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Journal = home > Archive >=20 Letter= > Full = Text

Letter

Nature Genetics 39, 673 - 677 (2007)
Published online: 1 April 2007 = | doi:10.1038/ng2003

Impaired microRNA processing enhances cellular = transformation and=20 tumorigenesis

Madhu S Kumar¹,=20 Jun Lu 2,³,=20 Kim L Mercer 1,=20 Todd R Golub 2,^3,⁴=20 & Tyler Jacks 1,⁵

Abstract

MicroRNAs (miRNAs) are a new class of small noncoding = RNAs that=20 post-transcriptionally regulate the expression of target mRNA = transcripts. Many=20 of these target mRNA transcripts are involved in proliferation, = differentiation=20 and apoptosis^1,=20²,=20 processes commonly altered during tumorigenesis. Recent work has shown a = global=20 decrease of mature miRNA expression in human cancers³.=20 However, it is unclear whether this global repression of miRNAs reflects = the=20 undifferentiated state of tumors or causally contributes to the = transformed=20 phenotype. Here we show that global repression of miRNA maturation = promotes=20 cellular transformation and tumorigenesis. Cancer cells expressing short = hairpin=20 RNAs (shRNAs) targeting three different components of the miRNA = processing=20 machinery showed a substantial decrease in steady-state miRNA levels and = a more=20 pronounced transformed phenotype. In animals, miRNA = processing=96impaired cells=20 formed tumors with accelerated kinetics. These tumors were more invasive = than=20 control tumors, suggesting that global miRNA loss enhances = tumorigenesis.=20 Furthermore, conditional deletion of Dicer1 enhanced tumor = development in=20 a K-Ras=96induced mouse model of lung cancer. Overall, these studies = indicate that=20 abrogation of global miRNA processing promotes tumorigenesis.

Introduction

In order to examine the role of miRNA processing in = tumorigenesis,=20 we designed shRNAs targeting the three established regulators of miRNA=20 processing: Drosha, DGCR8 and Dicer1. In mouse lung adenocarcinoma = (LKR13)=20 cells^4,=20⁵,=20 these hairpins achieved robust knockdown of target mRNA levels (Fig.= 1a)=20 and reduced steady-state miRNA levels (Fig.= 1b).=20 Of note, these shRNAs did not completely eliminate mature miRNAs. For = Dicer1 in=20 particular, we would expect incomplete knockdown, as Dicer1 is required = for the=20 generation of its target small interfering RNA. We then tested the = effect of=20 impaired miRNA processing on the transformation properties of these = cells. miRNA=20 processing=96impaired cells (hereafter referred to as 'miRKD') LKR13 = cells formed=20 substantially larger colonies than controls did when we plated them at = low=20 density (Fig.= 1c)=20 and had improved colony formation in soft agar (= Supplementary=20 Fig. 1 online). Additionally, miRKD cells formed more foci than = control=20 cells (= Supplementary=20 Fig. 1). These enhanced transformation properties seem to be a = consequence=20 of increased proliferation, as miRKD cells grew faster than controls and = showed=20 a higher percentage of cells entering S phase and a reduced number of=20 G₀/G₁ cells (= Supplementary=20 Fig. 1). Thus, defective miRNA maturation (or some other unknown = function of=20 these genes) alters the transformed state of mouse tumor cells, = accompanied by=20 enhanced proliferative capacity.

F= igure=20 1: Impaired miRNA processing promotes cellular = transformation.

