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Keywords:

  • miR-125b;
  • ovarian cancer;
  • proliferation;
  • BCL3

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Micro-RNAs (miRNAs) important for post-transcriptional gene expression as negative regulators are endogenous 21- to 23-nucleotide noncoding RNAs. Many miRNAs are expressed in ovarian cancer (OC). In this study, we reported that miR-125b was underexpressed in human OC specimens. Ectopic expression of miR-125b in OC cells induced cell cycle arrest and led to reduction in proliferation and clonal formation. This inhibitory effect on OC cell growth was mediated by miR-125b inhibition of the translation of an mRNA encoding a proto-oncogene, BCL3. Furthermore, expression of miR-125b suppressed the tumor formation generated by injecting OC cells in nude mice. Our results suggest that aberrantly expressed miR-125b may contribute to OC development.

Ovarian cancer (OC) is the leading cause of cancer deaths from gynecological malignancy in western countries.1 In 2009, there are 21,550 new cases and 14,600 deaths from OC in the United States, according to the national cancer statistics.2 Unfortunately, recurrences are common and the overall prognosis is poor after optimal primary treatment with surgery and systemic chemotherapy.3, 4 To understand the mechanism by which progression of OC is regulated could help in treatment of this disease.

miRNAs are a class of non-protein-coding, endogenous 21- to 23-nucleotide RNAs that play important regulatory roles in post-transcriptional gene expression by translational repression, mRNA cleavage and mRNA decay. Mature miRNAs silence gene expression by binding to the 3′-untranslated region (UTR) of target mRNA, and initiate translational repression or cleavage of cognate mRNA, depending on the degree of sequence complementary.5 Recent studies reveal critical functions of specific miRNAs in various biological processes, including proliferation, apoptosis and cell differentiation, which are known to be dysregulated in human malignancies.6 Altered expression of miRNA has been reported in leukemia,7 lung cancer8 and other carcinomas, suggesting that they may play a role as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. For example, let-7 family was shown to regulate RAS,9 HMGA210 and FOS.11 Overexpression of let-7 in lung cancer cells decreased their growth.12 Conversely, miR-20 and miR-106a were able to regulate the tumor suppressors TGFBR2 and RB, respectively.13 Moreover, miRNAs remain largely intact in routinely collected clinical tissues. Therefore, miRNAs could be optimal at classifying poorly differentiated tumors than previous mRNA expression, and distinguishing tumors from normal ones.14–16

Genome-wide miRNA expression profiling showed that miR-125b was aberrantly expressed in human OC.17 The mechanism that miR-125b might be involved in the pathological process of OC is unknown. In this study, we found that overexpression of miR-125b decreased proliferation of human OC cells, and suppressed growth of OC cells in nude mice. In addition, BCL3, a proto-oncogene was downregulated by miR-125b at the translational level. Our study suggests that aberrant expression of miR-125b is critical for the development of human OC.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

Antibody against BCL3 was from Santa Cruz (Santa Cruz, CA), antibodies against α-tubulin and β-actin from Sigma (St Louis, MO), antibodies against cdc2, phospho-cdc2, cyclin B1 from Cell Signaling (Danvers, MA). All other reagents were from Sigma. Anti-miR-125b and nonsense of anti-miR were from GenePharma (Shanghai, PR China). Clinical specimens were collected from the patients registered at The First Hospital of China Medical University (Shenyang, China) with patients' consent and ethical committee approval.

Quantitative RT-PCR

Total RNA isolated from clinical specimens or cells using Trizol Reagent (Invitrogen, Carlsbad, CA) were reverse-transcribed into cDNA according to the previous report.18 Real-time PCR was carried out using SYBR green PCR master mix (TaKaRa, Otus, Shiga, Japan). Amplification and detection were performed using ABI Prism 7700 system (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Primers used for expression analysis were as following: MiR-125b RT primer: 5′-gtcgtatccagtgcagggtccgaggtattcgcactggatacgactcac aa-3′; MiR-125b ST forward primer: 5′-gccctccctgagacctcaa-3′; ST reverse primer: 5′-gtgcagggtccgaggt-3′; BCL3-sense: 5′-aaagaattcatggacgaggggcccgtggac-3′; BCL3- antisense: 5′-aaactcgagtcagctgcctcctggagctgg-3′; 18s rRNA-sense: 5′-cgccgctagaggtgaaattc-3′; 18s rRNA-antisense: 5′-ttggcaaatgctttcgctc-3′; GAPDH-sense: 5′-tgcaccaccaactgcttag-3′; GAPDH-antisense: 5′-gacgcagggatgatgttc-3′.

