Regulation of the transcription factor NF-κB1 by microRNA-9 in human gastric adenocarcinoma

Background MicroRNAs (miRNAs) are a new class of naturally occurring, small, non-coding RNAs that regulate protein-coding mRNAs by causing mRNA degradation or repressing translation. The roles of miRNAs in lineage determination and proliferation, as well as the localization of several miRNA genes at sites of translocation breakpoints or deletions, have led to speculation that miRNAs could be important factors in the development or maintenance of the neoplastic state. Results We showed that miR-9 was downregulated in human gastric adenocarcinoma. Overexpression of miR-9 suppressed the growth of human gastric adenocarcinoma cell line MGC803 cell as well as xenograft tumors derived from them in SCID mice. Bioinformatics analysis indicated a putative miR-9 binding site in the 3'-untranslated region (3'UTR) of the tumor-related gene NF-κB1 mRNA. In an EGFP reporter system, overexpression of miR-9 downregulated EGFP intensity, and mutation of the miR-9 binding site abolished the effect of miR-9 on EGFP intensity. Furthermore, both the NF-κB1 mRNA and protein levels were affected by miR-9. Finally, knockdown of NF-κB1 inhibited MGC803 cell growth in a time-dependent manner, while ectopic expression of NF-κB1 could rescue MGC803 cell from growth inhibition caused by miR-9. Conclusion These findings indicate that miR-9 targets NF-κB1 and regulates gastric cancer cell growth, suggesting that miR-9 shows tumor suppressive activity in human gastric cancer pathogenesis.


Background
In recent years, a new class of naturally occurring, small, non-coding RNAs called microRNAs (miRNAs) have been discovered in plants and animals [1][2][3]. Unlike protein-coding mRNAs, miRNAs are first transcribed as long primary transcripts in the nucleus, and then cleaved to produce stem-loop-structured precursor molecules (pre-miRNAs) by Drosha [4]. Pre-miRNAs are then exported to the cytoplasm, where the RNase III enzyme Dicer further processes them into mature miR-NAs (~22 nucleotides). Mature miRNAs negatively regulate gene expression at the post-transcriptional level. Through specific targeting of the 3'-untranslationed regions (3'UTR) of multicellular eukaryotic mRNAs, miRNAs downregulate gene expression by degrading target mRNAs or repressing their translation [5,6]. Since miRNAs bind the 3'UTR of target genes through partial sequence homology, miRNAs could control as much as 30% of all protein-coding genes [7].
An accumulating body of evidence has revealed critical functions for miRNAs in diverse biological processes, such as proliferation, apoptosis, and cell differentiation [8,9]. Thus, deregulation of miRNA expression may lead to a variety of disorders including many types of cancers [10]. Previous studies have identified cancer-specific miRNAs in many kinds of cancers, including ovarian cancer [11], lung cancer [12], breast cancer [13], hepatocellular carcinoma [14], and brain cancer [15,16]. In addition, miRNA genes are frequently located near genomic breakpoints [16] or are affected by copy number alterations [17]. Also, factors involved in miRNA biosynthesis may be dysregulated in human tumors [18,19]. Furthermore, a recent study described aberrant hypermethylation as a mechanism for miRNA genes including miR-9 inactivation and downexpression in human breast cancer [20]. Meanwhile, another study provided evidence that miR-9 acts as a tumor suppressor gene in recurrent ovarian cancer [21].
Gastric cancer is the second most common cause of cancer death in the world [22], and adenocarcinoma is the most common type of gastric cancer. In this study, we detected differential expression of miR-9 in human gastric adenocarcinoma and adjacent normal tissues through quantitative RT-PCR, and hypothesized miR-9 as a tumor suppressor. Consistent with this hypothesis, we observed that overexpression of miR-9 inhibited the growth of the gastric adenocarcinoma cell line MGC803 in vitro and in vivo. Subsequently, we predicted and confirmed that the tumorrelated transcription factor NF-B1 was a direct target of miR-9 and was negatively regulated by miR-9 at the post-transcriptional level. Finally, in MGC803 cells, suppression of NF-B1 expression by specific small interfering RNA (siRNA) could also inhibit MGC803 cell growth, while ectopic expression of NF-B1 could rescue MGC803 cell from growth inhibition caused by miR-9.