<= IMG=20 class=3Dthumb=20 alt=3D"Figure 1 : Impaired miRNA processing promotes cellular = transformation."=20 src=3D"http://www.nature.com/ng/journal/v39/n5/thumbs/ng2003-F1.jpg">=

(a) Quantitative real-time PCR (Q-RT PCR) was = performed with=20 probes for DGCR8, Drosha, or Dicer1 in LKR13 cells infected with empty = vector or=20 shRNAs against DGCR8 (shDGCR8), Drosha (shDrosha) or Dicer1 (shDicer1) = (= Supplementary=20 Methods). Expression levels were initially normalized to TBP and=20 subsequently normalized relative to DGCR8, Drosha or Dicer1 levels in = LKR13=20 cells infected with empty vector. Values are mean =20 s.d. (n =3D 3) with propagated error. (b) Small RNA = blotting was=20 performed against let-7 and glutamine tRNA in LKR13 cells = infected with=20 empty vector, a control shRNA (= Supplementary=20 Methods) or shRNAs against DGCR8, Drosha or Dicer1. (c) LKR13 = cells=20 infected with empty vector or shRNAs against DGCR8, Drosha or Dicer1 = were plated=20 at low density (2500 cells per 10 cm plate), grown for 5 d and fixed and = stained=20 with crystal violet. (d) Small RNA blotting was performed as = above in=20 U2OS cells infected with shRNAs against Renilla luciferase, = DGCR8, Drosha=20 or Dicer1. We generated two independent shRNAs (numbered 1 and 2) = specific for=20 DGCR8, two specific for Drosha and two specific for Dicer1 (= Supplementary=20 Methods). (e) U2OS cells infected with shRNAs against = Renilla=20 luciferase, DGCR8, Drosha or Dicer1 were plated at low density as = described=20 above.

F= ull size=20 image (83 KB)

To determine if impaired miRNA processing affected mouse = and human=20 cancer cells similarly, we designed additional short hairpin RNAs = against the=20 human miRNA processing machinery. These hairpins also achieved knockdown = of=20 steady-state miRNA levels in three independent human cancer cell lines = as=20 assessed by RNA blotting (Fig.= 1d)=20 and miRNA profiling (= Supplementary=20 Table 1 online). Similar to the results from LKR13 cells, impaired = miRNA=20 processing enhanced both colony formation and growth in soft agar of = human=20 cancer cells (Fig= . 1e=20 and = Supplementary=20 Fig. 1). Notably, this effect was independent of the tissue of = origin; miRKD=20 cells derived from osteosarcomas (U2OS), colorectal carcinomas (HCA7) = and breast=20 carcinomas (MCF7) all showed enhanced transformation (Fig.= 1e=20 and = Supplementary=20 Fig. 1). Overall, these results suggest that global repression of = miRNAs=20 enhances transformation of cancer cells.

To determine if global repression of miRNAs enhances the = tumorigenic potential of cancer cells, we injected miRKD LKR13 cells=20 subcutaneously into immunocompromised mice. miRKD cells formed tumors = with=20 accelerated kinetics, with tumor volumes that were at least an order of=20 magnitude greater than in controls (Fig.= 2a).=20 Steady-state miRNA levels in miRKD tumors were lower than those in = controls (= Supplementary=20 Fig. 2 online). Earlier work suggested that the global loss of = miRNAs in=20 human cancers corresponds to altered differentiation in tumors relative = to=20 normal tissue³.=20 Thus, it was possible that miRKD tumors might be histologically distinct = from=20 controls. We were surprised to find that miRKD tumors were similar to = controls=20 by histological analysis; all appeared moderately differentiated, with = the clear=20 presence of glandular structures (= Supplementary=20 Fig. 2). Notably, however, miRKD tumors commonly invaded the = surrounding=20 normal tissue of the host, including skeletal muscle, adipose tissue and = nerve=20 sheaths (Fig.= 2b).=20 Careful histological analysis of control tumors did not show any such = invasion=20 (data not shown). Consistent with the invasive phenotype in miRKD = tumors, miRKD=20 cells demonstrated enhanced migration through a collagen matrix in = vitro,=20 suggesting that repression of miRNAs in cancer cells results in = increased=20 motility (Fig.= =20 2c).

F= igure=20 2: Impaired miRNA processing promotes tumorigenesis in=20 vivo.

