Modulation of mismatch repair and genomic stability by miR-155 Nicola Valeria,b,1, Pierluigi Gasparinia,1, Muller Fabbria,c,1, Chiara Braconid, Angelo Veronesea, Francesca Lovata, Brett Adaira, Ivan Vanninic, Francesca Faninic, Arianna Bottonia, Stefan Costineana, Sukhinder K. Sandhua, Gerard J Nuovoe, Hansjuerg Aldera, Roberta Gafaf, Federica Calorea, Manuela Ferracinf, Giovanni Lanzaf, Stefano Voliniaa,b, Massimo Negrinif, Michael A. McIlhattona, Dino Amadoric, Richard Fishela,g,2, and Carlo M. Crocea,f,2 aDepartment of Molecular Virology, Immunology and Medical Genetics, Ohio State University Medical Center and Comprehensive Cancer Center, Columbus, OH 43210; bDepartment of Embryology and Morphology, University of Ferrara, Ferrara 44121, Italy; cIstituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola 47014, Italy; dDepartment of Internal Medicine, Ohio State University Medical Center and Comprehensive Cancer Center, Columbus, OH 43210; eDepartment of Pathology, Ohio State University Medical Center, Columbus, OH 43210; fDepartment of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara 44121, Italy; and gDepartment of Physics, Ohio State University, Columbus, OH 43210 Contributed by Carlo M. Croce, February 25, 2010 (sent for review February 5, 2010). Inactivation of mismatch repair (MMR) is the cause of the common play MSI resulting from mutation or inherited epigenetic inac- cancer predisposition disorder Lynch syndrome (LS), also known as tivation of the core MMR genes (13–15). Most of the 10–40% of hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10– sporadic CRC, endometrial, ovarian, gastric, and urothelial 40% of sporadic colorectal, endometrial, ovarian, gastric, and uro- tumors that display MSI are a result of acquired hMLH1 pro- thelial cancers. Elevated mutation rates (mutator phenotype), moter methylation (16). Approximately 95% of MSI tumors can including simple repeat instability [microsatellite instability (MSI)] be accounted for, at least partially, by mutation and/or epigenetic are a signature of MMR defects. MicroRNAs (miRs) have been inactivation of the core MMR components (14). The remaining implicated in the control of critical cellular pathways involved in 5%, as well as a significant proportion of the biallelic MMR development and cancer. Here we show that overexpression of inactivation mechanism, remain poorly understood. miR-155 significantly down-regulates the core MMR proteins, MicroRNAs (miRs) are noncoding RNAs that play a role in hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and the posttranscriptional regulation of >30% of the human genes MSI. An inverse correlation between the expression of miR-155 controlling critical biological processes, including development, MEDICAL SCIENCES and the expression of MLH1 or MSH2 proteins was found in cell differentiation, apoptosis, and proliferation (17, 18). Over- human colorectal cancer. Finally, a number of MSI tumors with expression of miR-155 has been observed in CRC (19–21), and unknown cause of MMR inactivation displayed miR-155 overex- appears to be more frequent in MSI than MSS CRCs (22). These pression. These data provide support for miR-155 modulation of data are consistent with the hypothesis that miR-155 might MMR as a mechanism of cancer pathogenesis. regulate components of the MMR machinery and, consequently, rates of mutation and MSI. colorectal cancer | DNA repair | microRNA Results ismatched nucleotides may arise from polymerase mis- hMLH1, hMSH2, and hMSH6 Are Targets of miR-155. We used in silico Mincorporation errors, recombination between heteroallelic prediction models to identify potential binding sites for miR-155 parental chromosomes, or chemical and physical damage to the in the mRNA of the core MMR genes (23, 24). Two putative DNA (1). MutS homologs (MSH) and MutL homologs (MLH/ sites were found in hMLH1 using RNAhybrid (BiBiServ; NCBI PMS) are highly conserved proteins that are essential for the NM_000249.2), one in the 3′-UTR and the other in the coding mismatch repair (MMR) excision reaction (2). In human cells, sequence (CDS) (NCBI NM_000249.2). One site was found in hMSH2 and hMLH1 are the fundamental components of MMR. the 3′-UTR of hMSH2 using TargetScan (Whitehead Institute, The hMSH2 protein forms a heterodimer with hMSH3 or hMSH6 MIT; NCBI NM_000251.1), and one site was found in the CDS and is required for mismatch/lesion recognition, whereas the of hMSH6 using RNAhybrid (BiBiServ; NCBI NM_000179.2) hMLH1 protein forms a heterodimer with hMLH3 or hPMS2 and (Fig. 1A and Fig. S1). As a functional