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Mol Ther. Jun 2011; 19(6): 1116–1122.
Published online Mar 22, 2011. doi:  10.1038/mt.2011.48
PMCID: PMC3129804

Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice

Abstract

MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer.

Introduction

Lung cancer is a deadly disease with millions of victims worldwide each year. Non-small cell lung cancers (NSCLC) make up the majority of these deaths. Current therapies fail to treat this disease in the vast majority of cases, with <15%, 5 year survival rate.1 Novel therapies based on a better understanding of the disease are desperately needed to save more lives.

MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression to affect a multitude of biological processes including cell proliferation, differentiation, survival, and motility.2 In addition, miRNAs are often found misexpressed or damaged in many cancers and have been implicated causally in promoting proliferation and metastasis of tumor cells.3,4,5 Two classes of oncogenesis-associated miRNAs (oncomiRs) have been described, those that are overexpressed in tumors and act as oncogenes and those that are underexpressed in tumors and act as tumor suppressors.5 Two well-characterized families of tumor suppressor miRNAs are let-7 and miR-34. let-7 is normally expressed in differentiated tissues but frequently lost in cancer, notably, lung cancers.3,6 let-7 negatively regulates multiple cell cycle oncogenes, such as RAS, MYC, and HMGA26,7,8 and exogenous application of let-7 to human lung cancer cells reduces proliferation and radiosensitizes the cells.9,10 miR-34 is also lost in lung cancer and acts as a tumor suppressor by regulating multiple cell cycle and cell survival genes.11,12,13 miR-34 is directly transcribed by the p53 tumor suppressor gene and is required for a radiation response in vitro and in vivo.14,15

Delivery of endogenous tumor suppressor miRNAs as synthetic miRNA mimics has emerged as a promising approach to treat cancer.16 To date, several key miRNAs have been identified that inhibit tumor growth in mouse models of cancer. Among these are the tumor suppressors let-7, miR-16, miR-34, and miR-26a.17,18,19,20 In most of these cases, the miRNA was delivered directly by intratumoral injections or was expressed from a viral vector which—despite providing a means for successful miRNA delivery to the tumor in the particular mouse model—are delivery routes that are unlikely to succeed in the clinic. Intratumoral injections are merely amenable to a small number of easily accessible and localized tumors that have not yet metastasized. Similarly, expression from viral vectors is likely to show the same weaknesses encountered in gene therapy, such as limited infectivity as well as the need for nuclear translocation of a relatively large DNA vector, transcription and final maturation of the gene product.21,22 Since cancer cells frequently show deficiencies in the maturation of miRNA precursors, expression from a viral vector is a less preferable approach.23 Thus, systemic delivery of chemically synthesized miRNA mimics could facilitate the most efficacious dissemination to primary and advanced tumors.24

Recently, we enabled systemic delivery of miR-34a mimics using a neutral lipid emulsion (NLE) that has the potential to be translated into the clinic.20 Systemic delivery of miR-34a mimics led to an accumulation of miR-34a in tumor tissues, repression of direct miR-34a targets and robust inhibition of NSCLC xenografts in mice.20 However, since these lung tumors were grown subcutaneously, the clinical relevance of this novel lipid-based miRNA formulation remains unknown. Here, we explored the utility of the systemic delivery formulation in orthotopic mouse models of NSCLC. We demonstrate therapeutic delivery of synthetic RNA interference (RNAi) agents to both normal lung tissues as well as orthotopic lung tumors, and show tumor-inhibitory effects of our let-7 and miR-34 formulations in an autochthonous KRASG12D transgenic mouse model of lung cancer.