<= IMG=20 class=3Dthumb=20 alt=3D"Figure 2 : Impaired miRNA processing promotes tumorigenesis in = vivo."=20 src=3D"http://www.nature.com/ng/journal/v39/n5/thumbs/ng2003-F2.jpg">=

(a) LKR13 cells infected with empty vector or = shRNAs against=20 DGCR8, Drosha or Dicer1 were injected subcutaneously into = immunocompromised mice=20 (10⁵ cells/injection), and tumor growth was measured over = time.=20 Values are mean =20 s.e.m. (n =3D 6). (b) Hematoxylin/eosin staining of = tumors, showing=20 examples of tumor cells surrounding nerve sheaths and infiltrating into = host=20 adipose tissue and skeletal muscle of the host. Original magnification, = 40.=20 N, nerve sheath. A, adipose tissue. M, skeletal muscle. T, tumor cells. = Arrows=20 indicate infiltrating tumor cells. Arrowheads indicate host tissue = infiltrated=20 by tumor cells. (c) In vitro migration assays using LKR13 = cells=20 infected with empty vector or shRNAs against DGCR8, Drosha or Dicer1 = (see Met= hods).=20 Results are representative of three replicates. Values are mean =20 s.d. (n =3D 3).

F= ull size=20 image (41 KB)

Recent work with loss-of-function alleles of = Dicer1 in=20 mammals has described defects in processes potentially unrelated to = miRNAs, such=20 as silencing of heterochromatic regions and chromosome = segregation^6,=20^7,=20⁸.=20 We did not observe any changes in transcript levels from major satellite = repeats=20 (= Supplementary=20 Fig. 3 online); in addition, we noted similar levels of = phosphorylated=20 ribosomal S6 and eIF2 in = miRKD and control cells, suggesting that global translation is not = altered (= Supplementary=20 Fig. 3). However, it remained possible that miRKD cells had = undergone=20 irreversible changes (independent of miRNAs) that enhance = transformation. To=20 investigate this possibility, we generated a shRNA against Dicer1 = flanked by=20 loxP sites (pSico^R-shDicer1), allowing subsequent removal of = the=20 hairpin via Cre-mediated recombination⁹.=20 Upon delivery of adenoviral Cre to pSico^R-shDicer1 cells, we = observed=20 a concomitant restoration of miRNA processing (= Supplementary=20 Fig. 4 online). miRNA processing continued to be impaired in=20 pSico^R-shDicer1 cells infected with a control adenovirus. = Notably,=20 adenoviral Cre-mediated removal of the Dicer1 hairpin resulted in = reduced soft=20 agar growth relative to control adenovirus-treated cells. We did not = observe=20 this reversal in pSico^R-shluc cells (= Supplementary=20 Fig. 4 online). Thus, the enhanced transformation associated with = impaired=20 miRNA processing was reversible.

To determine if this enhanced transformation was = specific to=20 depletion of the miRNA processing machinery, we attempted to overcome = the effect=20 through expression of human cDNAs in miRKD LKR13 cells. As mouse shRNAs = do not=20 show full sequence complementarity with their human cDNA counterparts, = we=20 expected the human cDNAs to retain expression. In this context, miRNA = processing=20 would be maintained while the shRNA was expressed and functional. We = observed a=20 near-complete restoration of miRNA expression in cells containing the = human cDNA=20 in tandem with its target shRNA (= Supplementary=20 Fig. 4). Of note, shRNA-expressing cells lacking their corresponding = cDNA or=20 expressing a cDNA against a different miRNA processing component failed = to=20 restore mature miRNA levels, validating the specificity of the effect. = The cells=20 whose miRNA processing was rescued showed reductions in both low-density = colony=20 formation and growth in soft agar compared with miRKD cells (= Supplementary=20 Fig. 4), demonstrating that protection against impaired miRNA = processing was=20 sufficient to prevent the phenotype triggered by the shRNAs. Therefore, = the=20 enhanced transformation corresponding to defective miRNA maturation was = specific=20 to the miRNA processing machinery and was not a nonspecific effect of = shRNA=20 expression.