Cell culture

The human OC cell lines, SKOV3, ES2 were maintained in McCoy's 5a supplemented with 10% (v/v) fetal calf serum (Invitrogen, Carlsbad, CA). These cells were incubated at 37°C with 5% CO2.

Vector construction

Full-length human miR-125b was amplified from human genomic DNA and cloned into the pcDNA3.1 according to the previous report.19 The human wild-type BCL3 was generated by PCR from cDNA using primers as following and the PCR products were inserted into pcDNA3.1: BCL3 sense: 5-atgccccgatgccccgcgggggcca-3′; BCL3 antisense: 5-tcagctgcctcctggagctggggag-3′.

The 3′-untranslated mRNA sequences of BCL3 containing the miR-125b binding site were amplified by PCR from genomic DNA using the following primers: BCL3-3′-UTR sense: 5′-gccggccggtgcccccctccccagc-3′; BCL3-3′-UTR antisense: 5′- caaaagaaatccaaccaaaa-3′. PCR products were cloned into pGL3-control at XbaIsite. One construct was prepared by deleting 8 bp of the predicted miR-125b interaction sites in the BCL3 3′-UTR luciferase reporter construct using the Quick-Change kit (Stratagene, La Jolla, CA). Del had miR-125b site deleted and was constructed using the following primers: Del-sense: 5′-cccctccgagctggtggaccaacagccac-3′; Del-antisense: 5′-gtggctgttggtccaccagctcggagggg-3′.

Cell growth assay

Cell growth was estimated by determination of the cell number and the colony formation. The cells were transfected with miR-125b or anti-miR-125b. After culture for 24 hr, the cells were seeded at an initial density of 1 × 105 per 35-mm dish. The cells were then harvested at the indicated times and the numbers were counted using the COULTERTM (Beckman, Fullerton, CA).

A total of 500 cells from the cell lines stably transfected with miR-125b or empty vector (Ctrl) were seeded in 35-mm dishes separately in triplicate. The culture medium contained 600 mg/l G418 to maintain the miR-125b expression. Three weeks later, the colonies were fixed with 4% paraformaldehyde, permeated with 20% methanol and stained with crystal violet. The stained cells were eluted by 10% glacial acetic acid and the OD595 values were measured by spectrophotometer as the indicator of cell number.

Cell cycle analysis

Briefly, the cells harvested and fixed were incubated with 0.2 mg/ml RNase and 10 g/ml propidium iodide (PI). They were then assayed on FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ) and cell cycle distributions were analyzed by the CellQuest Pro software (Becton-Dickinson). All analyses were performed in triplicate.

Western blotting

Proteins were separated on SDS-PAGE 8–10%, transferred to PVDF membranes (Amersham, Buckinghamshire, UK) and probed with primary antibodies and secondary antibodies conjugated with horseradish peroxidase. The protein bands were visualized by the Amersham ECL system and scanned. Their densities were determined by ImageQuant 5.2 software (Amersham).

Immunostaining

The cells grown on chamber slides were fixed with 4% paraformaldehyde, washed with PBS and then incubated for 30 min with 5% normal goat serum in PBS. They were then exposed to primary antibodies and Alexa Fluor 488-goat anti-mouse secondary antibodies. Slides were mounted in medium containing Hoechst 33258 and imaged with a fluorescent microscope.

Luciferase reporter assays

For the relative luciferase reporter assay, 1.5 × 105 cells were seeded in 35-mm dishes. After 24 hr, 1 μg of wide-type BCL3 3′-UTR-luciferase construct, del and 0.25 μg of beta-galactosidase were transiently introduced into SKOV3 stable cells using FuGene6 (Roche, Diagnostics, Indianapolis, IN) following the manufacturer's protocol. Forty-eight hours after transfection, luciferase activity was measured using a commercially available system (Promega). The data were representative of at least three independent experiments in triplicate. We analyzed luciferase activity using a commercially available system (Promega).

