Long Non-coding RNA HOTAIR Is Targeted and Regulated by miR-141 in Human Cancer Cells*

Background: Silencing of long non-coding RNA (lncRNA) by microRNA (miRNA) has only been recently observed. Results: miR-141 binds to HOTAIR and suppresses its oncogenic function in Ago2 (Argounaute2). Conclusion: miR-141 targets and silences HOTAIR in an Ago2-dependent manner in cancer cells. Significance: Our results suggest that regulation of lncRNA expression by miRNA plays essential roles in gene expression and cellular functions. HOTAIR is a long non-coding RNA that interacts with the polycomb repressive complex and suppresses its target genes. HOTAIR has also been demonstrated to promote malignancy. MicroRNA-141 (miR-141) has been reported to play a role in the epithelial to mesenchymal transition process, and the expression of miR-141 is inversely correlated with tumorigenicity and invasiveness in several human cancers. We found that HOTAIR expression is inversely correlated to miR-141 expression in renal carcinoma cells. HOTAIR promotes malignancy, including proliferation and invasion, whereas miR-141 suppresses malignancy in human cancer cells. miR-141 binds to HOTAIR in a sequence-specific manner and suppresses HOTAIR expression and functions, including proliferation and invasion. Both HOTAIR and miR-141 were associated with the immunoprecipitated Ago2 (Argonaute2) complex, and the Ago2 complex cleaved HOTAIR in the presence of miR-141. These results demonstrate that HOTAIR is suppressed by miR-141 in an Ago2-dependent manner.

Non-coding RNAs have been demonstrated to have important roles in gene regulation, and a large number of long noncoding RNAs (lncRNAs) 2 have been discovered (1)(2)(3). lncRNAs are differentially expressed in various tissues and have important functions in gene regulation in normal and cancer cells (4 -6). lncRNAs function through diverse molecular mechanisms (7), and a number of lncRNAs associate with chromatinmodifying complexes affecting gene expression (8 -12). HOTAIR is an lncRNA localized in the HOXC gene cluster, and it interacts with PRC2 (polycomb repressive complex 2), which enhances H3K27 trimethylation to decrease expression of multiple genes (13). HOTAIR expression has been shown to promote cancer cell invasiveness (13,14) and to increase proliferation, cell cycle progression, and reduced apoptosis (15).
Here we show that HOTAIR promotes malignancy, including proliferation and invasion, whereas miR-141 suppresses malignancy in cancer cells. miR-141 was found to bind HOTAIR in a sequence-specific manner and suppress HOTAIR expression and function, including proliferation and invasion, in cancer cells. Suppression of HOTAIR expression by miR-141 correlated with alteration of HOTAIR function. miR-141 suppression of HOTAIR expression was found to be Ago2 (Argo-naute2)-dependent. Immunoprecipitation studies showed that HOTAIR was pulled down with miR-141 in the Ago2 complex, and HOTAIR was cleaved by Ago2 in the presence of miR-141. Our results demonstrate that HOTAIR is suppressed by miR-141 in an Ago2-dependent manner.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-Human renal carcinoma cells (786-O and ACHN cells), prostate cancer cells (DU145), colorectal adenocarcinoma cells (HT-29 cells), and normal HK-2 kidney cells were purchased from the American Type Culture Collection (Manassas, VA). 786-O cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). ACHN and DU145 cells were cultured in Eagle's minimum essential medium supplemented with 10% FBS. HT-29 cells were cultured in McCoy's 5A medium supplemented with 10% FBS. HK-2 cells were cultured in keratinocyte serum-free medium (Invitrogen) with bovine pituitary extract and human recombinant epidermal growth factor (EGF). ␣-Amanitin was purchased from Sigma-Aldrich. ␣-Amanitin was dissolved in water and added to 786-O and ACHN cells at a final concentration of 5 g/ml.
Cells were transfected with either 30 nM pre-miR negative control or pre-miR-141 (Applied Biosystems, Foster City, CA) or 30 nM siRNA control or HOTAIR siRNA (Sigma-Aldrich) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions.
For co-transfection, cells were initially transfected with 20 nM pre-miR negative control or pre-miR-141 (Applied Biosystems) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Subsequently, the cells were transfected with 20 nM siRNA control or HOTAIR siRNA using Lipofectamine 2000, according to the manufacturer's instructions.
