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. 2012 Jun;2(6):540-53.
doi: 10.1158/2159-8290.CD-11-0267. Epub 2012 Mar 30.

miR-23a promotes the transition from indolent to invasive colorectal cancer

Sohail Jahid  1 , Jian Sun, Robert A Edwards, Diana Dizon, Nicole C Panarelli, Jeffrey W Milsom, Shaheen S Sikandar, Zeynep H Gümüs, Steven M Lipkin
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miR-23a promotes the transition from indolent to invasive colorectal cancer

"V体育2025版" Sohail Jahid et al. Cancer Discov. 2012 Jun.

"V体育官网" Abstract

Colorectal cancer is a classic example of a tumor that progresses through multiple distinct stages in its evolution. To understand the mechanisms regulating the transition from indolent to invasive disease, we profiled somatic copy number alterations in noninvasive adenomas and invasive adenocarcinomas from Apc and DNA mismatch repair (MMR) mutant mouse models VSports手机版. We identified a recurrent amplicon on mouse chromosome 8 that encodes microRNA (miRNA) 23a and -27a (miR). miR-23a and -27a levels are upregulated in mouse intestinal adenocarcinomas, primary tumors from patients with stage I/II colorectal cancers, as well as in human colorectal cancer cell lines and cancer stem cells. Functionally, miR-23a promotes the migration and invasion of colorectal cancer cells and stem cells, whereas miR-27a primarily promotes proliferation. We computationally and experimentally validated that metastasis suppressor 1 (MTSS1) is a direct miR-23a target and similarly validated that the ubiquitin ligase FBXW7 is a direct miR-27a target. Analyses of computationally predicted target genes in microarray data sets of patients with colorectal cancers are consistent with a role for miR-23a, but not miR-27a, specifically in invasive colorectal cancers. .