In vivo tumorigenesis

SKOV3 cells transfected with miR-125b or anti-miR-125b separately. Each nude mouse was subcutaneously injected with 2 × 106 transfected SKOV3 cells. One month after injection, the mice were killed and the tumors were taken. Nude mice were subcutaneously injected with two stable cell lines separately. Tumor size was measured every 3–5 days. The longest and shortest diameter of the tumor was noted as d1 and d2. The tumor volume was calculated using the equation: V = d1 × d2 × d2/2. After one month, the mice were killed and dissected. Tumor weight was then measured.

Statistics

Multiple comparisons were assessed by one-way analysis of variance in nonparametric statistics; otherwise Student's unpaired t-test was used for statistical analysis. Results were considered significant difference at *p < 0.05; **p < 0.01.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Decreased expression of miR-125b in human OC specimens

It has been observed that the expression level of miR-125b was decreased in human malignancies, including breast carcinoma,20 prostate carcinoma20 and oral cancer,21 indicating a possible role of miR-125b in progression of the malignancies. Additionally, microarray assay showed that miR-125b was downregulated in human OC versus normal ovary.17 To explore the possible role of miR-125b in OC development, we first examined the expression of miR-125b in OC specimens by SYBR-Green stem-loop qRT-PCR, a real-time quantification of miRNAs with a high sensitivity, specificity and wide dynamic range.22 Stem-loop RT-PCR primers are better than conventional ones in terms of RT efficiency and specificity.18 We examined the expression of miR-125b in 28 tumor samples (see Supporting Information Table 1) and 11 normal ovaries. As shown in Figure 1, the expression levels of miR-125b in tumor samples were much lower than those in normal ovary samples. Together, these results provide us initial evidence that miR-125b may play a role in the development of human OC.

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Figure 1. The expression of miR-125b was decreased in OC samples. Quantitative analysis of the expression levels of miR-125b normalized to those of 18s rRNA by qRT-PCR. Data for each dot were mean value of one sample repeated in three independent experiments (normal, n = 11; tumor, n = 28). **p < 0.01 versus normal.

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Overexpressing miR-125b suppressed OC cell growth and inhibiting miR-125b promoted OC cell proliferation

We next examined whether miR-125b affects OC cell growth using SKOV3 and ES2 cells, two human OC cell lines, as models. As shown in Figures 2a and 2b (left panel), the growth ability of SKOV3 and ES2 cells was markedly reduced by overexpression of miR-125b. To provide further evidence that miR-125b was indeed involved in OC cell growth, we studied the effect of inhibitor of miR-125b on the cell growth. The proliferation of the cells transfected with anti-miR-125b, a sequence-specific and chemically modified, single-stranded nucleic acids designed to specifically bind to and inhibit endogenous miR-125b molecules, was increased compared with that of the cells transfected with nonsense-oligo (Figs. 2a and 2b, right panel). As shown in Supporting Information Figure 1, qRT-PCR revealed that anti-miR-125b reduced miR-125b level, suggesting that anti-miR-125b is efficiently introduced into the cells and knock down miR-125b. Moreover, stable expression of miR-125b in two SKOV3 cell clones (Stable 1 or Stable 2) reduced the growth of the cells about 37% or 26%, respectively (Fig. 2c, left panel). qRT-PCR analysis showed that miR-125b expression level was greatly increased in Stable 1 and Stable 2 cells (Fig. 2c, right panel). These results suggest that miR-125b indeed affected OC cell growth.

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Figure 2. MiR-125b suppressed OC cell growth. Growth curve of SKOV3 (a) or ES2 (b) cells transfected with miR-125b (left panel) or anti-miR-125b (right panel). Cell numbers were normalized to those in 0 hr. Data were mean ± s.e. of three independent experiments in triplicate. *p < 0.05, **p < 0.01 versus empty vector or nonsense. (c, left panel) The growth curves of Stable 1, Stable 2 and control (Ctrl) cells. Data were mean ± s.e. of three independent experiments in triplicate. **p < 0.01 versus Ctrl. (c, right panel) qRT-PCR determined the expression levels of miR-125b in Stable 1 and Stable 2 cells. Data were mean ± s.e. of three independent experiments in triplicate. **p < 0.01 versus Ctrl. (d, left panel) Representative colonies formed by control (Ctrl), Stable 1 or Stable 2 cells. (d, right panel) Quantification of the colonies formed by control (Ctrl), Stable 1 or Stable 2 cells. Data were mean ± s.e. of three independent experiments. **p < 0.01 versus Ctrl.

