ID	Duplex structure	Position	Score	MFE
1	miRNA 3' ggGUUGUUGUACUUUGAUGGAu 5' |||||||||||||||:||| Target 5' ccCAACAACATGAAACTGCCTa 3'	411 - 432	184.00	-35.50
2	miRNA 3' ggguuGUUGUACUUUGAUGGau 5' ||:| ||::|||:|| Target 5' -gcttCAGC-TGGGACTGCCag 3'	1 - 20	124.00	-15.20
3	miRNA 3' ggGUUGUUG------UAC-UUUGAUGGau 5' :||:|:| :|| :|:||||| Target 5' cgTAATAGCTTCTGTGTGTGAGCTACCgt 3'	172 - 200	118.00	-11.90

Mutant ID	Mutant Position	Mutant Source
COSN31578629	9	COSMIC
COSN30455548	10	COSMIC
COSN30143258	18	COSMIC
COSN30154940	34	COSMIC
COSN30155446	41	COSMIC
COSN26979214	43	COSMIC
COSN31505800	56	COSMIC
COSN17436237	135	COSMIC
COSN15219283	225	COSMIC
COSN22987713	265	COSMIC
COSN31484780	296	COSMIC
COSN8603315	363	COSMIC
COSN25516802	386	COSMIC
COSN20114128	460	COSMIC
COSN23289350	461	COSMIC
COSN30161442	472	COSMIC
COSN31779179	477	COSMIC
COSN20093304	559	COSMIC
COSN26554888	562	COSMIC
COSN8603314	579	COSMIC
COSN31571000	600	COSMIC
COSN20825152	732	COSMIC
COSN30166982	744	COSMIC
COSN26549468	752	COSMIC
COSN17183111	839	COSMIC

