7p14.2 (-)80 U CU U -GCCA UC A G GCUA UC CCUGA UUCUGAGCC AAUC CUU C |||| || ||||| ||||||||| |||| |||...
1
7p14.2 (-) 80 U CU U -GCCA UC A G GCUA UC CCUGA UUCUGAGCC AAUC CUU C |||| || ||||| ||||||||| |||| ||| CGAU AG GGACU AGGACUUGG UUAG GAG C C -- - GUUUA -- A A CUCCUGAGCCAUU CUGAGCCUC (22) M-17 12q14.1 (+) 100 C U U U UGU UGGC GAGGUAGUAGUUUGUGC GUU--------GG CGGGU G |||| ||||||||||||||||| ||| || ||||| AUCG-UUCCGUCAUCGAACGCG CAA UC-GCCCG A U UAGAGGUG UUA CUGCGCAAGCUAC UGCCUUGC (21) M-64 14q11.2 (-) 53 AC U UGAAAU UAAGUCC -GUU G UUU AGUUUGC-CA GA GCAUGU GU UCAG-CU A ||| ||||||| || || |||||| || |||| || AAA--UCAGACG GU-CU------CGUACA-------CA AGUC GA U A AAUU C C CCUGAUUAAACAC AUGCUCUGA (22) M-84 17q21.32 (-) 82 U C-C A G GCAG CA AGC U CCU CCGG GCCA GAU CU AGCCAC AGGGUG U ||| |||| |||| ||| || |||||| |||||| U GGA GGUC CGGU UUA----GA UCGGUG UCUCGC A CGU G G CC ACU G CCCGGAGCCAGGA UGCAGCUC (21) NM-84 6q25.3 (+) 90 CUGCAGA UG A UG GA CU C CCUG GG CC GCU-GC--AG GGG GGCA GC A |||| || || ||| || || ||| |||| || GGAC CC GG CGACCG UC CUC CCGU--CG G AAGAA-- GU A AU CU AC G GUGCCAGCUGCAG UGGGGGAG (21) M-94 Localizaci ón (DNA strand) Hairp in score a Predicted hairpin Cloned sequence (nt) miRNA a The hairpin score was calculated according to structural features of predicted hairpins using the on-line structure-based miRNA tool (http://tagc.univ-mrs.fr/mirna/) 40. Scores under 50 are considered inadequate substrates for RNAse III-dependent processing. Table 3. Novel miRNA candidates cloned Table 3. Novel miRNA candidates cloned from CLL cells. from CLL cells. Expression levels were assessed by northern blot assays from total RNA isolated from leukemic cells and normal peripheral B lymphocytes. Samples from CLL patients were segregated into “unmutated” (4 representative patients) and “mutated” (3 representative patients) according to the mutational status of IgVH genes. 5S rRNA detected by ethidium bromide staining of gels was used as loading control. Since normal B CD19+ lymphocytes were negative for miR-223, a control RNA from peripheral mononuclear cells were used as normal control (PBMC) to assess expression of this miRNA in CLL patients. Figure 1. Expression levels of selected Figure 1. Expression levels of selected mature miRNAs in CLL cells relative to mature miRNAs in CLL cells relative to normal CD19+ B lymphocytes. normal CD19+ B lymphocytes. CLONING-BASED ANALYSIS OF SMALL RNAs IN B- LYMPHOCYTES FROM PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA Marton, S.#; Garcia, R.#; Robello, C.◊; Persson, H.§; Trajtenberg, F.; Pritsch, O.‡; Rovira, C.§; Naya, H.●; Dighiero, G.; Cayota, A#. # Molecular Oncology Unit, Institut Pasteur, Montevideo, Uruguay; ◊ Molecular Biology Unit, Institut Pasteur, Montevideo, Uruguay; § Department of Oncology, Lund University. Lund, Sweden; Structural Biology Unit, Institut Pasteur Montevideo, Uruguay; ‡Protein Biophysics Unit, Institut Pasteur Montevideo, Uruguay; ● Bioinformatics Unit, Institut Pasteur, Montevideo, Uruguay; Immunohematology Unit Pasteur Institute. Paris, France INTRODUCTION INTRODUCTION B-chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world and is a significant cause of morbidity and mortality in the older adult population. Although the underlying cause of B-CLL remains poorly understood, CLL patients can be segregated in two main evolutive groups according to the mutational status of the immunoglobulin heavy-chain variable-region gene. MicroRNAs (miRNAs) are short non-coding RNAs that serve as post-transcriptional regulators of gene expression by binding to complementary sites on