Clinical Specimen and RNA Isolation
Nine pairs of gastric samples, including nine human gastric adenocarcinoma tissue samples and nine matched normal gastric tissue samples, were obtained from the Tumor Bank Facility of Tianjin Medical University Cancer Institute and Hospital and the National Foundation of Cancer Research (TBF of TMUCIH & NFCR) with patients' informed consent. The category of gastric samples was confirmed by pathological analysis. The large RNA and small RNA of tissue samples were isolated using mirVana™ miRNA Isolation Kit (Ambion) according to the manufacturer's instructions.

Cell Culture and Transfection
The human gastric adenocarcinoma cell line MGC803 was grown in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum, 100 IU/ml of penicillin and 100 μg/ml of streptomycin, incubated at 37°C in a humidified chamber supplemented with 5% CO 2 . Transfection was performed with Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer's protocol. Spiked RFP-expressing vector was used to monitor transfection efficiency.

Cell Proliferation Assay
Cells were seeded in 96-well plate at 4 000 cells per well the day before transfection. The cells were transfected with pri-miR-9 or control vector at a final concentration of 5 ng/μl as described above. To detect the dosedependent effects, we gradually increased concentration of pri-miR-9 from 0 ng/μl to 15 ng/μl. To examine the time-dependent effects of si-NF-B1 on MGC803 cells, MTT assay was used to measure the viable, proliferating cells at 24, 48, and 72 h after transfection. The absorbance at 570 nm was measured using a μQuant Universal Microplate Spectrophotometer (Bio-tek Instruments).

Colony Formation Assay
After transfection, cells were counted and seeded in 12well plates (in triplicate) at 100 cells per well. Fresh culture medium was replaced every 3 days. Colonies were counted only if they contained more than 50 cells, and the number of colonies was counted from the 6th day after seeding and then the cells were stained using crystal violet. The rate of colony formation was calculated with the equation: colony formation rate = (number of colonies/number of seeded cells) ×100%.

In vivo Tumor Xenograft Studies
To establish the stable miR-9-overexpression cell line and the control cell line, MGC803 cells were transfected with pcDNA3/pri-miR-9 (pri-miR-9) or pcDNA3 (control), followed by selection for 20-30 days in complete medium supplemented with 800 μg/ml of G418 (Invitrogen). Single colonies were picked and amplified, and the expression level of miR-9 was detected by real-time RT-PCR. The stable miR-9-overexpression MGC803 cells or control cells were inoculated with 4 × 10 6 cells per site bilaterally on the axillary fossae of female athymic nude mice aged 6-8 weeks. Tumor size was monitored by measuring the length and width with calipers, and volumes were calculated with the formula: (L × W 2) × 0.5, where L is the length and W is the width of each tumor. The mice used in this experiment were maintained under specific pathogen-free conditions and handled in accordance with NIH Animal Care and Use Committee regulations.

MiRNA Target Prediction
MiRNA predicted targets were predicted using the algorithms TargetScan, PicTar, and miRBase Targets. To identify the genes commonly predicted by the three different algorithms, results of predicted targets were intersected using MatchMiner.
MGC803 cells were transfected with pri-miR-9 or control vector pcDNA3 in 24-well plates, and then with the reporter vector pcDNA3/EGFP-NF-B1 3'UTR or pcDNA3/EGFP-NF-B1 3'UTRmut on the next day. The vector pDsRed2-N1 (Clontech) expressing RFP was spiked in and used for normalization. The intensities of EGFP and RFP fluorescence were detected with Fluorescence Spectrophotometer F-4500 (HITACHI).

Quantitative RT-PCR
To detect the relative level of NF-B1 transcript, realtime RT-PCR was performed. Briefly, a cDNA library was generated through reverse transcription using M-MLV reverse transcriptase (Promega) with 2 μg of the large RNA extracted from gastric tissue samples or MGC803 cells. The cDNA was used for the amplification of NF-B1 gene and the β-actin gene was used as an endogenous control for the PCR reaction. PCR was performed under the following conditions: 94°C for 4 min followed by 40 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 1 min. PCR primers were: NF-B1 sense and NF-B1 antisense were as above; β-actin sense, 5'-C GTGACATTAAGGAGAAGCTG-3'; and β-actin antisense, 5'-CTAGAAGCATTTGCGGTGGAC-3'.

