Depletion of Human Micro-RNA miR-125b Reveals That It Is Critical for the Proliferation of Differentiated Cells but Not for the Down-regulation of Putative Targets during Differentiation
2005; Elsevier BV; Volume: 280; Issue: 17 Linguagem: Inglês
10.1074/jbc.m412247200
ISSN1083-351X
AutoresYong Sun Lee, Hak Kyun Kim, Sangmi Chung, Kwangsoo Kim, Anindya Dutta,
Tópico(s)Cancer-related molecular mechanisms research
ResumoMicro-RNAs are small non-coding RNAs that regulate target gene expression post-transcriptionally through base pairing with the target messenger RNA. Functional characterization of micro-RNAs awaits robust experimental methods to knock-down a micro-RNA as well as to assay its function in vivo. In addition to the recently developed method to sequester micro-RNA with 2′-O-methyl antisense oligonucleotide, we report that small interfering RNA against the loop region of a micro-RNA precursor can be used to deplete the micro-RNA. The depletion of miR-125b by this method had a profound effect on the proliferation of adult differentiated cancer cells, and this proliferation defect was rescued by co-transfected mature micro-RNA. This technique has unique advantages over the 2′-O-methyl antisense oligonucleotide and can be used to determine micro-RNA function, assay micro-RNAs in vivo, and identify the contribution of a predicted micro-RNA precursor to the pool of mature micro-RNA in a given cell. miR-125b and let-7 micro-RNAs are induced, whereas their putative targets, lin-28 and lin-41, are decreased during in vitro differentiation of Tera-2 or embryonic stem cells. Experimental increase or decrease of micro-RNA concentrations did not, however, affect the levels of the targets, a finding that is explained by the fact that the down-regulation of the targets appears to be mostly at the transcriptional level in these in vitro differentiation systems. Collectively these results reveal the importance of micro-RNA depletion strategies for directly determining micro-RNA function in vivo. Micro-RNAs are small non-coding RNAs that regulate target gene expression post-transcriptionally through base pairing with the target messenger RNA. Functional characterization of micro-RNAs awaits robust experimental methods to knock-down a micro-RNA as well as to assay its function in vivo. In addition to the recently developed method to sequester micro-RNA with 2′-O-methyl antisense oligonucleotide, we report that small interfering RNA against the loop region of a micro-RNA precursor can be used to deplete the micro-RNA. The depletion of miR-125b by this method had a profound effect on the proliferation of adult differentiated cancer cells, and this proliferation defect was rescued by co-transfected mature micro-RNA. This technique has unique advantages over the 2′-O-methyl antisense oligonucleotide and can be used to determine micro-RNA function, assay micro-RNAs in vivo, and identify the contribution of a predicted micro-RNA precursor to the pool of mature micro-RNA in a given cell. miR-125b and let-7 micro-RNAs are induced, whereas their putative targets, lin-28 and lin-41, are decreased during in vitro differentiation of Tera-2 or embryonic stem cells. Experimental increase or decrease of micro-RNA concentrations did not, however, affect the levels of the targets, a finding that is explained by the fact that the down-regulation of the targets appears to be mostly at the transcriptional level in these in vitro differentiation systems. Collectively these results reveal the importance of micro-RNA depletion strategies for directly determining micro-RNA function in vivo. 