Mutant ID	Mutant Position	Mutant Source
rs767336984	6	dbSNP
rs908454758	8	dbSNP
rs867920442	12	dbSNP
rs1354941311	14	dbSNP
rs1267757263	17	dbSNP
rs1449864488	18	dbSNP
rs914865246	19	dbSNP
rs761784323	20	dbSNP
rs774175320	21	dbSNP
rs752741466	23	dbSNP
rs755861849	23	dbSNP
rs530602224	24	dbSNP
rs762950008	26	dbSNP
rs1219834233	31	dbSNP
rs775725748	35	dbSNP
rs1374643837	37	dbSNP
rs1310888939	42	dbSNP
rs1325376928	44	dbSNP
rs569491928	51	dbSNP
rs1484223842	54	dbSNP
rs368268515	54	dbSNP
rs1453463082	56	dbSNP
rs981448693	56	dbSNP
rs1406075325	60	dbSNP
rs1426663357	64	dbSNP
rs1335251772	66	dbSNP
rs970577837	67	dbSNP
rs1022999227	69	dbSNP
rs1355441839	74	dbSNP
rs986682300	79	dbSNP
rs887575327	80	dbSNP
rs1426421647	81	dbSNP
rs1026194409	85	dbSNP
rs955368262	90	dbSNP
rs901525344	93	dbSNP
rs568042475	94	dbSNP
rs1298938552	97	dbSNP
rs1344787219	100	dbSNP
rs1241203538	101	dbSNP
rs548302451	105	dbSNP
rs56052247	106	dbSNP
rs888896496	107	dbSNP
rs957416109	111	dbSNP
rs1032974577	114	dbSNP
rs1043493089	129	dbSNP
rs1192961965	134	dbSNP
rs949529691	135	dbSNP
rs1480847597	139	dbSNP
rs984870294	141	dbSNP
rs914714807	142	dbSNP
rs1053384748	143	dbSNP
rs1406232102	147	dbSNP
rs1250596123	152	dbSNP
rs1202136153	154	dbSNP
rs559349896	156	dbSNP
rs1440203855	166	dbSNP
rs1299011894	175	dbSNP
rs928891100	183	dbSNP
rs1225873991	188	dbSNP
rs1306047644	189	dbSNP
rs753643958	196	dbSNP
rs1228575360	198	dbSNP
rs1291526034	205	dbSNP
rs1466765050	208	dbSNP
rs967637572	212	dbSNP
rs1248572603	214	dbSNP
rs1466253453	215	dbSNP
rs1190921556	221	dbSNP
rs1372336950	224	dbSNP
rs1246828451	225	dbSNP
rs1472980909	226	dbSNP
rs1382043236	228	dbSNP
rs568752502	232	dbSNP
rs1449593126	237	dbSNP
rs552002840	239	dbSNP
rs45572038	242	dbSNP
rs1390905169	244	dbSNP
rs1381067290	256	dbSNP
rs868845324	260	dbSNP
rs1318207074	271	dbSNP
rs1341481790	273	dbSNP
rs1298005758	281	dbSNP
rs1450578893	292	dbSNP
rs894649867	294	dbSNP
rs1294764967	295	dbSNP
rs1460783778	299	dbSNP
rs1351268582	305	dbSNP
rs916000893	305	dbSNP
rs984808723	307	dbSNP
rs1203359789	311	dbSNP
rs1260899396	317	dbSNP
rs1442802159	318	dbSNP
rs1423483988	322	dbSNP
rs1055934831	324	dbSNP
rs1475034925	328	dbSNP
rs34679352	331	dbSNP
rs1160148826	332	dbSNP
rs1414328867	334	dbSNP
rs951892293	341	dbSNP
rs1158523675	344	dbSNP
rs1403466477	345	dbSNP
rs1001957463	351	dbSNP
rs1398164737	357	dbSNP
rs1026083156	358	dbSNP
rs1382798823	358	dbSNP
rs1413842442	359	dbSNP
rs1288555687	360	dbSNP
rs1337040260	363	dbSNP
rs898246120	366	dbSNP
rs201858397	367	dbSNP
rs939656891	374	dbSNP
rs993328480	381	dbSNP
rs759536472	382	dbSNP
rs563107503	384	dbSNP
rs767148471	386	dbSNP
rs908480755	394	dbSNP
rs1183195076	402	dbSNP
rs1252873902	411	dbSNP
rs1417421101	414	dbSNP
rs148900317	415	dbSNP
rs1378275560	421	dbSNP
rs1468083727	421	dbSNP
rs574601618	426	dbSNP
rs933893040	428	dbSNP
rs921102565	429	dbSNP
rs1172291344	430	dbSNP
rs562024540	431	dbSNP
rs147839497	438	dbSNP
rs1430756832	439	dbSNP
rs1175320516	440	dbSNP
rs1043504067	441	dbSNP
rs957488034	448	dbSNP
rs1389316323	451	dbSNP
rs1010636277	453	dbSNP
rs892200613	453	dbSNP
rs1053308355	460	dbSNP
rs867699969	466	dbSNP
rs1301104969	467	dbSNP
rs934835994	468	dbSNP
rs1205540164	471	dbSNP
rs534725867	473	dbSNP
rs1223455917	475	dbSNP
rs1195433331	476	dbSNP
rs140231017	477	dbSNP
rs1455620407	477	dbSNP
rs72829888	477	dbSNP
rs756712751	477	dbSNP
rs774199719	477	dbSNP
rs1491396371	479	dbSNP
rs201227589	479	dbSNP
rs1289748102	480	dbSNP
rs1297215788	481	dbSNP
rs1442400192	481	dbSNP
rs948734526	481	dbSNP
rs1318121765	482	dbSNP
rs1323826477	485	dbSNP
rs1370078932	485	dbSNP
rs1229051276	487	dbSNP
rs68041649	489	dbSNP
rs71356525	489	dbSNP
rs915886428	490	dbSNP
rs1390033852	491	dbSNP
rs967332742	492	dbSNP
rs984861401	497	dbSNP
rs919330381	503	dbSNP
rs952156191	503	dbSNP
rs552296206	506	dbSNP
rs1188778319	507	dbSNP
rs763080788	509	dbSNP
rs1169330669	510	dbSNP
rs1428246651	510	dbSNP
rs553413766	524	dbSNP
rs1462568443	526	dbSNP
rs1159999092	531	dbSNP
rs1162258412	534	dbSNP
rs1435790175	543	dbSNP
rs1290526369	545	dbSNP
rs540104142	551	dbSNP
rs577635730	553	dbSNP
rs1381392987	554	dbSNP
rs530923243	559	dbSNP
rs1156764687	561	dbSNP
rs556244855	562	dbSNP
rs1429676508	571	dbSNP
rs1231671037	575	dbSNP
rs568679830	579	dbSNP
rs760434374	584	dbSNP
rs1003942953	586	dbSNP
rs547979996	587	dbSNP
rs1240810142	594	dbSNP
rs1485752137	605	dbSNP
rs1050748528	607	dbSNP
rs185485263	612	dbSNP
rs921186240	625	dbSNP
rs773042869	641	dbSNP
rs1416580600	643	dbSNP
rs1260685238	648	dbSNP
rs1039545997	649	dbSNP
rs1343305782	651	dbSNP
rs565294499	667	dbSNP
rs1303271836	668	dbSNP
rs747802252	671	dbSNP
rs977493226	686	dbSNP
rs1292856348	687	dbSNP
rs1413209790	687	dbSNP
rs1351204474	696	dbSNP
rs1225476026	698	dbSNP
rs967456239	698	dbSNP
rs551768287	700	dbSNP
rs1282977356	702	dbSNP
rs1185388802	709	dbSNP
rs1256308716	713	dbSNP
rs1284368773	724	dbSNP
rs1052186905	728	dbSNP
rs999430297	739	dbSNP
rs1418479446	743	dbSNP
rs778479035	745	dbSNP
rs1432577155	746	dbSNP
rs992840286	747	dbSNP
rs370594191	749	dbSNP
rs532245081	751	dbSNP
rs1355261591	752	dbSNP
rs1444638192	752	dbSNP
rs768175577	753	dbSNP
rs1277919871	759	dbSNP
rs1372676058	760	dbSNP
rs1220357580	768	dbSNP
rs1046415163	770	dbSNP
rs9380	772	dbSNP
rs948952032	774	dbSNP
rs563588497	777	dbSNP
rs894714509	778	dbSNP
rs959248547	785	dbSNP
rs548389724	788	dbSNP
rs1467570442	792	dbSNP
rs930639670	796	dbSNP
rs1396747109	801	dbSNP
rs563219042	807	dbSNP
rs114859649	811	dbSNP
rs1419952702	812	dbSNP
rs1411918461	820	dbSNP
rs963559446	825	dbSNP
rs1327067809	837	dbSNP
rs559630857	837	dbSNP
rs1427479649	846	dbSNP
rs1015072789	847	dbSNP
rs780476048	854	dbSNP
rs1429220987	855	dbSNP