Western Blot
MGC803 cells were transfected and lysed 48 h later with RIPA lysis buffer and proteins were harvested. All proteins were resolved on 10% SDS denatured polyacrylamide gel and then transferred onto a nitrocellulose membrane. Membranes were incubated with anti-NF-B1 antibody or anti-GAPDH antibody with blotto overnight at 4°C. The membranes were washed and incubated with horseradish peroxidase (HRP) conjugated secondary antibody. Protein expression was assessed by enhanced chemiluminescence and exposure to chemiluminescent film. Lab Works™ Image Acquisition and Analysis Software (UVP) was used to quantify band intensities. Antibodies were purchased from Tianjin Saier Biotech and Sigma-Aldrich.

Statistical Analysis
Data are expressed as means ± standard deviation (SD), and P ≦ 0.05 is considered as statistically significant by Students-Newman-Keuls test.

Quantitative Analysis of miR-9 Expression in Human Gastric Adenocarcinoma
To test the expression of miR-9 in human gastric adenocarcinoma and adjacent normal tissues, real-time RT-PCR assay was performed in nine pairs of gastric tissue samples.
It was shown that miR-9 expression level was generally and significantly lower in gastric adenocarcinoma tissues than in matched normal gastric tissues (figure 1).

Suppression of Gastric Adenocarcinoma Cell Proliferation by Overexpression of miR-9 in vitro
To determine the role of miR-9 in tumor cell proliferation, a miR-9 overexpression vector, pcDNA3/pri-miR-9 (pri-miR-9), was constructed. The validity of miR-9 ectopic expression was confirmed by quantitative RT-PCR, which revealed a 13-fold increase of miR-9 expression in pri-miR-9-transfected cells than in the control group ( figure 2A). To test the effects of miR-9 overexpression on MGC803 cell proliferation, we investigated cell growth by MTT assay and found that pri-miR-9 could reduce MGC803 cell growth (figure 2B). Also, this effect showed a dose-dependent manner. At the concentration of 2.5 ng/μl and 15 ng/μl of pri-miR-9 plasmid, cell growth was inhibited by 15% and 45%, respectively (figure 2C). To further confirm the effect of miR-9 overexpression on the growth of gastric cancer cells, colony formation assay was performed. The colony formation rate of MGC803 cells transfected with pri-miR-9 was significantly lower than the control group ( figure 2D). These results indicated that overexpression of miR-9 showed an anti-proliferative effect.

Overexpression of miR-9 Inhibits Xenograft Tumor Growth in vivo
To further determine whether miR-9 is involved in tumorigenesis, we established a stable miR-9-overexpression cell line as well as a control cell line. Cells of either line were injected into the axillary fossa of SCID mice, and the tumor growth activity was measured. Tumors derived from pri-miR-9-treated MGC803 cells grew substantially slower compared with the control group during the whole tumor growth period ( figure  3B). When tumors were harvested, the average volume of tumors derived from the pri-miR-9 group was only half of that in the control group (figures 3A &3C). These results were consistent with the effects of miR-9 overexpression in vitro and strongly suggested that miR-9 could inhibit gastric cancer cell growth.

NF-B1 is a Candidate Target Gene of miR-9
MiRNAs can function as tumor suppressors or oncogenes, depending on whether they specifically target oncogenes or tumor suppressor genes [25][26][27]. Tumor suppressive miRNAs, such as miR-9, are usually underexpressed in tumors and may fail to control some of the oncogenic genes. Although underexpression of miR-9 in some types of tumors suggested its role in cancer development, the underlying mechanism is still unclear because little is known of miR-9 targets. Therefore, identification of miR-9-regulated targets is a necessary step to understand miR-9 functions. We used a threestep consequential approach to identify miR-9 target genes. First, target genes were predicted by bioinformatics analysis; second, target genes were validated by fluorescent reporter assay; and finally, the correlation of miRNA expression and target protein expression were detected to confirm the specific miRNA:mRNA interactions. The target genes of miR-9 were predicted using three programs, known as PicTar, TargetScan, and miR-Base Targets. Of the predicted target genes, the oncogene NF-B1, whose mRNA 3'UTR contained a putative binding site of miR-9, was identified. This analysis is consistent with the model in which tumor suppressor miRNAs inhibit tumor development by targeting and negatively regulating oncogenes.