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Up-regulation of lin-4 during the first larval stage (L1) triggers the subsequent developmental stages by translational repression of lin-14 and lin-28, both of which contain sites of partial complementarity to lin-4 in the 3′-untranslated region (19Feinbaum R. Ambros V. Dev. Biol. 1999; 210: 87-95Crossref PubMed Scopus (123) Google Scholar, 20Moss E.G. Lee R.C. Ambros V. Cell. 1997; 88: 637-646Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). Likewise, let-7 is induced during development and translationally repress lin-41 through partially complementary base-pairing between let-7 and target sequences in the 3′-untranslated region of lin-41 (17Reinhart B.J. Slack F.J. Basson M. Pasquinelli A.E. Bettinger J.C. Rougvie A.E. Horvitz H.R. Ruvkun G. Nature. 2000; 403: 901-906Crossref PubMed Scopus (3654) Google Scholar, 21Pasquinelli A.E. Reinhart B.J. Slack F. Martindale M.Q. Kuroda M.I. Maller B. Hayward D.C. Ball E.E. Degnan B. Muller P. Spring J. Srinivasan A. 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Recently, 2′-O-methyl antisense oligonucleotide against a miRNA was reported to specifically knock-down the miRNA (26Meister G. Landthaler M. Dorsett Y. Tuschl T. RNA. 2004; 10: 544-550Crossref PubMed Scopus (519) Google Scholar, 27Hutvagner G. Simard M.J. Mello C.C. Zamore P.D. PLoS Biol. 2004; 2: e98Crossref PubMed Scopus (556) Google Scholar). Despite the successful knock-down of a miRNA in vitro and in vivo, this method has several limitations. First, a direct measurement of the depletion of a miRNA is difficult, because 2′-O-methyl antisense oligonucleotide binds to the miRNA and sequesters it from its target rather than induces its degradation. In addition, even a slight contamination of 2′-O-methyl antisense oligonucleotide in an RNA preparation interferes with assays to measure the miRNA level, such as Northern blot, primer extension assay, or RNase protection assay. 2Y. S. Lee and A. Dutta, unpublished data. 2Y. S. Lee and A. Dutta, unpublished data. Therefore, the only possible way to confirm the decrease of a miRNA is to employ a very indirect and cumbersome assay, measuring the level of expression of a reporter gene containing the target sequence of the miRNA. In addition, although rescuing the depletion effect by adding back miRNA is the most stringent control against the nonspecific effects of miRNA depletion, the knock-down phenotype cannot be rescued in the presence of the 2′-O-methyl antisense oligonucleotide. In addition to 2′-O-methyl antisense oligonucleotides to miRNAs, we now show that siRNA against the loop region of a pre-miRNA can be used to specifically down-regulate miRNAs. The unique advantages of this method over 2′-O-methyl antisense oligonucleotide will be discussed. Using both these methods, we show that miR-125b, putative homolog of lin-4 in C. elegans (9Lagos-Quintana M. Rauhut R. Yalcin A. Meyer J. Lendeckel W. Tuschl T. Curr. Biol. 2002; 12: 735-739Abstract Full Text Full Text PDF PubMed Scopus (2694) Google Scholar), is critical for the proliferation of differentiated human cell lines, providing a new example of the biological role of a mammalian miRNA. In addition, we could restore miRNA function in the miRNA-depleted cells by the introduction of single-stranded RNA representing the miRNA sequence. Although the exact regulatory mechanism of mammalian lin-28 and lin-41 in in vitro differentiation systems or the role of the miRNAs in this regulation has not yet been described, a translational control of mammalian Lin-28 has been hypothesized similar to that seen in C. elegans (28Moss E.G. Tang L. Dev. Biol. 2003; 258: 432-442Crossref PubMed Scopus (244) Google Scholar, 29Sempere L.F. Freemantle S. Pitha-Rowe I. Moss E. Dmitrovsky E. Ambros V. Genome Biology. 