Subconfluent cells (60 -70% confluent) were treated with 25 M genistein (Sigma-Aldrich) dissolved in dimethyl sulfoxide, and cells treated only with dimethyl sulfoxide served as control. Cell media and genistein were changed every day, and cells were cultured for 4 days. Cell Viability and Proliferation Assays-Cell viability was measured using the CellTiter 96 Aqueous One Solution cell proliferation assay (MTS assay) (Promega, Madison, WI), a colorimetric assay that measures the activity of reductase enzymes. Cells were seeded at a density of 1.5 ϫ 10 3 cells/well in flat bottomed 96-well plates. At the indicated times, CellTiter 96 Aqueous One reagent was added to each well according to the manufacturer's instructions. Cell viability was determined by measuring the absorbance at 490 nm using a kinetic microplate reader (Spectra MAX 190; Molecular Devices Co., Sunnyvale, CA). A bromodeoxyuridine (BrdU) cell proliferation assay was performed using a BrdU cell proliferation kit (Millipore, Billerica, MA) according to the manufacturer's instructions. BrdU was incorporated into cells for 4 h, and the cells were fixed at the indicated times after transfection. Data are the mean Ϯ S.D. of three independent experiments.
Apoptosis Analysis-Apoptosis was measured using flow cytometry (Cell Lab Quanta SC, Beckman Coulter, Brea, CA) with Annexin-V-FITC/7-AAD labeling. Measurements were repeated independently three times.
Transwell Invasion Assay-Culture inserts of 8-m pore size (Transwell; Costar) were coated with Matrigel (BD Biosciences) (100 g/well) and placed into the wells of 24-well culture plates. In the lower chamber, 500 l of DMEM containing 10% FBS was added, and 1 ϫ 10 5 cells were seeded to the upper chamber. After 36 h of incubation at 37°C with 5% CO 2 , the number of cells that had migrated through the pores were fixed with 10% formalin and stained with 0.05% crystal violet. Crystal violet was solubilized with methanol, and absorbance at 540 nm was measured by a kinetic microplate reader (Spectra MAX 190; Molecular Devices). Data are the mean Ϯ S.D. of three independent experiments.
RNA Extraction and Quantitative Real-time PCR-Total RNA was isolated using the miRNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Reverse transcription reactions were performed using the iScript cDNA synthesis kit (Bio-Rad) or reverse transcription system kit (Applied Biosystems). Quantitative real-time PCR analysis was performed in triplicate with an Applied Biosystems Prism7500 fast sequence detection system using TaqMan universal PCR master mix according to the manufacturer's protocol (Applied Biosystems). Levels of RNA expression were determined using the 7500 Fast System SDS software version 1.3.1 (Applied Biosystems).
Plasmids-A putative HOTAIR target site was cloned into the PmeI-XbaI site of the dual luciferase pmirGLO plasmid (Promega). For mismatch constructs, eight mismatches, which are shown in boldface letters in the HOTAIR sequence, AAACA-GAGTCCGTTCAGTGTCA, in Fig. 4 were introduced in the putative target site, and the target site sequence was changed to AAACAGAGTAAGTTAGTGACCA. The HOTAIR cDNA was amplified from cDNA of 786-O cells by using primers carrying BamHI and XhoI restriction enzyme sites at the flanking ends. The amplified cDNA was sequenced and subcloned into the BamHI and XhoI restriction site of the pcDNA3.1(ϩ) plasmid (Invitrogen). A mutant HOTAIR expression plasmid was constructed with the eight mismatches in the sequence in the putative target site of HOTAIR in pmirGLO plasmid shown above by site-directed mutagenesis. Ago2 expression plasmid (pFRT/FLAG/HA-DEST-EIF2C2 (plasmid 19888)) (27) was purchased from Addgene (Cambridge, MA). A HOTAIR promoter region (Ϫ35 to Ϫ2286) (28) was cloned into the MluI-XhoI site of the pLightSwitch_Prom plasmid (SwitchGear Genomics, Carlsbad, CA).
Western Blot Analysis-Protein extracts were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Hybond-P, GE Healthcare), followed by incubation with the indicated primary and secondary antibodies conjugated to horseradish peroxidase (GE Healthcare). Signals were detected using the ECL detection system (Amersham Biosciences ECL Plus Western blotting detection system). Antibodies against Ago2, ZEB1, E-cadherin, vimemtin, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against FLAG and fibronectin were purchased from Sigma-Aldrich or Abcam (Cambridge, MA), respectively. An antibody against RNA polymerase II was purchased from Millipore.