Results

Systemically delivered miRNA biodistribution in vivo

Since the biodistribution profile of NLE-mediated delivery was unknown, we first investigated the accumulation of a NLE-delivered miRNA mimic in lung and other tissues upon intravenous tail-vein injection. miR-124 was chosen because it is primarily expressed by cells of the central nervous system and therefore allows the discrimination of delivered miRNA mimics from the endogenously expressed miRNAs in most other tissues. Mice were administered a single dose of 20 µg NLE-formulated miR-124 via tail-vein injections. This dose is equivalent to 1 mg per kg body weight, assuming that a mouse weighs on average 20 g. Whole blood, liver, kidney, and lung were collected 10 minutes after injection and subjected to RNA isolation and quantitative reverse transcriptase PCR (qRT-PCR). As shown in Figure 1a,b, increased miR-124 levels were detectable in all tissues tested. As anticipated, liver did not yield the highest miR-124 levels, in agreement with a report showing that neutral lipids—unlike cationic lipid particles—do not preferentially accumulate in the liver.25 To determine whether the miR-124 miRNA mimic was being taken up by cells or if it was simply present in the blood found in the tissues, organs from a separate group of animals were perfused with 0.9% saline prior to RNA isolation. Of note, perfusion with saline solution diminished miR-124 levels by ~70–80% in liver and kidney which suggests that the majority of miR-124 in these tissues remains in blood. In contrast, perfusion hardly affected the levels of miR-124 in lung. Thus, we hypothesize that the miRNA mimic is taken up by lung tissue and that NLE may be a useful vehicle to deliver therapeutic miRNAs and presumably other small RNAs to normal lung and lung tumors.

Figure 1
Biodistribution of systemically delivered microRNAs (miRNA) mimics. (a) A group of six mice was injected each with 20 µg neutral lipid emulsion-formulated miR-124. After killing, three mice were used to isolate total RNA from liver, kidney, ...

Delivery of siRNA to orthotopic lung tumors via NLE

This data suggested that NLE facilitates delivery of miRNA mimics to normal lung; however, it was unknown whether the miRNA was successfully internalized by lung cells and whether the miRNA is therapeutically active. In addition, lung tumors assume a different microenvironment than normal lung and therefore, delivery to normal lung is not necessarily indicative of delivery to lung tumors. To evaluate whether NLE provides a suitable tool for therapeutic delivery to lung tumors, we used an in vivo luciferase reporter system based on an orthotopic H460-luc xenograft. Orthotopic H460-luc lung tumors were initiated in NOD/SCID mice by endotracheal intubation.26 Endotracheal intubation facilitates efficient delivery of the cargo to lung bronchi and peripheral lung tissue, including distal alveoli, as shown by endotracheal delivery of green dye and India Ink (Supplementary Figure S1a,b). Using this method, inoculation of H460-luc xenografts leads to the formation of solid tumor masses 25–52 days after intubation of the xenograft (Supplementary Figure S1c,d). Tumor growth was monitored periodically by live animal imaging. Since H460-luc cells stably express luciferase, the luminescent signal directly correlates with viable tumor cells. Once mice developed readily detectable tumors, total luminescence was recorded as the total flux at 0 hours (Figure 2a,b). Immediately after measuring luminescence, two mice received intravenous tail-vein injections of 20 µg luciferase siRNA formulated in NLE. As a negative control, two mice were given intravenous NLE formulations containing an siRNA composed of a scrambled sequence (negative control, NC). Forty-eight hours after injection of the formulated siRNAs, luminescence was measured again and expressed as percent relative to the total flux of each mouse at 0 hours (100%). As shown in Figure 2a,b, a single intravenous administration of luciferase siRNA led to a >95% reduction of luminescence 48 hours postinjection relative to baseline levels that were determined on the day of administration. In contrast, mice treated with the negative control siRNA showed increased luciferase activity 48 hours post-treatment, which is presumably due to continued tumor growth. Taken together, the data suggest that NLE-mediated RNAi delivery led to cellular entry into the majority of orthotopically grown lung tumor cells, loading into the RNAi-induced silencing complex and efficient repression of its intended target.

Figure 2
Systemic delivery of luciferase siRNA (si-luc) to orthotopic H460-luc lung tumors in mice. (a) IVIS images of mice carrying luciferase-expressing lung tumors were taken right before and 48 hours after intravenous injection of si-luc (animals number 3–4) ...