screen, we subcloned the forms a ternary complex with MSH heterodimers to complete the coding and 3′-regions including the predicted miR-155 seed excision repair reaction (2, 3). Human cells contain at least 10-fold regions of MLH1, MSH2,orMSH6 downstream of the luciferase more of the hMSH2-hMSH6/hMLH1-hPMS2 complex, which gene and recorded luciferase protein activity (25). We used repairs single nucleotide and small insertion-deletion loop (IDL) MMR-proficient Colo-320 DM CRC cells for these studies, mismatches, compared with the hMSH2-hMSH3/hMLH1-hMLH3 because they contain a low basal level of miR-155 (26). The complex, which primarily repairs large IDL mismatches (4–6). In Colo-320 DM cells were transfected with the luciferase reporter addition to MMR, the hMSH2-hMSH6/hMLH1-hPMS2 compo- constructs and with a miR-155 precursor (premiR-155) or con- nents have been uniquely shown to recognize lesions in DNA and to signal cell cycle arrest and apoptosis (7, 8). Mutations in the hMSH2, hMSH6, hMLH1, and hPMS2 core Author contributions: N.V., P.G., M. Fabbri, R.F., and C.M.C. designed research; N.V., P.G., MMR genes have been linked to Lynch syndrome (LS), also M. Fabbri, A.V., F.L., B.A., I.V., F.F., A.B., S.C., S.S., G.J.N., R.G., F.C., M. Ferracin, G.L., S.V., M.N., M.M., and D.A. performed research; H.A. contributed new reagents/analytic tools; known as hereditary nonpolyposis colorectal cancer (HNPCC) N.V., P.G., M. Fabbri, C.B., A.V., G.J.N., M.M., R.F., and C.M.C. analyzed data; and N.V., (9). These observations have provided considerable support for P.G., M. Fabbri, C.B., R.F., and C.M.C. wrote the paper. MSH MLH/ the mutator hypothesis, because defects in the and The authors declare no conflict of interest. PMS fi genes signi cantly increase cellular mutation rates, which Freely available online through the PNAS open access option. then may drive the evolution of numerous oncogene and tumor- 1N.V., P.G., and M. Fabbri contributed equally to this work. suppressor gene mutations found in cancers (10). The most 2To whom correspondence may be addressed. E-mail: [email protected] or rfishel@ obvious signature of a mutator phenotype is instability of simple osu.edu. repeat sequences or microsatellite DNA [ie, microsatellite This article contains supporting information online at www.pnas.org/cgi/content/full/ instability (MSI)] (11, 12). Virtually all LS/HNPCC tumors dis- 1002472107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1002472107 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 M S H 6 A 5 ’ ML H 1 3 ’ 5 ’ M S H 2 3 ’ 5 ’ ’3 19 7 6 1 9 9 4 23 6 8 2 3 9 5 29 4 6 2 9 6 3 39 3 7 3 9 5 1 B p re -m iR co n t r o l M L H1 p r e - m i R - 15 5 M SH 2 M SH 6 12 0 12 0 12 0 10 0 * * 10 0 10 0 * 80 * 80 80 * 60 60 60 40 40 40 relative to control to relative relative to control to relative relative to control to relative dual luciferase activity, 20 20 20 dual luciferase activity, dual luciferase activity, 0 0 0 5 5 5 5 5 T 15 1 5 15 T 5 T 5 1 W T- H 1 T - 1 T - s W T - 1 W T - M U L M U L H M U i te M U 2 U 6 M M h s SH M SH T R D S ot M M 3' U C b Fig. 1. hMLH1, hMSH2, and hMSH6 are direct targets of miR-155. (A) Locations of the target sites of miR-155 in the 3′ UTRs and/or the CDS of the indicated genes (see also Fig. S1). The base position is counted from the first nucleotide in the CDS. (B) Colo-320 DM cells were transfected with the phRL-SV40 construct as a control and either the luciferase construct WT or MUT-155 and with premiR-155 or premiR control. After 24 h, dual luciferase assays were performed.*P < 0.005 relative to premiR control. trol precursor (premiR control). We observed luciferase activity process, miRs cannot modulate a target protein. Both MLH1 and reductions of 37%, 38%, and 24% in the presence of premiR-155 MSH6 contain CDS seed regions (Fig. 1 and Fig. S1). To with constructs containing the miR-155 seed regions of MLH1, examine the possibility of miR-RISC displacement, we cotrans- MSH2, and MSH6, respectively (P < 0.001) (Fig. 1B; see WT for fected cell lines lacking MLH1 (HCT116) or MSH6 (DLD1) each). No changes in luciferase activity were seen when the with a mammalian expression vector containing the corre- constructs contained a deletion of the miR-155 seed region (Fig. sponding full-length cDNA with or without the miR-155 pre- 1B; see MUT-155 for each construct). Interestingly, the effect of cursor and examined protein expression (Fig. S2). We deleted the individual miR-155–binding sites in the hMLH1 gene the CDS miR-155 target site in the hMLH1 and hMSH6 cDNA appeared to be additive (Fig.