In order to test if impaired miRNA processing was = sufficient to=20 transform noncancerous cells, we expressed shRNAs against the miRNA = processing=20 machinery in Kras^G12D-expressing mouse embryonic = fibroblasts=20 (MEFs), which have been shown to be incompletely transformed^10<=
/A>.=20 Compared with control Kras^G12D MEFs, miRKD=20 Kras^G12D MEFs had an elevated growth rate (= Supplementary=20 Fig. 5 online). However, impaired miRNA processing was not = sufficient to=20 fully transform Kras^G12D MEFs, as evidenced by the = inability=20 of the cells to form colonies in soft agar (data not shown). To further = address=20 the role of miRNA processing on proliferation, we infected wild-type = MEFs with=20 shRNAs against the miRNA processing machinery and examined long-term = population=20 growth. Notably, wild-type cells had a markedly lower long-term = proliferative=20 capacity compared with controls (= Supplementary=20 Fig. 5), consistent with observations from Dicer1-null MEFs = and=20 embryonic stem cells^6,=20^7,=20⁸.=20 Therefore, although defective miRNA biogenesis improves the = transformation of=20 cancer cells, it is not sufficient to promote de novo = transformation.

To begin to investigate the basis for the enhanced = transformation=20 associated with repression of miRNAs, we measured the expression of = several=20 oncogenes in miRKD LKR13 cells. We detected substantially higher protein = levels=20 of the oncogenes c-Myc and K-Ras without changes in other known = oncogenes such=20 as N-Ras, H-Ras and E2F-1 (Fig.= 3a).=20 K-Ras mRNA levels were slightly elevated in miRKD cells, whereas c-Myc = mRNA=20 levels were unchanged compared with controls (= Supplementary=20 Fig. 5). As Ras family members have been shown to stabilize the = c-Myc=20 protein post-translationally through activation of the MEK/ERK and Akt = signaling=20 pathways^11<=
/A>,=20 we investigated whether these pathways were altered in miRKD cells. = Notably,=20 there were no consistent changes in MEK1/2, ERK1/2 or Akt = phosphorylation in=20 response to impaired miRNA processing (= Supplementary=20 Fig. 5). These data suggest that miRNAs directly regulate c-Myc = expression=20 post-transcriptionally.

F= igure=20 3: Impaired miRNA processing increases expression of specific=20 oncogenes.

<= IMG=20 class=3Dthumb=20 alt=3D"Figure 3 : Impaired miRNA processing increases expression of = specific oncogenes."=20 src=3D"http://www.nature.com/ng/journal/v39/n5/thumbs/ng2003-F3.jpg">=

(a) Immunoblotting for K-Ras, c-Myc, -tub= ulin,=20 N-Ras, H-Ras and E2F-1 in LKR13 cells infected with empty vector or = shRNAs=20 against DGCR8, Drosha or Dicer1. (b) Luciferase assays were = performed in=20 293T cells transfected with pRL-TK containing bulged siCXCR4 binding=20 sites^28<=
/A>=20 or mouse K-Ras and c-Myc 3' UTRs. pGL3 was used as a transfection = control.=20 Expression w as normalized to pGL3 levels and subsequently normalized = relative=20 to pRL-CXCR4 expression. Values are mean =20 s.d. (n =3D 6) with propagated error. (c) LKR13 cells were = transfected with 2'-O-methyl oligonucleotides complementary to = either=20 miR-181a or let-7g and were then plated at 5,000 cells per = well.=20 Cells were counted over time in triplicate. Values are mean =20 s.e.m. (n =3D 3).