Systemic delivery of let-7 or miR-34 inhibits tumor growth in the K-ras autochthonous NSCLC mouse model

We have previously reported that let-7 and miR-34 interfere with tumor growth in mouse models of NSCLC.19,20,27 However, the therapeutic effects of systemic delivery of their respective miRNA mimics in an orthotopic tumor model has not yet been investigated. To explore this experimentally, we used the KRASG12D autochthonous NSCLC mouse model.28 This model is based on oncogenic KRASG12D that is expressed from a Cre recombinase dependent allele (LSL-KRAS G12D) containing native 5′and 3′ untranslated regions. One hundred percent of LSL-KRAS G12D heterozygous animals develop lung tumors when treated intranasally with adenovirus expressing Cre (Ad-cre). Six-week-old LSL-Kras-G12D mice were administered 5 × 108 plaque-forming units of Ad-Cre intranasally to activate LSL-K-ras G12D (Supplementary Figure S2b,c) and maintained for 10 weeks. At 10 weeks postinfection, synthetic let-7b, miR-34a or negative control (miR-NC) miRNAs conjugated with NLE were introduced via tail-vein injections into groups of five animals every other day for a total of eight injections at a concentration of 1 mg/kg each time. Forty-eight hours after the last treatment, mice were killed and lung tissues were harvested and lung morphology, cell proliferation, and apoptosis were assessed by immunohistochemistry.

Mice treated with miR-NC showed extensive diffuse hyperplasia and adenomas (Figure 3b). Four of the five mice that were injected with NLE-formulated let-7b had significantly lower tumor burden than those injected with miR-NC (Figure 3a,d). qRT-PCR revealed that the lungs from let-7b-treated animals had significantly higher levels of let-7b than did the lungs from animals that were treated with miR-NC (Figure 3e). Consistent with our previous finding,19 TdT-mediated dUTP nick end labeling (TUNEL), measuring apoptosis, as well as Ki-67 staining, measuring proliferation, showed that systemically delivered let-7b reduced proliferation without affecting apoptosis (Figure 3c). The mean value of the Ki-67 index obtained as a percentage of 1,000 background cells was 13.6 for let-7b treated mice compared to 51.5 for miR-NC treated mice (P = 0.01). Interestingly, the single animal that did not respond to treatment with let-7b (Supplementary Figure S2a) showed increased let-7b levels in the lungs, yet failed to show a decrease in proliferation as indicated by Ki-67 staining. It remains unclear why lung tumors from this animal did not respond to let-7b treatment.

Figure 3
Systemic delivery of let-7b mimic reduces lung tumor burden in a Kras activated non-small cell lung cancer model. Whole lungs and tumor histologies (H&E) are shown. (a) Mice treated with let-7b display a significant reduction in lung lesions (arrows) ...

In mice treated with miR-34a, we observed significantly reduced tumor burden compared to those treated with miR-NC (a 60% reduction in tumor area) (Figure 4a, (second row); Figure 4c; Supplementary Figure S3). This corresponded to a significant increase of miR-34a levels in the lung as measured by quantitative reverse transcriptase PCR (Figure 4d). Lung tissues from miR-34a treated mice showed reduced expression of Ki-67 with an index of 20.2 compared to 51.5 for miR-NC treated mice (P = 0.04) and an increase in TUNEL-positive cells when compared to miR-NC treated mice (16.6% versus 2.4%, respectively) (Figure 4b). These results show that systemic delivery of miR-34a mimics can effectively cause reduction of advanced lung tumors in a Kras activated NSCLC mouse model through inhibition of proliferation and induction of apoptosis.

Figure 4
miR-34a mimics reduce lung tumor burden in an autochthonous non-small cell lung cancer model. Whole lungs and tumor histologies (H&E) are shown. (a) LSL-K-ras G12D mice tumors were allowed to develop for 10 weeks. Then, synthetic miR-34a or miR-NC ...

Discussion

In summary, our data provide strong evidence that miRNA mimics delivered systemically via NLE is a viable therapeutic approach for the treatment of lung cancer. Since a single miRNA can target a number of different genes, restoring the loss of a miRNA in cancer can potentially affect multiple cellular pathways to induce a therapeutic response. Support for this hypothesis is presented here: miR-34a is fully capable of inhibiting a KRAS-dependent tumors despite the fact that KRAS is not predicted to be directly repressed by miR-34a. Thus, the repression of other cancer related genes, presumably downstream of oncogenic RAS, is likely to induce the tumor-inhibitory effects of miR-34a in this mouse model. Although let-7 and miR-34 share a few common targets, such as CDK6 and MYC,8,9,13,29 many of their targets remain distinct, suggesting that their mechanisms of tumor inhibition are also distinct. This might be reflected by our observation that miR-34 treated tumors displayed reduced proliferation markers and showed increased apoptosis, while let-7 treated tumors showed reduced proliferation only. Given that both, let-7 and miR-34a are frequently downregulated in human lung tumors, and that they might affect distinct cancer pathways, a let-7/miR-34a combination might yield superior therapeutic effects than any of the miRNAs used alone.