target mRNAs. Many miRNA genes are located at fragile sites, minimal loss of heterozygosity regions, minimal regions of amplification, or common breakpoints in human cancers, suggesting that miRNA might play an important role in the pathogenesis of human cancer. In the present work we analyzed by cloning the small RNA populations in pooled samples of total RNA from B-CLL cells from patients with and indolent course (mutated status; Mut) and compared them with those from patients with an aggressive evolution (unmutated status; NM). Our results showed a global reduction in miRNA expression levels in CLL cells associated to a consistent underexpression of miR-181a, let-7a and miR-30d. We observed overexpression of miR-155 and a set of five miRNAs that are differentially expressed between patients with different clinical outcomes. Five novel miRNA candidates cloned from leukemic cells are reported. Surprisingly, predicted mRNA targets for these novel miRNA revealed a high proportion of targets located in a small region of chromosome 1 which is frequently altered in human cancer. Additionally, several targets were shared by at least two of miRNA candidates. Predicted targets included several genes recently described as tumor suppressors. These data could afford new avenues for exploring innovative pathways in CLL biology and therapy. CONCLUSIONS CONCLUSIONS Small RNAs cloned from B-CLL cells show a low proportion of miRNAs when compared to those obtained in other human tissues Small RNAs cloned from B-CLL cells show a low proportion of miRNAs when compared to those obtained in other human tissues Different miRNA profiles distinguish between normal B lymphocytes and B-CLL cells with either unmutated (indolent evolution) or mutated Different miRNA profiles distinguish between normal B lymphocytes and B-CLL cells with either unmutated (indolent evolution) or mutated (aggressive evolution) Ig VH genes (aggressive evolution) Ig VH genes Low expression levels of let-7a and miR-181a suggest an undifferentiated phenotype of B-CLL cells Low expression levels of let-7a and miR-181a suggest an undifferentiated phenotype of B-CLL cells The altered pattern of miRNA expression and the finding of new putatives miRNAs associated to B-CLL cells could provide news insights The altered pattern of miRNA expression and the finding of new putatives miRNAs associated to B-CLL cells could provide news insights into the pathogenesis of chronic lymphocytic leukemia into the pathogenesis of chronic lymphocytic leukemia The experimental confirmation of involvement of these putative targets including several tumor suppressors could provide new avenues The experimental confirmation of involvement of these putative targets including several tumor suppressors could provide new avenues for exploring innovative pathways in CLL biology and therapy for exploring innovative pathways in CLL biology and therapy Table 1. Small RNAs populations cloned from Table 1. Small RNAs populations cloned from CLL groups CLL groups 41 (14% ) 157 (55%) 28 (10% ) 25 (9%) 4 (1%) 32 (11 %) 287 (100%) M 24 (10% ) 132 (55.5%) 28 (12% ) 28 (12%) 1 (0.5%) 24 (10%) 237 (100%) NM NDE d nc RNAs c mRNA Sens/ antisens non coding RNA transcripts b Novel miRNAs candidates a miRNAs Total Abbreviations: M, mutated; NM, unmutated; ncRNA, non coding RNAs; NDE, non database entry. a sequences for which flanking form predicted hairpin precursors. b sequences matching the human genome but did not forming miRNA-related hairpin precursors. c includes ribosomal, transfer, small nuclear, small nucleolar and