F= ull size=20 image (37 KB)

To examine potential miRNA-mediated regulation, we used = the miRNA=20 target prediction program miRanda^12<=
/A>=20 and observed that the 3' UTRs of mouse K-Ras and c-Myc were both targets = of the=20 let-7 family of miRNAs. To evaluate whether elevated K-Ras and = c-Myc=20 protein levels were due to let-7-mediated derepression, we = selectively=20 overexpressed let-7g and measured the effects on luciferase-based = reporter constructs carrying the 3' UTRs of K-Ras and c-Myc (= Supplementary=20 Fig. 5). Consistent with previous studies^13<=
/A>,=20 we observed robust repression of the K-Ras 3' UTR as a result of = let-7=20 overexpression (Fig.= 3b).=20 In addition, the c-Myc 3' UTR was also repressed in this setting, = suggesting=20 that both oncogenes are let-7 targets. Similarly, = 2'-O-methyl=20 oligonucleotides complementary to let-7g caused derepression of = the 3'=20 UTRs of K-Ras and c-Myc (= Supplementary=20 Fig. 5). Moreover, both oncogenes are direct let-7 targets, = as=20 mutation of the let-7 binding sites in the K-Ras and c-Myc 3' = UTRs caused=20 derepression comparable to let-7 inhibition (= Supplementary=20 Fig. 5). To examine the role of let-7 family members in=20 transformation, LKR13 cells were transfected with 2'-O-methyl=20 oligonucleotides complementary to let-7g. We observed an = elevation in=20 growth rate with let-7 inhibition (Fig.= 3c).=20 Moreover, overexpression of let-7g in miRKD LKR13 cells led to=20 substantial reduction in low-density colony-forming ability (= Supplementary=20 Fig. 5). Finally, in miRKD human cancer cells, we noted an = enrichment of=20 let-7 family members among the miRNAs whose repression correlates = best=20 with heightened transformation (= Supplementary=20 Table 2 online). However, it is also possible that other miRNAs = whose=20 repression correlates with enhanced transformation might function in a = similar=20 way.

To understand the significance of these findings in = vivo,=20 we assessed the role of impaired miRNA processing in a mouse model of = lung=20 tumorigenesis, using the conditional, activatable allele of Kras, = LSL-Kras^G12D(ref. 10<= /A>).=20 Intranasal infection of LSL-Kras^G12D mice with = adenovirus=20 expressing Cre has been shown to induce non=96small cell lung cancer in = these=20 mice, with complete penetrance and a defined time course^14<=
/A>.=20 By crossing LSL-Kras^G12D mice with mice bearing a = conditional=20 knockout allele of Dicer1 (ref. 6), we=20 generated LSL-Kras^G12D mice that were wild-type, = heterozygous=20 or homozygous for the conditional allele for Dicer1 (hereafter = referred=20 to as LSL-Kras^G12DDicer1^+/+,=20 LSL-Kras^G12DDicer1^flox/+ and=20 LSL-Kras^G12DDicer1^flox/flox,=20 respectively).

We infected this cohort of mice with a Cre-expressing = adenovirus,=20 and 12 weeks later, we observed an increase in tumor burden in the=20 LSL-Kras^G12DDicer1^flox/+ and=20 LSL-Kras^G12DDicer1^flox/flox mice = compared=20 with the LSL-Kras^G12DDicer1^+/+ mice. = This=20 tumor burden was reflected in substantial increases in the ratio of = tumor area=20 to lung area (Fig.= 4a),=20 tumor size (Fig.= 4b)=20 and tumor number (Fig.= 4c).=20 We also performed histological grading analysis of tumors from=20 LSL-Kras^G12DDicer1^+/+,=20 LSL-Kras^G12DDicer1^flox/+ and=20 LSL-Kras^G12DDicer1^flox/flox mice. = Overall,=20 we noted a slight shift in grade in the Dicer1 mutant mice, with = the=20 highest-grade lesions only present in the=20 LSL-Kras^G12DDicer1^flox/+ and=20 LSL-Kras^G12DDicer1^flox/flox mice (Fig.= 4d).=20 These lesions had areas of nuclear pleomorphism, prominent nucleoli and=20 significant vacuolation (Fig.= 4e).=20 In addition, the = LSL-Kras^G12DDicer1^flox/+=20 and LSL-Kras^G12DDicer1^flox/flox mice = showed=20 more extensive bronchiolar hyperplasia than=20 LSL-Kras^G12DDicer1^+/+ mice (Fig.= 4f),=20 further demonstrating the enhanced tumor burden in Dicer1 mutant = mice. In=20 total, these results suggest that impaired miRNA processing can enhance=20 tumorigenesis in vivo through an enhanced tumor burden.

