was generated with a reverse transcription system kit (Applied Biosystems, Foster, CA). Random primers were used for mRNA reverse transcription, whereas specific primers were used for miR-26a and U6 reverse transcription. Real-time PCR was performed following a standard SYBR-Green PCR kit protocol on a StepOne Plus system (Applied Biosystems). β-Actin or U6 was used as an endogenous control to normalize the amount of total mRNA or miRNA in each sample. The real-time PCR were performed in triplicate and included no-template controls. Relative expression was calculated with the comparative CT method. Primer sequences are shown in Supplementary Table S2.

Cell proliferation and cell cycle assay. E2 (Sigma-Aldrich, Shanghai, China) and Fulvestrant (ICI 182,780) (Sigma-Aldrich) were dissolved in dimethyl sulfoxide (Sigma-Aldrich) and added to Dulbecco's minimum essential medium to different final concentrations before cell treatment. The proliferative potential of cells was analyzed according to the protocol of CCK-8 (Dojindo, Kumamoto, Japan).

For cell cycle assays, cells were trypsinized, collected after washing twice with phosphate-buffered saline, fixed in 70% cold ethanol, incubated with propidium iodide and then analyzed by FACS (Miltenyi, Bergisch Gladbach, Germany).

Western blot analysis. Cells or tissues were lysed and western blotted as previously described.31 Supplementary Table S3 lists the antibodies used in this research.

Luciferase reporter assay. Cells (2 × 105/well) in a 24-well plate were co-transfected with pGL3-ERα-WT (400 ng), pRL-TK (400 ng, internal control), and different concentrations of miR-26a mimics or a negative control using Lipofectamine 2000 (Invitrogen). A luciferase activity assay was performed 48 hours after transfection with the dual luciferase reporter assay system (Promega). The relative luciferase activity was normalized to Renilla luciferase activity.

Immunofluorescence assay. Huh-7 cells (5 × 104) stably transfected with pATM or its control vector pAT were plated into each well of a 24-well plate. When the cells reached a confluency of 50–60%, E2 (1 × 10−7 mol/l) and Fulvestrant (5 × 10−6 mol/l) were added to homologous growth medium. After 8 hours, an immunofluorescence assay was performed as previously described.32 DAPI (100 ng/ml; Wako, Tokyo, Japan) was used to stain nuclei.

In vivo tumorigenesis assay. Briefly, Huh-7 cells were transfected with pATM or its control vector, pAT. Stable cell lines were obtained as described above. Four-week-old male BALB nude mice (n = 5) were each inoculated with exponentially growing cells (1.0 × 107) in a total volume of 200 µl of phosphate-buffered saline by subcutaneously injection into their flanks. To avoid individual differences between mice, the two stable cell lines were injected into either flank of the same mouse; cells transfected with pATM were injected into the left side, whereas cells transfected with the control vector pAT were injected into the right side. The mice were maintained in a specific pathogen-free environment for 5 weeks and then sacrificed. We analyzed primary tumor growth by measuring tumor length (L) and width (W) and calculated tumor volume according to V = 0.4 × LW2. The animal studies were approved by the Institutional Animal Care and Use Committee of the Second Military Medical University.

Tumor-specific expression of miR-26a for HCC therapy. We prepared a human HCC-bearing mouse model (SMMC-LTNM) by transplanting histologically intact fresh human HCC tissues in male 4-week-old BALB nude mice, which subsequently formed subcutaneous transplantation tumors, and then continuously maintained this model with subcutaneous passage.33 Two weeks later, when the tumors were palpable, the mice were randomly divided into two groups before starting gene therapy. Group 1 mice (n = 5) were used as controls and received twice-weekly intratumoral injections of the control vector pAT (20 µg/mouse) with liposome (Escort Transfection Reagent E9770; Sigma-Aldrich) (plasmid: liposome 1:1) for 2 weeks. Group 2 mice (n = 5) received intratumoral injections of the pATM (20 µg/mouse) and liposome mixture twice weekly for 2 weeks. Tumor size was measured as described above. Animals were sacrificed at the end of the 2-week period, tumors were excised, and photos were taken before the tissues were processed for protein and histological analyses.