2004; (http://genomebiology.com/2004/5/1/reviews/R13)PubMed Google Scholar, 30Nelson P.T. Hatzigeorgiou A.G. Mourelatos Z. RNA. 2004; 10: 387-394Crossref PubMed Scopus (169) Google Scholar). The hypothesis is based on the following observations; 1) the evolutionary conservation of lin-4 and let-7 miRNAs and their targets lin-28 and lin-41, 2) the presence of putative target sequences with imperfect complementarity to miR-125b and let-7 within the 3′-untranslated region of lin-28 and lin-41, and 3) induction of miR-125b and let-7 and the attendant decrease of Lin-28 protein during differentiation. This assumption was supported by an experiment with artificial luciferase construct fused to the let-7 target sequence that is naturally found in the 3′-untranslated region of mammalian lin-28 mRNAs (30Nelson P.T. Hatzigeorgiou A.G. Mourelatos Z. RNA. 2004; 10: 387-394Crossref PubMed Scopus (169) Google Scholar). Using the methods developed above to decrease or increase miR-125b or let-7, we, however, find that the decrease of lin-28 and lin-41 during differentiation appears independent of the levels of these miRNAs, demonstrating the importance of miRNA depletion strategies to identify real targets of miRNAs in vivo. The negative result is probably explained by the fact that the decrease of lin-28 and lin-41 is mainly at the transcriptional stage, suggesting a variation in the paradigm of lin-28 and lin-41 regulation during differentiation of mammalian cells in vitro. RNA Isolation, Northern Blotting, and Primer Extension Assay— Total RNAs from various cell lines were isolated with Trizol reagent (Invitrogen) according to the manufacturer's instructions. Total RNAs from human cervix, prostate, and brain were purchased from Ambion. Multiple tissue Northern blot (human MTN blot II) was purchased from Clontech. Primer extension assay (31Hutvagner G. McLachlan J. Pasquinelli A.E. Balint E. Tuschl T. Zamore P.D. Science. 2001; 293: 834-838Crossref PubMed Scopus (2130) Google Scholar) was performed with Superscript II reverse transcriptase (Invitrogen) as per the manufacturer's instructions, with minor modifications. 20 μl of extension reaction contained 0.1 pmol of the γ-32P-labeled primer and 10 μg of total RNA. In the case of the 5 S rRNA extension reaction, 1 μg of total RNA was used. Extension reaction at 50 °C for 50 min was quenched by the addition of 20 μl of sequencing gel loading buffer (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Extension products were resolved from the primer by electrophoresis in an 18% polyacrylamide gel with 7 m urea. The sequences of the primers are as follows: let-7a-3-ext, 5′-GCCCCAAACTATACAACCTACTAC-3′; miR-125a-ext, 5′-TCACAGGTTAAAGGGTCTCA-3′; miR-125b-ext, 5′-CATCACAAGTTAGGGTCTCA-3′; 5 S rRNA-ext, 5′-GATCGGGCGCGTTCAGGGTGGTAT-3′; miR-16-ext, 5′-ACTACGCCAATATTTACGTGCT-3′; miR-15a-ext, 5′-AATCCACAAACCATTATGTGCT-3′. siRNAs, 2′-O-Methyl Oligonucleotides, miRNA, and Transfections— siRNA duplexes and 2′-O-methyl oligonucleotides were synthesized by Dharmacon. Transfection was performed with Oligofectamine reagent (Invitrogen) as per the manufacturer's instructions. Unless indicated, 960 nm of siRNA duplexes designed against the loop regions of miRNAs was used for the transfection into Tera-2 or PC-3 cell lines (ATCC number CRL-1435). In the case of 2′-O-methyl antisense oligonucleotides against miR-125b or GL2, 160 nm oligonucleotides were transfected into PC3 or HeLa cells. The target sequences for siRNA duplexes are as follows: siRNA against miR-125b-1 (si125b-1), 5′-AACCGUUUAAAUCCACGGGUU-3′; siRNA against miR-125b-2 (si125b-2), 5′-AAGGUAUUUUAGUAACAUCAC-3′; siRNA against miR-16–1 (si16–1), 5′-AAGAUUCUAAAAUUAUCUCCA-3′; siRNA against miR-16–2 (si16–2), 5′-GGCGUAGUGAAAUAUAUAU-3′; siRNA against miR-15a (si15a), 5′GUGGAUUUUGAAAAGGUGC3′. RNA oligonucleotides of mature let-7a-3 and miR-125b sequences were synthesized by Dharmacon. Transfection was performed as described above but at the final concentration of 2 μm. During long-term experiment with multiple transfections, we performed the first three transfections every day and the transfections from the fourth and later every other day. Cell Proliferation Assay—Cell growth was measured with CellTiter 96 non-radioactive cell proliferation assay kit (Promega). Cell Culture, Retinoic Acid Treatment, and Stem Cell Differentiation—Tera-2 cells (ATCC number HTB-106) were treated with 5 μm all-trans-retinoic acid (Sigma) on a gelatin-coated tissue culture dish (33Pertovaara L. Tienari J. Vainikka S. Partanen J. Saksela O. Lehtonen E. Alitalo K. Biochem. Biophys. Res. Commun. 