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Figures

Figure 1
Figure 1
Array-CGH analysis of Apc/MMR dual deficient Gl tumors. A, depiction of aCGH hybridization signal from a representative adenocarcinoma (additional tumors are shown in supplemental figure 1). The arrow indicates a region with gain of signal on chromosome 8. B,C, quantitative PCR of miR-23a and miR-27a levels in cDNA from MMR-deficient adenomas and Apc/MMR dual deficient adenocarcinomas. Levels are shown in fold relative to matched normal tissue. * denotes Student t test p value<0.001. D, representative images of a pre-invasive (adenoma, carcinoma in situ) (top), invasive (stage I, II) (middle) and metastatic (stage III and IV)(bottom) tumor stained with H&E. E, miR-23a level in pre-invasive tumors and primary CRCs from patients with invasive and metastatic disease. Pre-invasive (adenoma, carcinoma in situ), invasive (stage I, II) and metastatic (stage III and IV). * denotes Student t test p value<0.0001 in comparisons of pre-invasive vs. invasive levels or invasive vs. metastatic CRC expression level. Adenoma CiS n=31, stage I/II n=31, stage III/IV n=31. F, miR-27a levels in pre-invasive, invasive and metastatic human CRCs. Pre-invasive (adenoma, carcinoma in situ), invasive (stage I, II) and metastatic (stage III and IV). * denotes Student t test p value<0.005 in comparisons of pre-invasive vs. invasive levels or invasive vs. metastatic CRC expression levels. Adenoma CiS n=31, stage I/II n=31, stage III/IV n=31. G, array based microRNA profiling shows that miRs-23a and 27a are among the most highly expressed in colon cancer stem cells (left arrow miR-23a, right arrow miR-27a). The full list of microRNAs profiled is given in Supplemental Table 1.
Figure 2
Figure 2
FBXW7 is a direct target of miR-27a. JUN and MYC are downstream targets. A, schematic of miR-27a binding sites in FBXW7 mRNA 3’UTR sequences at nt (502–508 and 1378–1384) (top), miR-27a binding activity luciferase reporter assay in colon cancer stem cells CCSC (bottom). CCSC were transfected with a plasmid containing the FBXW7 miR-27a binding site fused to the 3' UTR of Firefly luciferase and co-transfected with plasmids driving expression of pre-miR-27a or a control insert sequence. Luciferase protein levels and activity are suppressed when a miR binds specifically to the 3' UTR target sequence. B, analysis of FBXW7, phospho and total c-JUN, phospho and total c-MYC protein levels. C, schematic of FBXW7 downstream targets under normal cellular conditions. D, qRT-PCR of NOTCH signaling downstream targets HES1 and HES5. E, bioassay of MUC2 immunocytochemistry+ cells. MUC2+ cells (brown HRP stain) indicate suppression of NOTCH signaling in CCSC. F, table with quantification of MUC2+ cells in miR-27a or control shRNA knockdown CCSC.
Figure 3
Figure 3
miR-27a increases CRC cell proliferation, clonogenicity and xenograft tumor volume. A, MTT assay of colon cancer stem cell (CCSC), LoVo, and SW480 cells infected with lentivirus expressing either anti-miR-27a or scrambled control shRNA. In all three lines, anti-miR-27a shRNA significantly reduced the number of viable cells. B, representative photos of CCSC lentiviral transduced with either anti-miR-27a or control shRNA sequences and plated in 3D collagen culture. Top, light microscopy at 10X. Bottom, hematoxylin and eosin staining of CCSC colony at 40X. C, Reduced volume of collagen 3D culture colony size in anti-miR-27a vs. scrambled control sequence lentiviral transduced CCSC, p<0.001. D, photos of xenograft tumors from CCSC transduced with either control shRNA (left) or anti-miR-27a (right). E, growth curve of subcutaneous xenograft tumor volume in mice injected with anti-miR-27a expressing (red) or control sequence (blue) CCSC, p<0.001 by Student t test. F, growth curve of subcutaneous xenograft tumor volume in mice injected with anti-miR-27a expressing (red) or control sequence (blue) LoVo cells, p<0.001 by Student t test. 95% confidence interval error bars are shown.
Figure 4
Figure 4
miR-23a knockdown increases MTSS1 protein levels and inhibits CRC cell and CCSC migration and invasion. A, crystal violet staining of a transwell migration assay of CCSC transduced with lentivirus containing anti-miR-23a shRNA or control sequences. B,C, anti-miR-23a or control sequence lentiviral transduced LoVo, SW480, and CCSC migration and invasion, 95% CI error bars are shown. For all comparisons p <0.002 by Student t test. D, miR-23a binding site in MTSS1 mRNA 3’UTR (nt 1949–1955) (top), bottom, miR-23a binding activity luciferase reporter assay in CCSC. CCSC were transfected with a plasmid containing the MTSS1 miR-23a binding site fused to the 3' UTR of Firefly luciferase and co-transfected with plasmids driving expression of pre-miR-23a or a control insert sequence. Luciferase protein levels and activity are suppressed when a miR binds specifically to the 3' UTR target sequence. E, MTSS1 protein levels in CCSC infected with lentivirus expressing a control or anti-miR-23a shRNA.
Figure 5
Figure 5
Filopodia formation is dramatically decreased following knockdown of miR-23a in CCSC. A, light microscope image of CCSC transduced with lentivirus containing control 10X (left; inset (40X) indicate the presence of filopodia) or anti-miR-23a shRNAs 10X (right, inset (40X) showing absence of filopodia). B, F-actin staining (phalloidin) of CCSC with control (left, inset @ 40X showing filopodia formation) or anti-miR-23a lentiviral knockdown (right, inset @ 40X with no filopodia formation). C, mean number of filopodia per colony of CCSC infected with anti-miR-23a or control lentiviral shRNA. (Student t test p<0.001). D, RHOB levels are increased post restoring of MTSS1 protein by knockdown of miR-23a. E, Rac1 activity is decreased by 25% in CCSC expressing anti-miR-23a, tested using the G-LISA assay (Student t test p<0.05). F, Phosphorylated pSrc416 levels are decreased in the miR-23a KD cells.
Figure 6
Figure 6
Knockdown of miR-23a or miR-27a reduces tumor burden and increases survival. A, gross image of lung from mice with CCSC injected into tail vein, left is CCSC expressing control sequence, middle anti-miR-23a, right anti-miR-27a CCSC. B, Lung tumor multiplicity in tail vein metastasis assay using CCSC expressing control shRNA, anti-miR-23a or anti-miR-27a. C, image of H&E stained lung tumor from tail vein injected mice. Left is control shRNA, middle is anti-miR-23a, and right is anti-miR-27a. D, Lung tumor volume from either CCSC expressing control shRNA, anti-miR-23a or anti-miR-27a. E, Kaplan-Meir survival curve for tail vein injected mice with either CCSC expressing Ctrl shRNA (black line) or anti-miR-23a (red line). Control scrambled shRNA sequence. F, Kaplan-Meir survival curve for tail vein injected mice with either CCSC cells expressing Ctrl shRNA (black line) or anti-miR-27a (red line). Control scrambled shRNA sequence.
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