In addition to inducing cell cycle arrest and apoptosis, overexpression of miR-34a has also been shown to cause cellular senescence in various cell lines13,14 and knockdown of miR-34a has been demonstrated to lead to the opposite effect of delayed onset cellular senescence.30 miR-34a's ability to induce apoptosis and senescence is potentially through a positive feedback loop with p53 involving a miR-34a direct target, involving a miR-34a direct target, silent information regulator 1 (SIRT1).31,32 SIRT1 is an NAD-dependent deacetylase that regulates apoptosis, cellular senescence, and limits longevity31,33 and one of its molecular targets is p53.34 miR-34a overexpression seems to have different effects in different cell types resulting in cellular senescence in some cell lines and inhibition of cell proliferation and apoptosis in others. These different outcomes might be due to potentially unique cellular factors that interact with miR-34a or availability of specific targets within different cell types. Indeed, the complexity of miR-34a function is seen in another level of regulation involving p53-independent upregulation of miR-34a in Caenorhabditis elegans and mammalian cells following exposure to DNA damaging agents and cell differentiation factors.15,29 p53-independent upregulation of miR-34a is driven from an alternative promoter element (currently uncharacterized), ~20 kilobasepairs upstream of the characterized p53 promoter element.29 The potential for the existence of different pathways involving miR-34a highlights the importance of this microRNA as a tumor suppressor.

Systemic delivery of miRNA mimics to orthotopic lung tumors was achieved with NLE, a novel lipid-based delivery vehicle that previously facilitated in vivo delivery of miR-34a mimics to subcutaneous tumors in mice.20 Unlike most lipid-based delivery systems, NLE does not contain cationic lipids, and therefore, may bypass some of the shortcomings that can be attributed to charge. For instance, particles based on neutral lipids are less likely to form aggregates in biofluids, be filtered by the liver, adhere to the endothelium or be taken up by scavenging macrophages.25 In accordance, NLE did not lead to a preferential accumulation of miRNA in liver, but instead, displayed excellent delivery to normal lung and orthotopic lung tumors. In addition, neutral lipids delivery systems may be less toxic than those containing cationic lipids in agreement with our previous observation that miR-34a formulated with NLE did not lead to elevated liver and kidney enzymes in serum nor induce a nonspecific immune response in mice.20 Hence, successful delivery to orthotopic lung tumors, a positive therapeutic response in a clinically relevant mouse model and a favorable safety margin further support the development of these let-7 and miR-34 formulations as novel targeted therapies for lung cancer patients.

Materials and Methods

miRNA biodistribution via NLE. NLE (MaxSuppressor in vivo RNALancerII) was purchased from BIOO Scientific, (Austin, TX). NLE consists of 1,2-dioleoyl-sn-glycero-3-phosphocholine, squalene oil, polysorbate 20, and an antioxidant that—in complex with synthetic miRNAs—forms nanoparticles in the nanometer diameter range. All animal experiments were performed in accordance with currently prescribed guidelines and under a protocol approved by the Institutional Animal Care and Use Committee at BIOO Scientific. Twelve female Balb/c mice ~10 weeks of age were given a single dose of 20 µg synthetic microRNA-124 (Dharmacon, Lafayette, CO) formulated with NLE according to the manufacturer's instructions by intravenous tail-vein injection. After 10 minutes, blood was drawn from animals via the orbital sinus and animals were subsequently killed by CO2 asphyxiation. To investigate blood associated delivery of miRNA-124, half the animals were perfused with 0.9% saline solution (NaCl) to remove circulating blood. To perfuse the animals, the peritoneum, pleural cavity and pericardium were exposed, the descending vena cava was nicked with tweezers and 20 ml 0.9% saline solution was injected directly into the left ventricle of the heart. Liver, lung, and kidney were removed from all animals and were snap frozen in liquid nitrogen. RNA was isolated from all tissues and whole blood following the mirVana miRNA Isolation Kit Procedure (Ambion/Life Technologies, Austin, TX). A miRNA-124 primer/probe set (Applied Biosystems, Foster City, CA/Life technologies) was used to analyze miRNA-124 levels in tissues and blood by qRT-PCR. To assess total copies per cell of microRNA-124, all samples were plotted against a standard curve made up of known copy numbers of synthetic miRNA-124.