F= igure=20 4: Impaired miRNA processing enhances the in vivo tumor = burden in a=20 mouse model of lung cancer.

<= IMG=20 class=3Dthumb=20 alt=3D"Figure 4 : Impaired miRNA processing enhances the in vivo tumor = burden in a mouse model of lung cancer."=20 src=3D"http://www.nature.com/ng/journal/v39/n5/thumbs/ng2003-F4.jpg">=

(a) LSL-Kras^G12D mice, either = wild-type,=20 heterozygous or homozygous conditional for Dicer1=20 (LSL-Kras^G12DDicer1^+/+ ('KD +/+'),=20 LSL-Kras^G12DDicer1^flox/+ ('KD = flox/+'), and=20 LSL-Kras^G12DDicer1^flox/flox ('KD=20 flox/flox'), respectively), were intranasally infected with adenovirus=20 expressing Cre. We killed animals 12 weeks after infection and = quantified tumor=20 and lung areas with Bioquant software. (b) Tumor sizes were = quantified=20 for the animals described above with Bioquant software. (c) Tumor = numbers=20 were quantified for the animals described above with Bioquant software. = Values=20 are mean =20 s.e.m. (n =3D 11 for=20 LSL-Kras^G12DDicer1^+/+, n =3D = 15 for=20 LSL-Kras^G12DDicer1^flox/+ and = n =3D 10=20 for LSL-Kras^G12DDicer1^flox/flox).=20 ^*, P < 5 =20 10^-5, ^**, P < 0.05; ^***, = P=20 < 0.005. (d) Tumors from animals of the genotypes described = above were=20 scored for histological grade using a grading scheme described = previously (see=20 Met= hods).=20 Values are mean =20 s.e.m. (n =3D 5 for each genotype). (e) Hematoxylin/eosin = staining=20 of a Grade III tumor from a=20 LSL-Kras^G12DDicer1^flox/+ animal. = Original=20 magnification, 20.=20 Black arrows indicate areas of nuclear pleomorphism. Arrowheads indicate = areas=20 with prominent nucleoli. White arrows indicate areas of vacuolation. = (f)=20 Hematoxylin/eosin staining of bronchiolar hyperplasia from a=20 LSL-Kras^G12DDicer1^flox/flox animal. = Original=20 magnification, 20 .

F= ull size=20 image (89 KB)

These studies demonstrate that impaired miRNA biogenesis = accelerates oncogenic transformation through the deregulation of target=20 oncogenes. Previous work has shown that both human and mouse cancers = show a=20 global reduction of mature miRNA levels compared with normal = tissues³.=20 Our work demonstrates that this reduced mature miRNA expression can = promote=20 tumorigenesis. Notably, the loss of miRNA processing, though incomplete, = causes=20 a marked change in the transformed phenotype of cancer cells. = Furthermore, our=20 results are consistent with observations correlating reduced Dicer1 = expression=20 in a subset of non=96small cell lung cancers with poor prognosis^15<=
/A>.=20 These findings are also consistent with work showing the let-7 = family as=20 a negative regulator of the Ras family of oncogenes^13<=
/A>,=20 while extending its role in oncogenic transformation to c-Myc. Of note, = previous=20 studies have implicated specific miRNAs as oncogenes, as their = overexpression=20 accelerates tumorigenesis^16,=
=20^17,=
=20^18,=
=20^19,=
=20^20<=
/A>.=20 Our study does not directly contradict these findings but rather = suggests that=20 overall repression of miRNAs can enhance tumorigenesis. Recent work has = also=20 shown that the global repression of miRNAs in human cancers does not = coincide=20 with reductions in the primary miRNA transcripts, suggesting that = altered=20 regulation of the miRNA processing machinery occurs in human = cancers^21<=
/A>.=20 Taken as a whole, these investigations highlight a functional role for = miRNA=20 biogenesis in tumorigenesis.