mitochondrial RNAs. d sequences that did not match with any known human sequence from public databases. Table 2. Cloned miRNAs from CLL Table 2. Cloned miRNAs from CLL cells cells a a . . miRNAs - 2 4 4 8 - 3 3 2 2 2 1 1 M 3 5 4 - 2 3 - - - - 4 3 - NM 191 155 150 142 5p 142 3p 30d 29a 26a 24 22 16 15a let7i 3p Abbreviations: M, mutated; NM, unmutated a For each miRNA the number of confirmed sequences are showed. Table 4. Selected relevant mRNA targets for novel Table 4. Selected relevant mRNA targets for novel miRNA candidatesa miRNA candidatesa. M-94 M-94 M-94 M-94 M-94 M-94 NM-84 NM-84 NM-84 NM-84 miRNA candida te PR domain-containing protein 16 chromodomain helicase DNA-binding protein 5 hairy/enhancer of split, drosophila, homolog of, 3 runt-related transcription factor 3 SRY-box 13 wingless-type mmtv integration site family, member 3A PR domain-containing protein 16 chromodomain helicase DNA-binding protein 5 hairy/enhancer of split, drosophila, homolog of, 3 ephrin receptor EphB2 Target Gene Name a PRDM16 CHD5 HES3 RUNX3 SOX13 WNT3A PRDM16 CHD5 HES3 EPHB2 Target Gene Symbol 1p36.32 1p36.31 1p36.31 1p36.11 1q32.1 1q42.13 1p36.32 1p36.31 1p36.31 1p36.12 Locati on 1644 1500 1467 1319 1272 1239 1277 1015 1003 942 Scor e M-17 M-17 M-64 M-64 M-64 M-64 M-64 M-84 M-84 M-84 miRNA candida te hairy/enhancer of split, drosophila, homolog 3 ephrin receptor (receptor tyrosine kinases; rtks) PR domain-containing protein 16: PR domain hairy/enhancer of split, drosophila, homolog 2 wingless-type mmtv integration site family, member 3A Sry-Box 11 plexin A1 chromodomain helicase DNA-binding protein 5 glutathione s-transferase, Mu-1 regulator of G protein signaling 2 Target Gene Name a HES3 EPHB2 PRDM16 HES2 WNT3A SOX11 PLXNA1 CHD5 GSTM1 RGS2 Target Gene Symbol 1p36.31 1p36.12 1p36.32 1p36.31 1q42.13 3q21.2 1p36.31 1p13.3 1q31.2 Locati on 1359 1049 859 674 584 498 176 148 Scor e 2p25.2 539 131 dicted target mRNAs were identified through the miRanda algorithm. of these genes have been predicted to function either directly or indirectly as tumor suppressors.
-
Upload
gordon-clarke -
Category
Documents
-
view
213 -
download
0
Transcript of 7p14.2 (-)80 U CU U -GCCA UC A G GCUA UC CCUGA UUCUGAGCC AAUC CUU C |||| || ||||| ||||||||| |||| |||...
7p14.2 (-) 80U CU U -GCCA UC A G GCUA UC CCUGA UUCUGAGCC AAUC CUU C |||| || ||||| ||||||||| |||| ||| CGAU AG GGACU AGGACUUGG UUAG GAG C C -- - GUUUA -- A A
CUCCUGAGCCAUUCUGAGCC
UC(22)
M-17
12q14.1 (+) 100C U U U UGU UGGC GAGGUAGUAGUUUGUGC GUU--------GG CGGGU G |||| ||||||||||||||||| ||| || ||||| AUCG-UUCCGUCAUCGAACGCG CAA UC-GCCCG AU UAGAGGUG UUA
CUGCGCAAGCUACUGCCUUG
C(21)
M-64
14q11.2 (-) 53 AC U UGAAAU UAAGUCC -GUU GUUU AGUUUGC-CA GA GCAUGU GU UCAG-CU A||| ||||||| || || |||||| || |||| || AAA--UCAGACG GU-CU------CGUACA-------CA AGUC GA U A AAUU C C
CCUGAUUAAACACAUGCUCU
GA(22)
M-84
17q21.32 (-) 82U C-C A G GCAG CA AGC U CCU CCGG GCCA GAU CU AGCCAC AGGGUG U ||| |||| |||| ||| || |||||| |||||| U GGA GGUC CGGU UUA----GA UCGGUG UCUCGC A CGU G G CC ACU G
CCCGGAGCCAGGAUGCAGCU
C(21)
NM-84
6q25.3 (+) 90 CUGCAGA UG A UG GA CU CCCUG GG CC GCU-GC--AG GGG GGCA GC A|||| || || ||| || || ||| |||| ||GGAC CC GG CGACCG UC CUC CCGU--CG G AAGAA-- GU A AU CU AC G
GUGCCAGCUGCAGUGGGGGA
G(21)
M-94
Localización (DNA strand)
Hairpin
scorea
Predicted hairpin
Cloned sequence
(nt)
miRNA
a The hairpin score was calculated according to structural features of predicted hairpins using the on-line structure-based miRNA tool (http://tagc.univ-mrs.fr/mirna/) 40. Scores under 50 are considered inadequate substrates for RNAse III-dependent processing .