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The notion was further tested in the clonal formation assay. The number and size of the colonies formed were markedly reduced in Stable 1 and Stable 2 cells (Fig. 2d). Together, these results suggest that miR-125b was indeed involved in the negative regulation of OC cell growth.

Upregulating miR-125b arrested OC cells at G2 phase

To elucidate the mechanism by which overexpression of miR-125b suppresses OC cell proliferation, we studied the effect of overexpression of miR-125b on cell cycle. We examined cell cycle profile of SKOV3 cells stably expressed miR-125b. As shown in Figures 3a and 3b, the percentage of cells found in G2/M phase was substantially increased with a concomitant reduction in the percentage of cells in G0/G1 phase.

Western blot analysis further showed that in Stable 1 and Stable 2 cells, the level of phosphorylated form of Cdc2 (Tyr 15) was markedly increased without affecting the total level of Cdc2, and the level of cyclin B1 was increased comparing with the control cells (Fig. 3c). Moreover, staining the cells with phospho-histone H3 (Ser10) antibody and Hochest showed that the percentage of mitotic cells was 2.63 ± 0.24, 1.25 ± 0.17 or 1.78 ± 0.39% in Ctrl, Stable 1 or Stable 2 cells, respectively (Fig. 3d and Supporting Information Fig. 2).These results suggest that upregulating miR-125b induces G2 phase arrest in OC cells.

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Figure 3. Upregulating miR-125b arrested OC cells at G2/M phase. (a) Flowcytometric analysis of the Stable 1, Stable 2 or control (Ctrl) cells. Quantitative analysis of the data was shown in (b). Data were mean ± s.e. of three independent experiments. **p < 0.01 versus Ctrl. (c) Western blot analysis of total cell lysates isolated from Ctrl, Stable 1 or Stable 2 cells using the indicated antibodies. The phosphorylation of Cdc2 at Tyr 15 and the level of cyclin B1 were increased in Stable 1 or Stable 2 cells. (d) Mitotic index of Ctrl, Stable 1 and Stable 2 cells. The mitotic index was presented as the percentage of phospho-histone H3 (Ser10) positive cells in total cells counted. A total of 4,000 cells were counted for each experiment. Data were mean ± s.d of three independent experiments. **p < 0.01 versus Ctrl.

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BCL3 was a target gene of miR-125b in OC cells

We then investigated the mechanisms by which miR-125b inhibits OC cell proliferation. Online search of miR-125b targeting genes by miRTarAS revealed that BCL3, a proto-oncogene associated with increased proliferation,23 could be a potential target of miR-125b. To verify whether BCL3 is a target of miR-125b, we set up a luciferase reporter assay. A portion of the 3′-UTR of BCL3, including miR-125b target sites, was cloned into the downstream of pGL3-control plasmid (Fig. 4a). The SKOV3-stable 1 cells were cotransfected with the reporter vector and beta-galactosidase. As shown in Figure 4b, the luciferase activity of 3′-UTR of BCL3 in Stable 1 cells was much lower than that in control cells. We also constructed another reporter vectors containing the deletional 3′-UTR of BCL3, del (Fig. 4a). The luciferase activity of del was rescued in Stable 1 cells compared with control cells (Fig. 4b). These results suggest that 3′-UTR of BCL3 transcript is a target of miR-125b.

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Figure 4. BCL3 was a target of miR-125b. (a) BCL3 mRNA 3′-UTR putative sites targeted by miR-125b. Lines indicate the deleted nucleotides. (b) Stable 1 cells were transfected with BCL3 3′-UTR reporter vector or 3′-UTR del. Data were mean ± s.e. of three independent experiments. **p < 0.01 versus Ctrl cells. (c) Western blot analysis of total cell lysates extracted from indicated transfected cells using the indicated antibodies. Quantitative analysis of the relative protein levels of BCL3 normalized to those of β-actin was shown at the right panel. Data were mean ± s.e. of three independent experiments.*p < 0.05, **p < 0.01 versus Ctrl cells or nonsense. (d) Overexpression of BCL3 increased the growth of Stable 1 or Stable 2 cells 72 hr after transfection. Cell numbers were normalized to Ctrl cells. Data were mean ± s.e. of three independent experiments in triplicate. **p < 0.01 versus Ctrl cells.