Orthotopic H460-luc xenografts. Orthotopic lung tumor xenografts were generated by endotracheal intubation of human NCI-H460 NSCLC cells stably expressing the firefly luciferase gene (H460-luc) following a modified protocol described in Brown et al.26 To demonstrate that endotracheal intubation facilitates delivery deep into the lungs, BALB/cJ mice (stock # 651; The Jackson Laboratory, Bar Harbor, ME) were intubated with a catheter to disperse either India ink (Becton, Dickinson and Company, Franklin Lakes, NJ) or green dye (Waterman, Paris, France). Lungs were dissected to identify stained areas macro- and microscopically. For orthotopic lung cancer xenografts, NOD/SCID mice (stock #1303; The Jackson Laboratory) were inoculated by endotracheal intubation using a 22G × 1” catheter to deliver 2 × 106 H460-luc cells directly to the lungs in 20 µl of RPMI growth media (Gibco, Invitrogen, Carlsbad, CA) with 0.01 mol/l EDTA. Mice were regularly monitored by a live animal imaging system (IVIS; Xenogen, Caliper Life Sciences, Hopkinton, MA) measuring luminescence (luciferase activity) following an intraperitoneal injection of 200 µl at 15 mg/ml of the luciferase substrate -luciferin (catalog #122796; Xenogen, Caliper Life Sciences, Hopkinton, MA). Once mice developed lung tumors that are detectable by IVIS measurements and show an increase in luminescence expression over time, mice were injected intravenously with 20 µg of either a synthetic luciferase-specific siRNA (si-luc; Ambion) or a negative control oligonucleotide (NC; Dharmacon) formulated with NLE (MaxSuppressor in vivo RNALancerII) according to the manufacturer's instructions. Luminescence was measured immediately prior to injection of formulated oligonucleotide (0 hours) and again 48 hours thereafter, and expressed as percent relative to the total flux of each mouse at 0 hours (100%).

Quantitative reverse transcriptase PCR. Total RNA from mouse tissues was isolated using the mirVANA PARIS RNA isolation kit (Ambion) following the manufacturer's instructions. For RT-PCR detection of let-7b and miR-34a oligonucleotides, 10 ng purified RNA was heat-denatured at 70 °C for 2 minutes and reverse transcribed using the let-7b and miR-34a TaqMan miRNA Assay (Applied Biosystems) with MMLV-RT (Invitrogen). Quantification of miRNA levels was performed using the Taqman miRNA Assay (Applied Biosystems, per standard protocol).

In vivo adenoviral infection and systemic delivery of let-7b and miR-34a to LSL-K-ras G12D mice. Six-week-old LSL-Kras-G12D mice were administered 5 × 108 plaque-forming units of Ad-Cre intranasally and maintained for 10 weeks. At 10 weeks postinfection, each mouse was injected with 20 µg synthetic let-7b, miR-34a or negative control miRNA (miR-NC) (Dharmacon) conjugated with NLE by intravenous tail-vein injections. Five animals per group were used. Formulations were given every other day for a total of eight injections at a concentration of 1 mg/kg each time. At 48 hours after the last treatment, mice were killed and lung tissues were harvested. Lungs were prepared for histological analysis to examine tumor burden by fixing the tissue in 4% paraformaldehyde overnight, embedded in paraffin and stained with hematoxylin and eosin. Ad-Cre recombination was tested using a PCR assay to verify that K-ras G12D mice had undergone Cre-mediated removal of the stop element at the mutant K-ras locus as previously described.28 Lung and tumor areas were quantified using ImageJ software in manual measurement mode as described previously.27 The overall tumor burden was measured as a ratio of total tumor area to total lung area by taking the average of every seventh slide section throughout the lung. Ki-67 and TUNEL staining was performed by the Pathology core (Yale, New Haven, CT) and quantified based on four individual samples/group. The Ki-67 index is defined as percent of positive cells per 1,000 surrounding cells.