Table 3. Novel miRNA candidates cloned from CLL Table 3. Novel miRNA candidates cloned from CLL cells.cells.
Expression levels were assessed by northern blot assays from total RNA isolated from leukemic cells and normal peripheral B lymphocytes. Samples from CLL patients were segregated into “unmutated” (4 representative patients) and “mutated” (3 representative patients) according to the mutational status of IgVH genes. 5S rRNA detected by ethidium bromide staining of gels was used as loading control. Since normal B CD19+ lymphocytes were negative for miR-223, a control RNA from peripheral mononuclear cells were used as normal control (PBMC) to assess expression of this miRNA in CLL patients.
Figure 1. Expression levels of selected mature Figure 1. Expression levels of selected mature miRNAs in CLL cells relative to normal CD19+ B miRNAs in CLL cells relative to normal CD19+ B lymphocytes.lymphocytes.
CLONING-BASED ANALYSIS OF SMALL RNAs IN B-LYMPHOCYTES FROM PATIENTS WITH CHRONIC
LYMPHOCYTIC LEUKEMIA Marton, S.#; Garcia, R.#; Robello, C.◊; Persson, H.§; Trajtenberg, F.; Pritsch, O.‡; Rovira, C.§; Naya, H.●; Dighiero, G.; Cayota, A#.
# Molecular Oncology Unit, Institut Pasteur, Montevideo, Uruguay; ◊ Molecular Biology Unit, Institut Pasteur, Montevideo, Uruguay;§ Department of Oncology, Lund University. Lund, Sweden; Structural Biology Unit, Institut Pasteur Montevideo, Uruguay; ‡Protein Biophysics Unit, Institut Pasteur Montevideo, Uruguay; ● Bioinformatics Unit, Institut Pasteur, Montevideo, Uruguay;
Immunohematology Unit Pasteur Institute. Paris, France
INTRODUCTIONINTRODUCTIONB-chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world and is a significant cause of morbidity and mortality in the older adult
population. Although the underlying cause of B-CLL remains poorly understood, CLL patients can be segregated in two main evolutive groups according to the mutational status of the immunoglobulin heavy-chain variable-region gene.
MicroRNAs (miRNAs) are short non-coding RNAs that serve as post-transcriptional regulators of gene expression by binding to complementary sites on target mRNAs. Many miRNA genes are located at fragile sites, minimal loss of heterozygosity regions, minimal regions of amplification, or common breakpoints in human cancers, suggesting that miRNA might play an important role in the pathogenesis of human cancer.
In the present work we analyzed by cloning the small RNA populations in pooled samples of total RNA from B-CLL cells from patients with and indolent course (mutated status; Mut) and compared them with those from patients with an aggressive evolution (unmutated status; NM).
Our results showed a global reduction in miRNA expression levels in CLL cells associated to a consistent underexpression of miR-181a, let-7a and miR-30d. We observed overexpression of miR-155 and a set of five miRNAs that are differentially expressed between patients with different clinical outcomes.
Five novel miRNA candidates cloned from leukemic cells are reported. Surprisingly, predicted mRNA targets for these novel miRNA revealed a high proportion of targets located in a small region of chromosome 1 which is frequently altered in human cancer. Additionally, several targets were shared by at least two of miRNA candidates. Predicted targets included several genes recently described as tumor suppressors. These data could afford new avenues for exploring innovative pathways in CLL biology and therapy.