1993; 191: 149-156Crossref PubMed Scopus (22) Google Scholar). Culture medium with retinoic acid was refreshed every other day. The cells were cultured in the presence of retinoic acid for 20 days, with subsequent cultivation for an additional 40 days without retinoic acid. Neural induction from mouse embryonic stem (ES) cell line D3 (ATCC number CRL-11632) was maintained and differentiated using the five-stage procedure as described (34Lee S.H. Lumelsky N. Studer L. Auerbach J.M. McKay R.D. Nat. Biotechnol. 2000; 18: 675-679Crossref PubMed Scopus (1115) Google Scholar). Briefly, undifferentiated ES cells were cultured on gelatin-coated dishes in supplemented Dulbecco's modified Eagle's medium (Invitrogen) and differentiated into embryoid bodies (EBs) on nonadherent bacterial dishes. The EBs were then plated onto an adhesive tissue culture surface, and neuronal precursor (NP) cells were selected in serum-free medium. After 6–10 days of selection, cells were trypsinized, and nestin+ NP cells were expanded in N2 medium supplemented with 1 μg/ml laminin (Sigma) and 10 ng/ml bovine fibroblast growth factor (R & D Systems). After expansion for 4 days, bovine fibroblast growth factor was removed to induce differentiation to neuronal phenotypes. Nuclear Run-on Assay—ORFs of lin-28 and lin-41 were PCR-amplified from Tera-2 cDNA. 0.5 μg of each fragment was slot-blotted onto a positively charged Nylon membrane (Nytran from Schleicher & Schuell). Nucleus isolation and run-on transcription reaction were performed as described (35Greenberg M.E. Bender T.P. Struhl K. Short Protocols in Molecular Biology. 1. John Wiley & Sons, Inc., New York2002: 4-29Google Scholar) with modifications. After the transcription reaction, Trizol reagent (Invitrogen) was added, and an RNA probe was prepared according to the manufacturer's instructions for Trizol reagent. Membranes were probed with 32P-labeled run-on RNA for 16 h at 68 °C in hybridization buffer containing 0.25 m Na2HPO4 (pH 7.2), 1 mm EDTA, and 7% (w/v) SDS. Membranes were washed twice in 2× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate) at room temperature for 15 min each followed by a wash in 0.2× SSC and 1% (w/v) SDS at 65 °C for 20 min. The blot was exposed to a PhosphorImager Storm 860 (Amersham Biosciences) and quantitated. Recombinant Lin-28 and Antiserum—cDNA containing full-length human Lin-28 protein (209 amino acids) was cloned into pET-23a(+) (Novagen). Recombinant Lin-28 with hexahistidine tag was expressed in BL21(DE3), purified through nickel-charged chelating Sepharose column (Amersham Biosciences), and used for raising antiserum in rabbit. Antibody was proven specific by the following criteria. It recognized the recombinant Lin-28 and a single protein band of expected size of Lin-28 in Tera-2 cell extract, whereas preimmune serum did not detect a protein of appropriate size (data not shown). In addition, it failed to detect protein in the extract of PC-3 cells where lin-28 mRNA was not detectable. Expression of let-7 and miR-125b in Differentiated Cells— Primer extension assays were employed to quantitate miR-125a and miR-125b, miRNAs that are the putative lin-4 homologs in mouse and human (9Lagos-Quintana M. Rauhut R. Yalcin A. Meyer J. Lendeckel W. Tuschl T. Curr. 