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=20

Methods

Cell culture.

Human cancer cell lines (HCA7, MCF7, U2OS) were = originally=20 obtained from ATCC. LKR13 cells were generated from a mouse lung=20 adenocarcinoma^4,=20⁵.=20 Wild-type and LSL-Kras^G12D MEFs were generated as = described=20 previously^10<=
/A>.=20 Cells were maintained using standard conditions.

Transformation, immortalization, growth and = tumorigenesis=20 assays.

Transformation, immortalization, growth and = tumorigenesis=20 assays were performed essentially as described^22<=
/A>.

5-bromodeoxyuridine (BrdU) and cell-cycle profile = analysis.

Anti-BrdU and 7-amino-actinomycin D (AAD) staining = was=20 analyzed by flow cytometry according to the manufacturer's instructions = (BD=20 Biosciences).

Invasion assays.

Invasion assays were performed using a 24-well=20 collagen-based cell invasion assay according to the manufacturer's = instructions=20 (Chemicon).

shRNA design and infection.

shRNAs were designed and cloned, and cells were = infected=20 with them, as described previously^9,=20^23<=
/A>.=20 Subsequent adenovirus infection of shRNA-expressing cells was performed = as=20 described⁹.

RNA blotting.

Small RNA blotting was performed as = described⁹.

Immunoblotting assays.

Immunoblotting was performed as described^22<=
/A>.

Quantitative reverse transcription (Q-RT) PCR.

Q-RT PCR of mouse DGCR8, Drosha, Dicer1, K-Ras and = c-Myc was=20 performed as before^24<=
/A>.=20 Q-RT PCR of satellite repeat transcripts was performed as = described^25<=
/A>.

miRNA overexpression and repression assays.

We cloned let-7g miRNA and seed mutant = let-7e=20 miRNA into MSCV-neo (Clontech) with approximately 500 bp of flanking = sequence.=20 2'-O-methyl oligonucleotides complementary to let-7g and = miR-181a=20 were synthesized commercially (Integrated DNA Technologies). = Overexpression=20 vectors and 2'-O-methyl oligonucleotides were transfected into = cells=20 using previously described methods^26,=
=20^27<=
/A>.

Luciferase assays.

Mouse K-Ras (EMBL accession number BC004642)=20 and c-Myc (EMBL accession number X01023)=20 3' UTRs were cloned into Renilla luciferase vectors described = previously,=20 and luciferase assays were performed as before^28<=
/A>.

miRNA expression analysis.

miRNA expression profiling was performed using a = bead-based=20 miRNA detection platform as previously described with = modifications³=20 (= Supplementary=20 Methods online).

Mice.

LSL-Kras^G12D mice^10<=
/A>=20 were cross-bred to Dicer1^flox/flox mice⁶=20 to generate LSL-Kras^G12D Dicer1^flox/+ mice, = which=20 were subsequently interbred to produce the experimental cohort. Research = was=20 approved by the Committee for Animal Care of the Massachusetts Institute = of=20 Technology and was conducted in compliance with the Animal Welfare Act=20 Regulations and other federal statutes relating to animals and = experiments=20 involving animals and adheres to the principles set forth in the 1996 = National=20 Research Council Guide for Care and Use of Laboratory Animals = (institutional=20 animal welfare assurance number, A-3125-01).

Intranasal infection and tumor analysis.