CONCLUSIONSCONCLUSIONS Small RNAs cloned from B-CLL cells show a low proportion of miRNAs when compared to those obtained in other human tissues Small RNAs cloned from B-CLL cells show a low proportion of miRNAs when compared to those obtained in other human tissues Different miRNA profiles distinguish between normal B lymphocytes and B-CLL cells with either unmutated (indolent evolution) or mutated (aggressive evolution) Different miRNA profiles distinguish between normal B lymphocytes and B-CLL cells with either unmutated (indolent evolution) or mutated (aggressive evolution) Ig VH genesIg VH genes Low expression levels of let-7a and miR-181a suggest an undifferentiated phenotype of B-CLL cellsLow expression levels of let-7a and miR-181a suggest an undifferentiated phenotype of B-CLL cells The altered pattern of miRNA expression and the finding of new putatives miRNAs associated to B-CLL cells could provide news insights into the pathogenesis of The altered pattern of miRNA expression and the finding of new putatives miRNAs associated to B-CLL cells could provide news insights into the pathogenesis of chronic lymphocytic leukemiachronic lymphocytic leukemia The experimental confirmation of involvement of these putative targets including several tumor suppressors could provide new avenues for exploring innovative The experimental confirmation of involvement of these putative targets including several tumor suppressors could provide new avenues for exploring innovative pathways in CLL biology and therapypathways in CLL biology and therapy
Table 1. Small RNAs populations cloned from CLL Table 1. Small RNAs populations cloned from CLL groupsgroups
41
(14%)
157(55%)
28(10%)
25(9%)
4(1%)
32(11 %)
287
(100%)
M
24
(10%)
132(55.5%)
28 (12%)
28 (12%)
1(0.5%)
24 (10%)
237 (100%)
NM
NDEdnc RNAscmRNA
Sens/antisens non coding RNA transcriptsb
Novel miRNAs
candidatesa
miRNAsTotal
Abbreviations: M, mutated; NM, unmutated; ncRNA, non coding RNAs; NDE, non database entry.a sequences for which flanking form predicted hairpin precursors. b sequences matching the human genome but did not forming miRNA-related hairpin precursors.cincludes ribosomal, transfer, small nuclear, small nucleolar and mitochondrial RNAs.dsequences that did not match with any known human sequence from public databases.
Table 2. Cloned miRNAs from CLL Table 2. Cloned miRNAs from CLL cellscellsaa. .
miRNAs
-2448-3322211M
354-23----43-NM
191155150142 5p
142 3p
30d29a
26a24221615a
let7i 3p
Abbreviations: M, mutated; NM, unmutateda For each miRNA the number of confirmed sequences are showed.
Table 4. Selected relevant mRNA targets for novel miRNA Table 4. Selected relevant mRNA targets for novel miRNA candidatesacandidatesa.
M-94
M-94
M-94
M-94
M-94
M-94
NM-84
NM-84
NM-84
NM-84
miRNA candida
te PR domain-containing protein 16
chromodomain helicase DNA-binding protein 5hairy/enhancer of split, drosophila, homolog of, 3runt-related transcription factor 3
SRY-box 13
wingless-type mmtv integration site family, member 3APR domain-containing protein 16
chromodomain helicase DNA-binding protein 5hairy/enhancer of split, drosophila, homolog of, 3ephrin receptor EphB2
Target Gene Namea
PRDM16
CHD5
HES3
RUNX3
SOX13
WNT3A
PRDM16
CHD5
HES3
EPHB2
Target Gene
Symbol 1p36.32
1p36.31
1p36.31
1p36.11
1q32.1
1q42.13
1p36.32
1p36.31
1p36.31
1p36.12
Location
1644
1500
1467
1319
1272
1239
1277
1015
1003
942
Score
M-17
M-17
M-64
M-64
M-64
M-64
M-64
M-84
M-84
M-84
miRNA candida
te hairy/enhancer of split, drosophila, homolog 3
ephrin receptor (receptor tyrosine kinases; rtks)PR domain-containing protein 16: PR domain
hairy/enhancer of split, drosophila, homolog 2
wingless-type mmtv integration site family, member 3ASry-Box 11
plexin A1
chromodomain helicase DNA-binding protein 5glutathione s-transferase, Mu-1
regulator of G protein signaling 2
Target Gene Namea
HES3
EPHB2
PRDM16
HES2
WNT3A
SOX11
PLXNA1
CHD5
GSTM1
RGS2
Target Gene
Symbol 1p36.31
1p36.12
1p36.32
1p36.31
1q42.13
3q21.2
1p36.31
1p13.3
1q31.2
Location
1359
1049
859
674
584
498
176
148
Score
2p25.2 539
131
aPredicted target mRNAs were identified through the miRanda algorithm. Most of these genes have been predicted to function either directly or indirectly as tumor suppressors.