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The Depletion of miR-125b-2 in Differentiated Cancer Cells Inhibits Cell Proliferation—We first attempted to decrease miR-125b by siRNAs against the precursor of the mature miRNA. The hairpin-shaped pre-miRNA is generated in the nucleus by the action of Drosha (39Lee Y. Ahn C. Han J. Choi H. Kim J. Yim J. Lee J. Provost P. Radmark O. Kim S. Kim V.N. Nature. 2003; 425: 415-419Crossref PubMed Scopus (3875) Google Scholar) but is exported to the cytoplasm (40Lund E. Guttinger S. Calado A. Dahlberg J.E. Kutay U. Science. 2004; 303: 95-98Crossref PubMed Scopus (2016) Google Scholar), where it is processed by Dicer to release the mature miRNA (31Hutvagner G. McLachlan J. Pasquinelli A.E. Balint E. Tuschl T. Zamore P.D. Science. 2001; 293: 834-838Crossref PubMed Scopus (2130) Google Scholar, 41Ketting R.F. Fischer S.E. Bernstein E. Sijen T. Hannon G.J. Plasterk R.H. Genes Dev. 2001; 15: 2654-2659Crossref PubMed Scopus (1416) Google Scholar, 42Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. 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After transfection of these siRNAs into PC-3 cells, the miR-125b level was measured by a primer extension assay. miR-125b was greatly reduced by the si125b-2 but not si125b-1 (Fig. 2A, right panel). This observation suggests that most of the mature miR-125b is derived from the precursor miR-125b-2 in PC-3 cells. To test if this strategy to knock-down miRNAs can be applied to other miRNAs, siRNAs against additional pre-miRNAs were transfected into PC-3 cells. miR-16 has two predicted precursors, miR-16–1 and -2. Transfection of siRNA against miR-16–1 and -2 (si16–1 and -2) decreased the mature miRNA to 40 and 60%, respectively (Fig. 2B). Thus, ∼60% of mature miR-16 is derived from the precursor -1, and the rest is from precursor -2. Consistent with this interpretation, co-transfection of the two siRNAs against miR-16 decreased the level of miR-16 to ∼20%, which is close to the background level. Efficient knock-down of miR-15a was also achieved by the transfection of siRNA against the loop region of the corresponding pre-miRNA. Titration of siRNA dose revealed that siRNA concentration of 0.32–1 μm was required to decrease the miRNA levels (Fig. 2B). This dose is higher than the amount of siRNA required to knock down a typical mRNA (0.02–0.3 μm). The lower the efficiency of the siRNA-directed degradation of a miRNA precursor could be due to the short length and/or transient nature of miRNA precursors. Indeed, the precursors of miRNA were never detected in our primer extension assays, presumably because miRNA precursors are quickly processed into mature miRNA. The inefficiency of siRNA against pre-miRNA might account for the failure of this strategy in two other cell lines, HeLa and DU 145. Nevertheless, the results dictate that an siRNA can be used to deplete a miRNA in at least some cell lines like PC-3. To investigate the biological effect of miR-125b, cell proliferation was measured after depletion of miR-125b by RNA interference (Fig. 3A). si125b-2, but not si125b-1, reduced proliferation of PC-3 cells. Growth was restored by the co-introduction of a synthetic mature miR-125b that was designed not to anneal to si125b-2, demonstrating that growth suppression is specifically caused by the depletion of miR-125b. This result also indicates that miRNA function can be provided to cells by transient transfection of single-stranded RNA. Tera-2 EC cells did not express miR-125b, and accordingly, si125b-2 did not suppress growth of Tera-2 cells. To eliminate a trivial explanation, we demonstrated that the RNA interference machinery is intact in Tera-2 cells by transfecting siRNA against ORC2 in this cell line and measuring Orc2 protein level (Fig. 3B). Thus, the failure of si125b-2 to suppress Tera-2 cell proliferation is not due to absence of the siRNA machinery