NF-B1 Carries a Functional miR-9 Binding Site
The mRNA 3'UTR of candidate miR-9 target gene NF-B1 carries a putative miR-9 binding site (figure 4A). To confirm that this site was responsible for the negative regulation by miR-9, we cloned the putative 3'UTR binding site into the downstream of an enhanced green fluorescence protein (EGFP) reporter gene (EGFP-NF-B1 3'UTR) and co-transfected this vector with pri-miR-9 or the control vector into MGC803 cells. The intensity of EGFP fluorescence of cells transfected with pri-miR-9 was decreased for about 1.7-fold, which was a statistically significant reduction compared with the control group ( figure  4C). To further determine the function of the miR-9 binding site, we constructed another EGFP reporter vector containing the NF-B1 3'UTR but with a mutated miR-9 binding site (figure 4B). As a result, pri-miR-9 had no effect on the intensity of EGFP fluorescence in this 3'UTR mutant vector (figure 4C), highlighting the importance of this miR-9 binding site. These results suggested that NF-B1 is a direct target of miR-9.

MiR-9 Negatively Regulates NF-B1 at the Post-Transcriptional Level
Translational repression is regarded as a major mechanism for miRNA regulation of target gene expression [28].
To determine whether miR-9 suppresses endogenous NF-B1 through translational repression, MGC803 cells were transfected with pri-miR-9 and the expression of NF-B1 protein was examined by Western blot. It was shown that the amount of NF-B1 protein was decreased after overexpression of miR-9 (figure 4D), suggesting that miR-9 negatively regulates endogenous NF-B1 protein expression through translational repression mechanism. Furthermore, overexpression of miR-9 in MGC803 cells Figure 2 Overexpression of miR-9 suppresses tumor cell growth in vitro. (A) In pcDNA3/pri-miR-9 transfected MGC803 cells, the mature miR-9 level was significantly increased. (B) MGC803 cells were transfected with pri-miR-9, control vector or pri-miR-9 with pcDNA3/NF-B1, and cell growth activity was determined at 72 h post-transfection by MTT assay. Values are means ± SD of three duplications and the relative cell growth activity is shown (*P < 0.05). (C) MGC803 cells were transfected with gradually increasing concentrations of pri-miR-9 from 0 ng/μl to 15 ng/μl (final concentration) and the dose-dependent anti-proliferative effects were detected by MTT assay. (D) Cell independent growth activity was detected by colony formation assay. MGC803 cells were transfected with pri-miR-9, control vector or pri-miR-9 with pcDNA3/NF-B1, and then seeded in 12-well plates. The number of colonies was counted from the 6th day after seeding. The colony formation rate was calculated and is shown. (E) The validity of NF-B1 ectopic expression vector pcDNA3/NF-B1 was confirmed by Western blot assay. MGC803 cells were transfected with pri-miR-9 as well as pcDNA3/NF-B1 or control vector. The NF-B1 protein level was detected by Western blot assay. GAPDH protein was regarded as endogenous normalizer and the relative NF-B1 protein quantity is shown.
Wan et al. Molecular Cancer 2010, 9:16 http://www.molecular-cancer.com/content/9/1/16 could also decrease the endogenous NF-B1 mRNA level ( figure 4E). In the nine pairs of gastric tissues, the NF-B1 mRNA level showed a predominant up-regulation in gastric cancer tissues (figure 5). These data suggested that miR-9 negatively regulate the expression of NF-B1 through mRNA cleavage mechanism.

NF-B1 Promotes Cell Proliferation and Independent Growth in vitro
Previous studies have shown that the up-regulation of NF-B1 is an important feature of many transformed cells, and that NF-B1 functions as an oncogene. Accordingly, we detected whether NF-B1 affects MGC803 cell growth. We constructed the pSilencer/si-NF-B1 (si-NF-B1) plasmid and confirmed that the expression of NF-B1 protein was effectively suppressed by the siRNA expression vector (figures 6A). Then, the effect of NF-B1 knockdown on cell growth was evaluated by MTT assay. As a result, suppression of NF-B1 decreased cell growth in a time-dependent manner. At 72 h after transfection, we observed that cells transfected with the NF-B1 knockdown vector grew more slowly than the control group ( figure 6B). Also, the colony formation rate of the cells transfected with si-NF-B1 vector was markedly lower than the control group (figures 6C &6D). Moreover, ectopic expression of NF-B1 could rescue MGC803 cell from growth inhibition caused by miR-9, both in MTT assay (figure 2B) and colony formation assay ( figure 2D). The validity of pcDNA3/NF-B1 vector was confirmed by Western blot assay (figure 2E). These data, consistent with the previous findings [29], provided further evidence that NF-B1 is an oncogene. The oncogenic role of NF-B1 in gastric cancer may explain why overexpression of miR-9 can inhibit gastric cancer cell growth.

