Proliferation and tumorigenesis of a murine sarcoma cell line in the absence of DICER1

Abstract

MicroRNAs are a class of short ~22 nucleotide RNAs predicted to regulate nearly half of all protein coding genes, including many involved in basal cellular processes and organismal development. Although a global reduction in miRNAs is commonly observed in various human tumors, complete loss has not been documented, suggesting an essential function for miRNAs in tumorigenesis. Here we present the finding that transformed or immortalized Dicer1 null somatic cells can be isolated readily in vitro, maintain the characteristics of DICER1-expressing controls and remain stably proliferative. Furthermore, Dicer1 null cells from a sarcoma cell line, though depleted of miRNAs, are competent for tumor formation. Hence, miRNA levels in cancer may be maintained in vivo by a complex stabilizing selection in the intratumoral environment.

Figure 1

Characterization of Kras^G12D;Trp53^-/-;Dicer1^-/- sarcoma cells. A) Derivation scheme for Dicer1^-/- sarcoma cells. Hindlimb injection of Adeno-cre generates Kras^G12D;Trp53^-/-;Dicer1^f/- tumors. Clones isolated following Cre-ER integration and tamoxifen treatment were genotyped by PCR to identify Dicer1^-/- clones. B) miRNA expression (copies per cell). Per cell calculations are based on relative representation of each miRNA in Dicer1^f/- and Dicer1^-/- small RNA-seq libraries, normalized to quantitative Northern blot of miR-22 in Dicer1^f/- cells (shown in Fig. S1C). miR-21, miR-22, and let-7 family members are indicated. C) Northern analysis for precursor and mature miRNAs. Glutamine tRNA was used to control for loading, and a dilution series of Dicer1^f/- RNA (1:1 to 1:16) is provided for quantitation. D) Luciferase reporter assays for abundant miRNAs. The renilla luciferase reporter contains six bulged sites for the let-7 family, and two perfect sites for miR-16 and miR-17. Targeted renilla luciferase reporters were normalized to nontargeted firefly luciferase reporters. Renilla/firefly luciferase expression was normalized to expression in the Dicer1^f/- sarcoma cell line. E) Proliferation assay. F) Cell cycle distribution determined by BrdU labeling. G) Apoptosis determined by caspase-3 cleavage assay. All error bars represent the SEM (D-G). See also Figure S1 and Table S1.

Figure 2

Tumorigenesis of Kras^G12D;Trp53^-/-;Dicer1^-/- sarcoma cells in transplant assays. A) Injection of 2.5 × 10⁴ Dicer1 ^f/- and Dicer1^-/- sarcoma cells into the flanks of nude mice. Error bars represent the SEM. B) Hematoxylin and eosin section of Dicer1^f/- and Dicer1^-/- tumors. In C-F, each lane represents an independent tumor derived from one injection of the indicated Dicer1^-/- sarcoma cell line. Scale bar, 100 μm. C) PCR genotyping of Dicer1^-/- tumors. Recombined and floxed bands are derived from the injected tumor cells, while wild type bands derive from host tissue. D) Northern analysis of tumor tissue derived from sarcoma injections. E) PCR and F) Northern analysis following one round of in vitro passage of secondary tumors. In C-F, each sample ID contains a prefix identifying the injected sarcoma cell clone followed by a suffix identifying the tumor replicate (e.g. sample 1-3 corresponds to clone 1 and tumor replicate 3). See also Figure S2.

Figure 3

Derivation and characterization of Dicer1^-/- mesenchymal stem cells. A) Schematic of MSC preparation. Primary MSC cultures were prepared from the tibia, femur, and pelvic bones of a one-year old Dicer1^f/f mouse. The primary cells were then infected with retrovirus encoding SV40 large T-antigen. Monoclonal cultures were then isolated, infected with Adeno-Cre-GFP, sorted by FACS for GFP-positive cells, and plated at low density to isolate Dicer1-recombined clones. B) Cell surface marker expression in Dicer1^f/f (left) and Dicer1^-/- (right) MSCs. Cells were analyzed by flow cytometry with antibodies against CD49e and CD106. C) PCR genotyping of clonally isolated Dicer1^f/f or Dicer1^-/- MSCs. Clones 6.8 and 6.9 (lanes 3, 4) were derived from parental clone 6 (lane 2), and clones 12.2 and 12.4 (lanes 7, 8) were derived from parental clone 12 (lane 5). PCR genotyping of a Dicer1^f/- sarcoma cell line was used as a heterozygous control (lane 1). D) Expression of miRNAs in Dicer1^f/f and Dicer1^-/- MSCs. Total RNA was analyzed with a Qiagen miScript qPCR assay for let-7a, mir-24, -26, and -31). A representative qPCR experiment is shown. Error bars indicate standard deviation. E) Luciferase reporter assay for let-7g. The reporter contains 6 bulged sites. Targeted renilla luciferase reporters were normalized to nontargeted firefly luciferase reporters. Renilla/firefly luciferase expression was normalized to expression in the Dicer1^f/f MSC line. F) Proliferation assay. G) Cell cycle distribution determined by BrdU labeling. H) Apoptosis determined by caspase-3 cleavage assay. For (E-H), error bars represent the SEM. See also Figure S3.