SUPPLEMENTARY MATERIAL Figure S1. Orthotopic NSCLC xenografts. (a) Lungs from mice inoculated with green dye by endotracheal intubation. to, tongue; tr, trachea; lu, lung. (b) Micrograph showing lung tissue stained with hematoxylin and eosin from mice endotracheally intubated with India Ink. Arrowheads indicate India Ink in distal alveoli. (c) IVIS image of a mouse carrying an orthotopic H460-luc tumor 47 days post xenograft intubation. (d) Lung histology of the mouse shown in c) on day of sacrifice (day 52). Arrows indicate tumor cells within lung stroma. Figure S2. Delivery of let-7 mimics with outlier shown. (a) Quantitative analysis of tumor burden in LSL-K-rasG12D animals treated with Ad-Cre and let-7b (n=5) (including outlying animal) versus LSL-K-ras G12Danimals treated with Ad-Cre and miR-NC (n=5). The ratios of tumor area versus normal lung area are presented as a box-and-whisker plot. Boxes represent interquartile ranges. The total range, mean (•), and median (blank bar) are shown. Red circle denotes outlier. (b) Recombination of K-ras. The Ad-Cre recombination was tested using the PCR assay with lung DNA to verify that K-ras G12Dmice had undergone Cre-mediated removal of the stop cassette. Lane legend: mice treated with Ad-Cre/let-7b mimic that shows the removal of the stop cassette performed by Cre activity lanes 1-4 (c) Genomic DNA from animals treated with Ad-Cre and miR-34a mimic is shown (lane 6-10). Lane 11 presents a positive control. The expected band for the looped out stop cassette is at 315 bp (G12D), while the wild type band is 285 bp (WT). Figure S3. LSL-K-ras G12D mice treated with miR-34a (second column) display a reduced tumor burden (arrows).

Acknowledgments

We would like to thank Dr Robert Homer at Yale Pathology for help with the sections, Dr Paul Lammers for critical reading of this manuscript. P.T. was supported by an NRSA postdoctoral fellowship (F32CA130376); A.G.B. was supported by a grant from the NCI (1R43CA134071); J.W. and F.S. were supported by a grant from the NCI (1R01CA131301). J.F.W., C.D., M.O., D.B., and A.G.B. are employees of Mirna Therapeutics, Inc., which develops miRNA-based therapeutics. F.J.S. is scientific advisor for Mirna Therapeutics. All other authors declare no competing financial interests.

Supplementary Material

Figure S1.

Orthotopic NSCLC xenografts. (a) Lungs from mice inoculated with green dye by endotracheal intubation. to, tongue; tr, trachea; lu, lung. (b) Micrograph showing lung tissue stained with hematoxylin and eosin from mice endotracheally intubated with India Ink. Arrowheads indicate India Ink in distal alveoli. (c) IVIS image of a mouse carrying an orthotopic H460-luc tumor 47 days post xenograft intubation. (d) Lung histology of the mouse shown in c) on day of sacrifice (day 52). Arrows indicate tumor cells within lung stroma.

Figure S2.

Delivery of let-7 mimics with outlier shown. (a) Quantitative analysis of tumor burden in LSL-K-rasG12D animals treated with Ad-Cre and let-7b (n=5) (including outlying animal) versus LSL-K-ras G12Danimals treated with Ad-Cre and miR-NC (n=5). The ratios of tumor area versus normal lung area are presented as a box-and-whisker plot. Boxes represent interquartile ranges. The total range, mean (•), and median (blank bar) are shown. Red circle denotes outlier. (b) Recombination of K-ras. The Ad-Cre recombination was tested using the PCR assay with lung DNA to verify that K-ras G12Dmice had undergone Cre-mediated removal of the stop cassette. Lane legend: mice treated with Ad-Cre/let-7b mimic that shows the removal of the stop cassette performed by Cre activity lanes 1-4 (c) Genomic DNA from animals treated with Ad-Cre and miR-34a mimic is shown (lane 6-10). Lane 11 presents a positive control. The expected band for the looped out stop cassette is at 315 bp (G12D), while the wild type band is 285 bp (WT).

Figure S3.

LSL-K-ras G12D mice treated with miR-34a (second column) display a reduced tumor burden (arrows).

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