Luciferase Reporter Assay-Cells in 24-well plates were transfected with 30 nM pre-miR negative control or pre-miR-141 (Applied Biosystems) using Lipofectamin 2000 (Invitrogen), according to the manufacturer's instructions. Dual luciferase pmirGLO plasmid (Promega) was transfected using X-tremeGENE HP (Roche Applied Science) according to the manufacturer's instructions. Luciferase activity was assayed 48 h after transfection, using a Dual-Luciferase reporter assay system (Promega). The values were normalized to those obtained for pre-miR negative control transfection. All transfection experiments were performed in triplicate.
Cells in 24-well plates were transfected with 30 or 6 nM pre-miR negative control or pre-miR-141 (Applied Biosystems) using Lipofectamin 2000 (Invitrogen), according to the manufacturer's instructions. pLightSwitch_Prom plasmid harboring A HOTAIR promoter region or pLightSwitch_Prom plasmid only was transfected using X-tremeGENE HP (Roche Applied Science) according to the manufacturer's instructions. Luciferase activity was assayed 48 h after transfection, according to the manufacturer's instructions. All transfection experiments were performed in triplicate.
RNA Immunoprecipitation-786-O was transfected with FLAG-tagged Ago2 expression plasmid, HOTAIR expression plasmid, and pre-miR-141. RNA immunoprecipitation was performed using the Imprint RNA immunoprecipitation kit (Sigma-Aldrich). Anti-FLAG antibody (Sigma-Aldrich) and Protein G-agarose (Santa Cruz Biotechnology, Inc.) were used. Reverse transcription reactions were performed using iScript (Bio-Rad) and reverse transcription system kit (Applied Biosystems) HOTAIR and miR-141, respectively. Quantitative realtime PCR analysis was performed as described above.
Statistical Analysis-Data are shown as mean values Ϯ S.D. Student's t test was used to compare two different groups. p values of less than 0.05 were regarded as statistically significant (n ϭ 3) and denoted with an asterisk, compared with control unless otherwise specified.

Inverse Expression of HOTAIR and miR-141 in Renal
Carcinoma Cells-Real-time RT-PCR revealed that the expression level of HOTAIR was markedly higher in 786-O and ACHN compared with non-malignant HK-2 cells (Fig. 1A). However, the expression level of miR-141 was significantly lower in 786-O and ACHN cells compared with non-malignant HK-2 cells (Fig. 1B). We transiently transfected these cells with pre-miR control or pre-miR-141 (30 nM) to study the effect of miR-141 on HOTAIR expression. Expression of miR-141 after transient transfection is shown in Fig. 1C, and miR-141 significantly decreased expression of HOTAIR in these cells (Fig. 1E). In Fig.  1D, miR-141 expression after transfection was compared with that in HK-2 cells. A lower concentration of miR-141 (6 nM) also suppressed HOTAIR expression (Fig. 4D). These results indicate that there is a strong inverse correlation between expression of HOTAIR and miR-141.
Genistein (4Ј,5,7-trihydroxyisoflavone) is a soy isoflavone that has been reported to inhibit NF-B and Akt signaling pathways. Genistein has been shown to induce apoptosis and cell cycle arrest and to inhibit cancer cell proliferation (29) and invasion (30). To investigate the effects of genistein on HOTAIR and miR-141, we treated 786-O and ACHN cells with 25 M genistein for 96 h. Genistein treatment decreased HOTAIR expression and increased miR-141 expression (Fig. 1F), indicating that genistein had opposing effects on their expression.
Antagonistic Effect of HOTAIR and miR-141 on Cell Proliferation and Invasion-HOTAIR function has never been studied in renal carcinoma cells. To study the effect of HOTAIR on the proliferation and invasion of renal carcinoma cells, we transiently transfected 786-O and ACHN cells with siRNA control or HOTAIR siRNA. Transfection of HOTAIR siRNA decreased HOTAIR levels in renal carcinoma cells ( Fig DU145 prostate cancer cells have a relatively higher expression level of HOTAIR and miR-141 compared with non-malignant RWPE-1 prostate cells (31). miR-141 has been reported to function as a tumor suppressor in DU145 cells (32). We have also reported that HOTAIR promotes proliferation, reduces apoptosis, and promotes invasion in DU145 cells (33). We transiently transfected DU145 cells with anti-miR control or anti-miR-141 to study the effect of miR-141 on the growth of DU145 cells. Transient transfection of anti-miR-141 decreased miR-141 levels in DU145 cells to about 5%, and MTS ( Fig. 2D) and BrdU ( Fig. 2E) assays showed that anti-miR-141 increased proliferation of DU145 cells by about 20% at 96 h. These results also indicate that miR-141 inhibits DU145 cell proliferation.