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We next examined whether miR-125b could regulate endogenous BCL3 expression. Compared with control, endogenous BCL3 mRNA level was not changed in Stable 1 cells (data not shown). We then used SKOV3 and ES2, two human OC cell lines, for further study. These cells were transfected with miR-125b or anti-miR-125b, respectively. The BCL3 protein level was downregulated or upregulated when these cells were transfected with miR-125b or with anti-miR-125b, respectively (Fig. 4c). Together, these results suggest that miR-125b regulates endogenous BCL3 protein expression through translational repression pathways.

MiR-125b controlled the proliferation of OC cells through BCL3

It has been shown that BCL3 is a prime candidate for inducing cell proliferation because it binds to NF-κB p50 and p52 dimers in the nucleus, resulting in expression of a variety of cellular genes, which are involved in proliferation.24 Given the fact that miR-125b was involved in proliferation of OC cells and BCL3 was a target of miR-125b, we next tested whether overexpression of BCL3 could rescue inhibition of proliferation by miR-125b in OC cells. As shown in Figure 4d, proliferation of Stable 1 and Stable 2 cells were decreased compared with control cells, whereas growth of Stable 1 or Stable 2 cells transfected with BCL3 (Supporting Information Fig. 2), were not different from that of control cells. These results indicate that miR-125b controlled proliferation of OC cells through regulation of BCL3 expression.

Expression of miR-125b inhibited the development of tumor in nude mice

To provide direct evidence that miR-125b was responsible for OC development, we subcutaneously injected SKOV3 cells transfected with either miR-125b or anti-miR-125b into the flank of nude mice. A week after implantation, xenografted tumors could be seen. We then measured tumor sizes every 3–5 days within 1 month post-implantation. Tumors formed by the cells transfected with miR-125b grew much slower than those formed by the cells transfected with empty vector (Fig. 5a), and tumors formed by the cells transfected with anti-miR-125b grew much faster than those formed by the cells transfected with nonsense (Fig. 5b). After 4 weeks, all nude mice were killed and the tumors were taken out and weighed. At the macroscopic observation, the differences in tumor size and volume among the two groups were indicated. The average weight of the tumor generated from empty vector was 1.498 ± 0.645, whereas the average weight of the tumor generated from miR-125b was 0.7472 ± 0.367 (gram, p < 0.01). The average weight of the tumor generated from nonsense was 0.557 ± 0.172, whereas the average weight of the tumor generated from anti-miR-125b was 0.944 ± 0.284 (gram, p < 0.01) (Fig. 5c), respectively. We then further confirmed these findings using Stable cells. We subcutaneously injected Ctrl, Stable 1 and Stable 2 cells into the nude mice to detect the tumorigenic abilities of these cells. As shown in Figure 5c, the tumor volumes determined at different times after the injection in Ctrl group were greater than those in Stable 1 and Stable 2 group. The average weight of the tumor generated from Ctrl was 0.486 ± 0.138, whereas the average weight of the tumor generated from Stable 1 was 0.219 ± 0.071 (gram, p < 0.01) and Stable 2 was 0.355 ± 0.176 (gram, p < 0.05) (Fig. 5e), respectively. These results suggest that miR-125b was critical for the development of OC in vivo.

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Figure 5. MiR-125b suppressed the development of tumor in nude mice. Tumor formation generated by the SKOV3 cells. (a) Transfected with miR-125b or anti-miR-125b. (b) Nude mice were subcutaneously injected with 2 × 106 transfected cells. Representative images (left) and quantification analysis (right) of the tumors formed. The tumor sizes were measured and calculated every 3–5 days as described in Material and Methods section. Data were mean ± s.d. of eight to nine mice. *p < 0.05, **p < 0.01 versus empty vector or nonsense. (c) Determination of the tumor growth. Nude mice were subcutaneously injected with Ctrl (n = 5), Stable 1 (n = 5) or Stable 2 (n = 5) at 2 × 106 cells per mice separately. Data were mean ± s.d. of five mice. *p < 0.05, **p < 0.01 versus Ctrl cells. (d) Measurement of the final tumor weight. Data were mean ± s.e. for eight to nine mice. **p < 0.01 versus empty vector or nonsense. (e) Measurement of the final tumor weight. Data were mean ± s.e. for five mice. *p < 0.05, **p < 0.01 versus Ctrl cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we have shown that miR-125b plays a critical role in OC development. Several lines of evidence support this conclusion. First, expression level of miR-125b was greatly decreased in human OC samples. Second, expression level of miR-125b affected OC cell growth in culture. Third, overexpression of miR-125b arrested OC cells at G2 phase. Forth, BCL3 was a target for miR-125b in OC cells. Finally, expression level of miR-125b affected tumor formation in nude mice. Therefore, our results are consistent with an idea that miR-125b is critical for the development of human OC.