Discussion
In the last few years, several studies have shown the dysregulation of miRNAs in various types of cancers [30][31][32]. Identification of cancer-specific miRNAs and their targets is critical for understanding their role in tumorigenesis and may be important for defining novel therapeutic targets [33][34][35]. Here, we focused on the role of miR-9 in the pathogenesis of human gastric adenocarcinoma. First, we examined miR-9 expression in gastric adenocarcinoma and matched normal gastric tissues by real-time RT-PCR assay as previously described [24]. This method uses a stem-loop reverse transcription primer and specially designed PCR primers to ensure the specificity of miRNA amplification. Meanwhile, U6 snRNA was also detected for normalization of Figure 3 Suppression of growth activity of xenograft gastric adenocarcinoma derived from MGC803 cells in SCID mice by overexpression of miR-9. (A) The graphs show the differences between tumor volume in pri-miR-9 group and control group. (B) Tumor volume was measured after injection of MGC803 cells stably expressing pri-miR-9, and the tumor volume growth curve was drafted. (C) The difference of tumor volume between the pri-miR-9 group and control group was statistically significant (*P < 0.05). expression in different samples. We discovered that from a total of nine pairs of matched advanced gastric adenocarcinoma tissue samples, the level of miR-9 was downregulated in tumor tissues compared to the matched normal tissues.
Several studies on the role of miR-9 deregulation in human oncogenesis have been reported. The aberrant hypermethylation study was done using the comprehensive methylation analysis approach, with breast cancer, normal breast tissues, and breast cancer cell lines as references. They found that the epigenetic inactivation of miR-9-1 in human breast cancer was due to aberrant hypermethylation, and found a strong correlation between hypermethylation of miR-9-1 and concomitant downregulation [20]. Interestingly, taking advantage of miRNA expression analysis and real-time TaqMan PCR, it was also found that miR-9 expression was decreased in recurrent ovarian cancer tissues compared to primary cancer tissues [21]. In addition, miR-9, miR-148a, and miR-34b/c were also underwent specific hypermethylation-associated silencing in cancer cells compared with normal tissues [36]. Hence, the Figure 4 NF-B1 is a direct target of miR-9. (A) The predicted miR-9 binding site on NF-B1 mRNA 3'UTR is shown. (B) Deletion mutation at the miR-9 "seed region" binding site on the NF-B1 mRNA 3'UTR is shown. Four nucleotides were deleted on the mutated 3'UTR. (C) MGC803 cells were transfected with the wild type of EGFP-NF-B1 3'UTR (Wt. UTR) or mutated EGFP-NF-B1 3'UTR (Mut. UTR) reporter vector as well as pri-miR-9 or control vector. Although pri-miR-9 suppressed the EGFP fluorescence intensity of EGFP-NF-B1 3'UTR, mutation of the miR-9 binding site abolished the effect of miR-9 on the EGFP fluorescent intensity. (D) MGC803 cells were transfected with pri-miR-9 or control vector, and the NF-B1 protein (P105 and P50) expression level was evaluated by Western blot. GAPDH protein was regarded as endogenous normalizer and the relative NF-B1 protein quantity is shown. (E) MGC803 cells were transfected with pri-miR-9 or control vector, and expression of NF-B1 mRNA was measured by quantitative RT-PCR. β-actin mRNA was regarded as an endogenous normalizer and the relative NF-B1 mRNA expression level is shown. (*P < 0.05) high frequency of aberrant regulation of miR-9 in different types of cancer tissues and cells suggests that downregulation of miR-9 might play an important role in oncogenesis.
It has been presumed that miRNAs suppressed in cancers may normally function as tumor suppressor genes. Therefore, we hypothesized that miR-9 is a growth inhibition factor in human gastric adenocarcinoma. Since miR-9 expression is decreased in cancer tissues, we expected that overexpression of miR-9 would result in the arrest of cell growth. Using the MTT assay, we found that MGC803 cells transfected with the miR-9 overexpression vector (pri-miR-9) exhibited decreased growth compared to control cells. We also found that the anti-proliferative activity of pri-miR-9 transfection was dose-dependent. Cell independent growth activity, which is inconspicuous in normal cells, is a typical characteristic of malignant transformed cells. In colony