Anti-miR-141 has been reported to function as an oncogene in HT-29 colorectal cancer cells (34). Therefore, we transiently transfected HT-29 cells with anti-miR control or anti-miR-141 to study the effect of miR-141 on the growth of HT-29 cells. Transient transfection of anti-miR-141 decreased miR-141 levels in HT-29 cells to about 3%, and MTS ( Fig. 2D) and BrdU ( Fig. 2E) assays showed that anti-miR-141 increased proliferation of HT-29 cells by about 15% at 96 h. These results also indicate that miR-141 inhibits HT-29 cell proliferation.
Because knockdown of HOTAIR suppressed 786-O, ACHN, and HT-29 cell proliferation, we performed apoptosis assays using flow cytometry with Annexin-V and 7-AAD. We found that HOTAIR siRNA increased apoptosis to about 2 times that of controls in 786-O and ACHN cells and to about 1.5 times that of controls in HT-29 cells (Fig. 2F), demonstrating that HOTAIR reduces apoptosis in these cells. We also performed apoptosis assays with miR-141-transfected cells and observed that miR-141 increased 786-O and ACHN cell apoptosis to about 3 times that of controls (Fig. 2G). Anti-miR-141 also reduced apoptosis about 30% compared with control in DU145 and HT-29 cells, demonstrating that miR-141 promotes apoptosis in DU145 and HT-29 cells (Fig. 2G).
Next, we performed a transwell invasion assay using Matrigel to investigate the effect of transiently transfected HOTAIR siRNA on the invasive ability of 786-O and ACHN cells. The results clearly revealed that HOTAIR siRNA decreased cell invasion to about 40% (HOTAIR siRNA-1) and 50% (HOTAIR siRNA-2) in 786-O cells and to about 50% (HOTAIR siRNA-1) and 60% (HOTAIR siRNA-2) in ACHN cells compared with controls (Fig. 3A). We have previously reported that HOTAIR siRNA reduced DU145 cell invasion (33). We also performed a transwell invasion assay using Matrigel to investigate the effect of transiently transfected miR-141 or anti-miR-141 on the invasive ability of 786-O, ACHN, and DU145 cells. The results show that miR-141 reduced invasion of 786-O and ACHN cells to 70% and anti-miR-141 increased invasion of DU145 cells by 25% compared with controls (Fig. 3B). These results show the opposing effects of HOTAIR and miR-141 in cell proliferation and invasion.
miR-141 Binds to and Suppresses HOTAIR Expression-We examined the seed sequence of miR-141 in HOTAIR and found a predicted binding site for miR-141 (Fig. 4A). We cloned the putative miR-141 target binding sequence into a luciferase construct. Luciferase reporter assays with miR-141-overexpressing 786-O, ACHN, DU145, and HT-29 cells showed that miR-141 repressed luciferase activity. Mutation of the putative miR-141 target sites decreased the response to miR-141 (Fig. 4B), indicating that miR-141 binds to HOTAIR in a sequence-specific manner.
We also examined whether miR-141 transcriptionally suppresses HOTAIR because miR-141 regulates transcription factors, such as ZEB1 and ZEB2 (23,35,36). We treated 786-O and ACHN cells with ␣-amanitin, which suppresses RNA polymerase II expression and activity (37, 38) and examined HOTAIR expression after transfection of miR-141. ␣-Amanitin treatment significantly reduced RNA polymerase II expression to about 5% in both cell lines (Fig. 4C). However, the treatment did not significantly affect suppression of HOTAIR by miR-141 (Fig.  4D). We also measured luciferase activity with the HOTAIR promoter (28) in 786-O ACHN, DU145, and HT-29 cells after miR-141 transfection and found that miR-141 had no significant effect on luciferase activity (Fig. 4E). These results indicate that miR-141 suppresses HOTAIR by binding to HOTAIR in a sequence-specific manner and may not reduce HOTAIR transcription.