Micro-RNAs are known to regulate the expression of genes involved in the control of development, proliferation, apoptosis and stress response.25 Recent studies showed a direct link between miRNAs and human cancers.26, 27 Since a common set of miRNAs may be deregulated in many tumor types, it is tempting to speculate that these miRNAs have anti-tumorigenic properties.28 Relevant targets for cancer miRNAs can be either recessive (tumor suppressor) or dominant (oncogene) cancer genes. As previously shown, the important cancer genes are regulated by aberrant expression of miRNAs, such as let-7 and Ras,9miR-16 and Bcl-27 and miR-17-5p and E2F1.29 In fact, the miRNA signatures obtained comparing different histotypes of OC with the normal tissue are overlapping in most cases. Recent studies identified a number of miRNAs altered in human OC and probably was involved in the biology of this malignancy.15, 19, 30–32MiR-125b has a lower expression in human OC compared with the normal ones.17 Our results obtained from gain-of-function and loss-of-function approaches indicated that miR-125b suppressed proliferation and inhibition of miR-125b promoted OC cell proliferation. As shown in Supporting Information Figure 4, the activity of caspase 3 was no different in control, Stable 1 and Stable 2 cells. These results suggest that ectopic expression of miR-125b did not produce apoptosis pathway.

The function of miRNAs is to regulate their target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation. Computational algorithms are focused on predicting and validating miRNA gene targets.33 Through analysis using miRTarAS and mirScan, we identified among the regulated genes, BCL3, as a possible target of miR-125b. Moreover, BCL3 is associated with increased proliferation as a proto-oncogene. Interestingly, two different interaction sites were predicted for miR-125b, positions (1,628–1,649)-for miR-125b* and (1,710–1,731)-for miR-125b, on the BCL3 3′-UTR (NM_005178.4). As shown in Supporting Information Figure 5, the deletion of positions (1,628–1,649) could also rescue the luciferase activity in Stable 1 cells compared with control cells. These results suggest that BCL3 was a target of miR-125b* and miR-125b. BCL3 has been found as a proliferative factor.23, 34 BCL3 is able to form a complex with the NF-kB p50 and p52 isoforms to lead activation of cyclin-D1 promoter which is responsible for increase in cell proliferation and enhancement in tumor growth.35 BCL3 is also able to block apoptosis in IL-4-deprived cells.36 Although we found that overexpression of BCL3 rescued miR-125b-suppressed OC cell growth, we did not find that its expression affected OC cell survival. Therefore, the critical role that miR-125b plays in OC growth may be coupled to its regulation of BCL3.

In conclusion, overexpression of miR-125b suppressed OC cell growth in culture and in nude mice. MiR-125b controlled G2 phase progression and affected proliferation of OC cells by targeting BCL3. An important implication of current study is that miR-125b might be a potential target for therapeutic intervention to human OC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr. YZ Wang for critical review of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_25575_sm_suppinfofig1.tif96KSupporting Information Figure 1
IJC_25575_sm_suppinfofig2.tif487KSupporting Information Figure 2
IJC_25575_sm_suppinfofig3.tif136KSupporting Information Figure 3
IJC_25575_sm_suppinfofig4.tif167KSupporting Information Figure 4
IJC_25575_sm_suppinfofig5.tif143KSupporting Information Figure 5
IJC_25575_sm_suppinfotable1.doc84KSupplemental Table 1. OC patient's clinicopathologic characteristics
IJC_25575_sm_suppinfo.doc27KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.