formation assay, we observed that the colony formation activity of MGC803 cells transfected with pri-miR-9 was significantly inhibited. Furthermore, we  Relative cell growth activity was determined by MTT assay at the indicated times. Values are means ± SD of three duplications and the relative cell growth activity is shown (*P < 0.05). (C) Cell independent growth activity was detected by colony formation assay. MGC803 cells were transfected with si-NF-B1 or control vector at a final concentration of 5 ng/μl (final concentration) and then seeded in 12-well plates. The number of colonies was counted on the 9th day after seeding. The colony formation rate was calculated and is shown (**P < 0.01). (D) The cells of colony formation assay were stained by crystal violet and the representative pictures of colonies are shown.
found that the growth rate of tumors derived from MGC803 cells transfected with pri-miR-9 in SCID mice was lower than that of control tumors. Hence, these results indicate that gastric adenocarcinoma cells transfected with pri-miR-9 showed deletion of malignant phenotypes, suggesting a role for miR-9 in the growth suppression of cancer cells.
It was unclear as to how miR-9 affects cell growth and proliferation, because little is known about the physiologic targets of miR-9. Although bioinformatic tools may help to reveal putative mRNA targets of miRNAs, experimental procedures are required for their validation. Only a few studies have identified oncogenes whose level of expression is regulated by miRNAs. For instance, members of the let-7 miRNA family can negatively regulate all three members of the RAS oncogene family [27], and miR-15a/miR-16-1 can target and regulate BCL2 in B-cell CLL cells [25]. These findings support the idea that miRNA dysregulation may be involved in cancer pathogenesis. In this study, we show that miR-9 targets the NF-B1 mRNA, thus revealing a potential mechanism associated with gastric tumorigenesis.
Experimental evidence indicated that NF-B1 is a target of miR-9. First, the ability of miR-9 to regulate NF-B1 expression is likely direct, because it binds to the 3'UTR of NF-B1 mRNA with complementarity to the miR-9 seed region. Second, the EGFP fluorescence intensity of EGFP-NF-B1-UTR was specifically responsive to miR-9 overexpression. Third, mutation of the miR-9 binding site abolished the effect of miR-9 on the regulation of EGFP fluorescence intensity. Fourth, we observed an inverse correlation between the expression of miR-9 and NF-B1 in gastric adenocarcinoma tissues. Finally, endogenous NF-B1 expression, both mRNA and protein, is decreased in pri-miR-9-treated MGC803 cells, suggesting that miR-9 may regulate NF-B1 protein expression by inducing mRNA degradation and/or translational suppression.
The NF-B1 gene encodes a 105 kD protein, which can undergo cotranslational processing by the 26S proteasome to produce the 50 kD protein. In fact, NF-B1 is a member of the Rel/NF-B transcription factor family, which plays important roles in the regulation of immune responses, embryo and cell lineage development, cell-cycle progression, inflammation, and oncogenesis [37][38][39]. Moreover, the positive rate of NF-B1 in diffuse large Bcell lymphoma (DLBCL) was 63% (39/62), but none was expressed in reactive proliferation lymph node. However, NF-B1 can positively regulate the expression of VEGF in DLBCL [40]. In addition, suppression of NF-B expression can downregulate the expression of VEGF, thereby inhibiting the invasion and metastasis of tumors [41]. In this study, knockdown of NF-B1 suppressed the growth of MGC803 cells, which was consistent with the results of miR-9 overexpression. Ectopic expression of NF-B1 could also rescue MGC803 cells from growth inhibition caused by miR-9. However, the underlying mechanisms by which NF-B1 affects gastric cancer cell growth remain to be established.

Conclusions
In summary, we show that miR-9 expression is decreased in gastric adenocarcinoma tissues and that pri-miR-9 inhibits gastric cancer cell growth in vitro and in vivo. Furthermore, the expression of NF-B1 can be negatively regulated by miR-9. This study extends our knowledge about the regulation of NF-B1, a tumorrelated protein. Thus, in addition to epigenetic regulation, NF-B1 is also regulated at the post-transcriptional level by miRNA. It may suggest that miR-9 plays important roles in diverse biological processes by regulating NF-B1.