We co-transfected ACHN and DU145 cells with HOTAIR siRNA and anti-miR-141 to study the effects of miR-141 on cell invasion mediated by HOTAIR (Fig. 6C). We observed that anti-miR-141 promoted (Fig. 6C, lane 2 compared with lane 1) and HOTAIR siRNA suppressed cell invasion (Fig. 6C, lanes  3 and 5 compared with lane 1). Co-transfection of anti-miR-141 and HOTAIR siRNA also showed that anti-miR-141 increased cell invasion suppressed by HOTAIR siRNA (Fig. 6C, lanes 4  and 6 compared with lanes 3 and 5, respectively). Fig. 6D shows representative images of invaded ACHN and DU145 cells with transient co-transfection of HOTAIR siRNA and anti-miR-141 in the transwell invasion assay.
We co-transfected 786-O cells with FLAG-tagged-Ago2 and HOTAIR expression plasmids and miR-141 to determine if HOTAIR and miR-141 are present in the Ago2 immunocomplex (Fig. 9D). The Ago2 immunocomplex pulled down using an anti-FLAG antibody contained miR-141 or HOTAIR when the cells were transfected with miR-141 or HOTAIR. Co-transfection of miR-141 and HOTAIR showed an increased amount of HOTAIR in the immunocomplex, indicating that miR-141 recruits HOTAIR to the immunocomplex (Fig. 9D). We also investigated whether miR-141 recruits HOTAIR to the Ago2 immunocomplex using in vitro transcribed HOTAIR. In vitro transcribed HOTAIR was incubated with the immunocomplex. The transfection of miR-141 increased the amount of in vitro transcribed HOTAIR in the immunocomplex, indicating that miR-141 recruits in vitro transcribed HOTAIR to the immunocomplex (Fig. 9E). These results also suggest that miR-141 silences HOTAIR in an Ago2-dependent manner.
We performed cleavage assays to investigate whether Ago2 cleaves HOTAIR in the present of miR-141. We co-transfected 786-O cells with FLAG-tagged-Ago2 and HOTAIR expression plasmids with and without miR-141 and pulled-down the Ago2 immunocomplex using an anti-FLAG antibody. Incubation of in vitro transcribed HOTAIR with the Ago2 immunocomplex from cells co-transfected with miR-141 degraded HOTAIR into small segments (Fig. 9F, lane 3). These results indicated that HOTAIR is degraded and silenced by the Ago2 complex.

DISCUSSION
In this study, we have shown that HOTAIR is strongly and negatively regulated by miR-141 through silencing of HOTAIR. Interaction of miRNA with lncRNA has been reported previously. The 3Ј-UTR of PTENP1, a pseudogene of tumor suppressor gene PTEN, has been reported to function as a decoy of PTEN-targeting miRNAs (42). The lncRNA HULC is highly up-regulated in liver cancer and interacts with miR-372, which leads to reduced translational repression of its target gene (43). While we were preparing this manuscript, miR-21 was reported to target lncRNA GAS5 (44). Here we have presented strong evidence that HOTAIR is silenced and negatively regulated by miR-141 in cancer cells.
HOTAIR is well studied among lncRNA and has been shown to have important functions in normal and cancer cells. HOTAIR stimulates H3K27 trimethylation to decrease expression of multiple genes through its interaction with the PRC2 (13) and promotes various types of cancers, such as breast cancer, hepatocellular carcinoma, nasopharyngeal carcinoma, and gastrointestinal stromal tumors (13)(14)(15)(45)(46)(47)(48)(49). However, HOTAIR function has never been studied in renal carcinoma cells. We found that HOTAIR promotes tumorigenicity in renal cell carcinoma, which is consistent with findings in other cell types. We found that HOTAIR and miR-141 expression are inversely correlated in renal carcinoma cells. Genistein has potent biological activity, regulates several signaling pathways in cancer cells, and promotes cancer cell death (29,30,50,51), and we have found that genistein regulates miRNAs (33,(52)(53)(54). We also found that genistein decreases HOTAIR expression while stimulating miR-141 expression, showing an inverse effect.