Immunohistochemistry. Immunohistochemistry was done as previously described.31 The proteins included AFP, CEA, cyclin D2, cyclin E2, ERα, PR, P53, PCNA, and Ki-67. For the proliferation index calculation, five fields per section were randomly examined at a higher magnification (×400). Two investigators examined the samples microscopically in a blinded fashion. The number of PCNA- or Ki-67-positive cells was used to determine the proliferation index. Supplementary Table S3 lists the antibodies used in this research.

Statistical analysis. Data are represented as the mean ± SD. Comparisons between two groups were performed using an unpaired Student's t-test or Mann–Whitney rank sum test. The differences in the mortality were tested using the χ2-test. P < 0.05 was considered statistically significant.

SUPPLEMENTARY MATERIAL Figure S1. Basic expression of cyclin D2 and cyclin E2 in three cell lines. Figure S2. The expression of ERα in Huh-7 cells transfected with pcDNA3.1 and pcDNA3.1-Erα. Figure S3. Expression of ERα, miR-26a, and snail in HCC patient samples. Figure S4. “Seed sequence” of mature miR-26a and 3′-UTR of PR mRNA. Figure S5. No significant difference between cells transfected with pAT, scrambled sequence or untreated cell. Table S1. Patient characteristics. Table S2. Primers used in this research. Table S3. Antibodies used in this research.

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (Project No. 30471676) and 973 project (2010CB933902). The authors who have taken part in the research of this paper declared that they do not have a relationship with the manufacturers of the materials involved either in the past or present, and they did not receive funding from the manufacturers to carry out their research. The authors thank Dr Lou for providing valuable assistance.

Supplementary Material

Figure S1.

Basic expression of cyclin D2 and cyclin E2 in three cell lines.

Click here for additional data file. (38.9KB, pdf)
Figure S2.

The expression of ERα in Huh-7 cells transfected with pcDNA3.1 and pcDNA3.1-Erα.

Click here for additional data file. (25.4KB, pdf)
Figure S3.

Expression of ERα, miR-26a, and snail in HCC patient samples.

Click here for additional data file. (200.8KB, pdf)
Figure S4.

“Seed sequence” of mature miR-26a and 3′-UTR of PR mRNA.

Click here for additional data file. (33.7KB, pdf)
Figure S5.

No significant difference between cells transfected with pAT, scrambled sequence or untreated cell.

Click here for additional data file. (84.2KB, pdf)
Table S1.

Patient characteristics.

Click here for additional data file. (33KB, doc)
Table S2.

Primers used in this research.

Click here for additional data file. (35KB, doc)
Table S3.

Antibodies used in this research.

Click here for additional data file. (36KB, doc)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Basic expression of cyclin D2 and cyclin E2 in three cell lines.

Click here for additional data file. (38.9KB, pdf)
Figure S2.

The expression of ERα in Huh-7 cells transfected with pcDNA3.1 and pcDNA3.1-Erα.

Click here for additional data file. (25.4KB, pdf)
Figure S3.

Expression of ERα, miR-26a, and snail in HCC patient samples.

Click here for additional data file. (200.8KB, pdf)
Figure S4.

“Seed sequence” of mature miR-26a and 3′-UTR of PR mRNA.

Click here for additional data file. (33.7KB, pdf)
Figure S5.

No significant difference between cells transfected with pAT, scrambled sequence or untreated cell.

Click here for additional data file. (84.2KB, pdf)
Table S1.

Patient characteristics.

Click here for additional data file. (33KB, doc)
Table S2.

Primers used in this research.

Click here for additional data file. (35KB, doc)
Table S3.

Antibodies used in this research.

Click here for additional data file. (36KB, doc)

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