and provides an additional control against nonspecific effects of si125b-2 in PC-3 cells. To confirm that the proliferation defect in Fig. 3A is specifically caused by miR-125b depletion and to test if this defect is seen in cell lines other than PC-3, the experiment was repeated after transfection of 2′-O-methyl antisense oligonucleotide against miR-125b. Again, depletion of miR-125b by 2′-O-methyl oligonucleotide resulted in the reduction of proliferation, whereas the transfection of 2′-O-methyl oligonucleotide against GL2 did not (Fig. 3C). In addition, this proliferation defect was observed not only in PC-3 cells but also in HeLa cells. Because identical results were obtained from two independent methods and each experiment was performed with stringent controls, it is clear that miR-125b is essential for the proliferation of differentiated cells. miR-125b and let-7 Increase, Whereas lin-28 and lin-41 Decrease at mRNA Levels during Differentiation—Taken together with the expression profiles of the human miRNAs in Fig. 1, the previous results in C. elegans (17Reinhart B.J. Slack F.J. Basson M. Pasquinelli A.E. Bettinger J.C. Rougvie A.E. Horvitz H.R. Ruvkun G. Nature. 2000; 403: 901-906Crossref PubMed Scopus (3654) Google Scholar, 19Feinbaum R. Ambros V. Dev. Biol. 1999; 210: 87-95Crossref PubMed Scopus (123) Google Scholar, 20Moss E.G. Lee R.C. Ambros V. Cell. 1997; 88: 637-646Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 21Pasquinelli A.E. Reinhart B.J. Slack F. Martindale M.Q. Kuroda M.I. Maller B. Hayward D.C. Ball E.E. Degnan B. Muller P. Spring J. Srinivasan A. Fishman M. Finnerty J. Corbo J. Levine M. Leahy P. Davidson E. Ruvkun G. Nature. 2000; 408: 86-89Crossref PubMed Scopus (1807) Google Scholar, 22Slack F.J. Basson M. Liu Z. Ambros V. Horvitz H.R. Ruvkun G. Mol. Cell. 2000; 5: 659-669Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 23Vella M.C. Choi E-Y. Lin S.-Y. Reinert K. Slack F.J. Genes Dev. 2004; 18: 132-137Crossref PubMed Scopus (369) Google Scholar) suggest that let-7 and miR-125b miRNAs are induced during differentiation, and this up-regulation of the miRNAs post-transcriptionally repress their putative targets, which contain partially complementary target sequences in their 3′-untranslated region. To test whether this regulation could be recapitulated in mammalian in vitro differentiation systems, we induced the differentiation of Tera-2 cells in the neuronal direction by retinoic acid (45Thompson S. Stern P.L. Webb M. Walsh F.S. Engstrom W. Evans E.P. Shi W.K. Hopkins B. Graham C.F. J. Cell Sci. 1984; 72: 37-64Crossref PubMed Google Scholar). miR-125b became pronounced after 20 days of retinoic acid treatment (Fig. 4, left panel). let-7 increased to a level barely detectable after 20 days and to a higher level after 60 days. This time pattern was reminiscent of C. elegans in which lin-4 is expressed earlier than let-7 during development (17Reinhart B.J. Slack F.J. Basson M. Pasquinelli A.E. Bettinger J.C. Rougvie A.E. Horvitz H.R. Ruvkun G. Nature. 2000; 403: 901-906Crossref PubMed Scopus (3654) Google Scholar, 19Feinbaum R. Ambros V. Dev. Biol. 1999; 210: 87-95Crossref PubMed Scopus (123) Google Scholar). In contrast to the miRNAs, Lin-28 protein decreased to barely detectable levels after 20 days. Also, the mRNA of lin-28 and lin-41 disappeared completely by 20 days of retinoic acid (Fig. 4, left panel). Thus, the regulation of the miRNAs and their targets was recapitulated in the differentiation of EC cells in vitro. To confirm the changes in expression of miRNAs and their targets in a second differentiation system, we induced the differentiation of mouse ES cells to the neuronal fate using the five-stage in vitro differentiation procedure (34Lee S.H. Lumelsky N. Studer L. Auerbach J.M. McKay
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