Luciferase assays indicate that miR-141 reduces HOTAIR expression through the putative miR-141 binding site in HOTAIR (Fig. 4A). We also examined whether miR-141 alters the transcription activities on the HOTAIR promoter because miR-141 regulates transcription factors such as ZEB1 and ZEB2 (23,35,36). Treatment with ␣-amanitin, an inhibitor of RNA polymerase II, did not significantly affect miR-141 suppression of HOTAIR in 786-O and ACHN cells (Fig. 4, C and D), and miR-141 also did not have significant effects on luciferase activity using a HOTAIR promoter (Fig. 4D). These results demonstrate that miR-141 did not transcriptionally repress HOTAIR. Our results show that miR-141 significantly targets and suppresses HOTAIR through the putative binding site, although we cannot exclude the possibility of transcriptionally suppression or other unknown mechanisms.
Functional analyses with co-transfection of HOTAIR siRNA or a HOTAIR expression plasmid and miR-141 or anti-miR-141 clearly show that miR-141 suppressed HOTAIR expression and function and that miR-141 function is mediated by HOTAIR. miR-141 significantly repressed wild-type HOTAIR, but a mutated putative HOTAIR binding site (Fig. 4) did not completely abolish the repression by miR-141 (Fig. 8), suggesting that miR-141 may bind to other sequences in HOTAIR.
HOTAIR has been reported to induce ABL2, Snail, LAMB3, and LAMC2 and repress JAM2, PCDH10, and MDA-MB-231 cells (13). We found that HOTAIR siRNA and miR-141 reduced ABL2 and induced PCDH10 in 786-O cells, and we observed that HOTAIR siRNA and miR-141 induced PCDH10 in ACHN cells. The difference in effects on HOTAIR target genes may be due to the difference in 786-O and ACHN cell type. 786-O cells are derived from a primary clear cell adenocarcinoma, and ACHN cells are derived from a metastatic renal adenocarcinoma. 786-O cells contain loss of heterozygosity of the VHL (von Hippel-Lindau) tumor suppressor, whereas ACHN cells harbor wild-type VHL. HOTAIR was found to regulate the miR-141 target gene ZEB1 in both 786-O and ACHN cells. HOTAIR siRNA reduced Snail1, whereas anti-miR-141 induced Snail expression in DU145 prostate cancer cells. HOTAIR siRNA reduced ABL2, whereas anti-miR-141 induced ABL2 expression in HT-29 colorectal adenocarcinoma cells. miR-141 may regulate these genes, which regulate metastasis and invasion, and the results of the co-transfection assays support the conclusion that miR-141 regulate genes by targeting HOTAIR.
miRNAs guide complementary target mRNAs to the RNAinduced silencing complex that contains Argonaute family proteins. Ago2 is the main protein that cleaves target transcripts directly in mammalian cells (39 -41). In this study, an Ago2 siRNA was found to prevent the decrease in HOTAIR expression by miR-141. HOTAIR and miR-141 were both found to be present in the Ago2 immunocomplex. Cleavage experiments show that HOTAIR is degraded into small segments in the presence of miR-141 (Fig. 9F). Thus, we have conclusively shown that miR-141 targets and cleaves HOTAIR in an Ago2-dependent manner similar to that by which miRNAs cause degradation of protein-coding mRNAs. However, our results do not exclude other mechanisms of lncRNA silencing by miRNAs.
Mammalian miRNA target sites are primarily in the 3Ј-UTR of mRNA transcripts, whereas most plant miRNAs target mRNA protein-coding regions. Studies have indicated that miRNA targets in protein-coding sequences regulate target genes (55)(56)(57)(58), and genome-wide analysis demonstrated that a large number of Ago binding sites are located in human protein-coding regions (59,60). However, it is believed that active mRNA translation interrupts miRNA from binding to its target site in a protein-coding region (61,62). It has also been demonstrated that 3Ј UTR sites are more effective in down-regulating gene expression than sites located in the protein-coding region (55,60,61). Because lncRNAs are not utilized for mRNA translation, its target sequence is readily accessible to miRNAs, as is the 3Ј-UTR of mRNA targeted by miRNAs. It is believed that a number of lncRNAs may be targets of miRNAs.
lncRNAs and miRNAs are essential regulators of gene expression, and it has been documented that these non-coding RNAs play important roles in diverse biological processes, such as development and disease. Interaction between lncRNA and miRNA provides another element of control in gene regulation. These results have provided strong evidence that shows that miR-141 targets and silences HOTAIR in an Ago2-dependent manner and regulates its function. They suggest that broad spectrum silencing by RNA interaction significantly regulates gene expression and cellular functions.