Mice were infected intranasally with adenovirus = expressing=20 Cre as described^14<=
/A>.=20 Twelve weeks after infection, mice were killed, and lungs were = collected, fixed=20 in formalin and stained for histology as described previously^14<=
/A>.=20 Lung and tumor areas were determined using Bioquant Image Analysis = software, as=20 described previously^29<=
/A>.

Tumor grading analysis.

Tumor grading was done without knowledge of = genotype, as=20 described previously^29<=
/A>.=20 Each tumor was given a score of 1 to 3 based on predetermined criteria = (= Supplementary=20 Methods).

Requests for materials.

e-mail: tjacks@mit.edu

Note: = Supplementary=20 information is available on the Nature Genetics = website.

Author contributions

M.S.K. and J.L. conceived the project. M.S.K., = J.L. and=20 K.L.M. carried out all experiments described. T.R.G. and T.J. supervised = the=20 experimental work and interpretation of data. The manuscript was = prepared by=20 M.S.K. and T.J.

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Acknowledgments

We thank R. Shiekhattar for the = Flag-Dicer1 cDNA=20 construct; V.N. Kim for the Flag-DGCR8 and Flag-Drosha cDNA constructs = and P.A.=20 Sharp for the pRL-TK and pGL3 constructs. We thank A. Ventura, P. Sandy, = M.=20 Winslow, H. Zhang and members of the Sharp laboratory for experimental = advice=20 and assistance. We also thank R. Bronson and M.E. McLaughlin for = histological=20 analysis. We acknowledge C. Bender Kim, M. Winslow, C. Reinhardt and S. = Kissler=20 for critical review of the manuscript. This work was supported by grant=20 2-PO1-CA42063-21 from the National Cancer Institute and by Cancer Center = Support=20 grant P30-CA14051 from the National Cancer Institute. M.S.K. is an NSF = Graduate=20 Research Fellow. T.J. is a Ludwig Scholar. T.R.G. and T.J. are = investigators of=20 the Howard Hughes Medical Institute.

Competing interests statement:

The authors declare no competing financial = interests.

Received 4 December 2006 | Accepted 12 February 2007 = |Published=20 online: 1 April 2007..

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MIT Center for Cancer Research, Cambridge, Massachusetts = 02139, USA.
Broad Institute of MIT and Harvard, Cambridge, = Massachusetts 02141,=20 USA.
Department of Pediatric Oncology, Dana-Farber Cancer = Institute and=20 Harvard Medical School, Boston, Massachusetts 02115, USA.
Howard Hughes Medical Institute, Harvard Medical School, = Boston,=20 Massachusetts 02115, USA.
Howard Hughes Medical Institute, Department of Biology,=20 Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, = USA.

Correspondence to: Tyler Jacks^1,⁵=20 e-mail: tjacks@mit.edu

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=09 // for each descendant of .leaderboard that isn't a

Letter

Impaired microRNA processing enhances cellular = transformation and=20 tumorigenesis

Abstract

Introduction

F= igure=20 1: Impaired miRNA processing promotes cellular = transformation.

F= igure=20 2: Impaired miRNA processing promotes tumorigenesis in=20 vivo.

F= igure=20 3: Impaired miRNA processing increases expression of specific=20 oncogenes.

F= igure=20 4: Impaired miRNA processing enhances the in vivo tumor = burden in a=20 mouse model of lung cancer.

Methods

Cell culture.

Transformation, immortalization, growth and = tumorigenesis=20 assays.

5-bromodeoxyuridine (BrdU) and cell-cycle profile = analysis.

Invasion assays.

shRNA design and infection.

RNA blotting.

Immunoblotting assays.

Quantitative reverse transcription (Q-RT) PCR.

miRNA overexpression and repression assays.

Luciferase assays.

miRNA expression analysis.

Mice.

Intranasal infection and tumor analysis.

Tumor grading analysis.

Requests for materials.

Author contributions

Acknowledgments

Competing interests statement:

References

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