- Science (New York, N.Y.), 2004

MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs, some of which are known to play important regulatory roles in animals by targeting the messages of protein-coding genes for translational repression. We find that miR-196, a miRNA encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. RNA fragments diagnostic of miR-196-directed cleavage of HOXB8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of HOXB8, HOXC8, HOXD8, and HOXA7 and supported the cleavage mechanism for miR-196-directed repression of HOXB8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of HOX gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting productive translation.

- Nucleic acids symposium series (2004), 2004

MicroRNAs (miRNAs) are endogenous small RNAs (approximately 21-25 nts) that can regulate gene expression at a post-transcriptional level. Recently a large number of miRNAs have been identified from plants and animals. In addition, it has been reported that these small RNAs can participate in cell differentiation of developmental timing. In this study, we show that microRNA-196 (miR-196) inhibits HOXB8 expression in myeloid differentiation of HL 60 cells. MiR-196 has perfect complementarity with target sequence of 3'-UTR in HOXB8 mRNA. Exogenous expressed miR-196 repressed expression of HOXB8 via cleaving mRNAs. Moreover miR-196 enhanced myeloid differentiation of HL60 cells. These results suggest that miR-196 participates in myeloid differentiation of HL60 cells via regulation of HOXB8 expression.

- PloS one, 2008

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense-strand siRNA elements, which possibly increase the assembly of antisense-strand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi.

- World journal of gastroenterology, 2009

AIM: To analyze the relevance of the microRNA miR-196a for colorectal oncogenesis. METHODS: The impact of miR-196a on the restriction targets HoxA7, HoxB8, HoxC8 and HoxD8 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) after transient transfection of SW480 cancer cells. The miR-196a transcription profile in colorectal cancer samples, mucosa samples and diverse cancer cell lines was quantified by RT-PCR. Transiently miR-196a-transfected colorectal cancer cells were used for diverse functional assays in vitro and for a xenograft lung metastasis model in vivo. RESULTS: HoxA7, HoxB8, HoxC8 and HoxD8 were restricted by miR-196a in a dose-dependent and gene-specific manner. High levels of miR-196a activated the AKT signaling pathway as indicated by increased phosphorylation of AKT. In addition, high levels of miR-196a promoted cancer cell detachment, migration, invasion and chemosensitivity towards platin derivatives but did not impact on proliferation or apoptosis. Furthermore, miR-196a increased the development of lung metastases in mice after tail vein injection. CONCLUSION: miR-196a exerts a pro-oncogenic influence in colorectal cancer.

- Cell, 2013

MicroRNAs (miRNAs) play key roles in gene regulation, but reliable bioinformatic or experimental identification of their targets remains difficult. To provide an unbiased view of human miRNA targets, we developed a technique for ligation and sequencing of miRNA-target RNA duplexes associated with human AGO1. Here, we report data sets of more than 18,000 high-confidence miRNA-mRNA interactions. The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, nonseed base pairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA base pairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding.

- Molecular bioSystems, 2014

Fragile X syndrome (FXS) is caused by the loss of expression of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that negatively regulates mRNA substrates. FMRP can regulate the translation via the cross-talk with the miRNA machinery, but the functional association among FMRP, miRNAs and mutual target mRNAs has rarely been studied. In this research, we find that HOXB8 mRNA is a target of FMRP associated with miR-196a-induced silencing, and discover that phosphorylation of FMRP promotes the miR-196a-mediated repression of HOXB8 without affecting the interaction between FMRP and mRNA. We further identify that the FMRP-binding site involved in the miR-196a-mediated repression of HOXB8 locates in the downstream neighbourhood of the miR-196a recognition element in the 3'UTR of HOXB8. Importantly, we reveal that FMRP faces toward the MID domain of AGO2 and interacts with a specific binding pocket (coordination with T544, K533 and K570) in the domain. Our research might provide new insights into both the cross-talk between FMRP and miRNA-mediated regulation of mRNA translation and the molecular pathogenesis of FXS.