Genome-wide microRNA and messenger RNA profiling in rodent liver development implicates mir302b and mir20a in repressing transforming growth factor-beta signaling
Open accessAbstract
MicroRNAs (miRNAs) are recently discovered small RNA molecules that regulate developmental processes, such as proliferation, differentiation, and apoptosis; however, the identity of miRNAs and their functions during liver development are largely unknown. Here we investigated the miRNA and gene expression profiles for embryonic day (E)8.5 endoderm, E14.5 Dlk1+ liver cells (hepatoblasts), and adult liver by employing Illumina sequencing. We found that miRNAs were abundantly expressed at all three stages. Using K-means clustering analysis, 13 miRNA clusters with distinct temporal expression patterns were identified. mir302b, an endoderm-enriched miRNA, was identified as an miRNA whose predicted targets are expressed highly in E14.5 hepatoblasts but low in the endoderm. We validated the expression of mir302b in the endoderm by whole-mount in situ hybridization. Interestingly, mir20a, the most highly expressed miRNA in the endoderm library, was also predicted to regulate some of the same targets as mir302b. We found that through targeting Tgfbr2 , mir302b and mir20a are able to regulate transforming growth factor beta (TGFβ) signal transduction. Moreover, mir302b can repress liver markers in an embryonic stem cell differentiation model. Collectively, we uncovered dynamic patterns of individual miRNAs during liver development, as well as miRNA networks that could be essential for the specification and differentiation of liver progenitors.
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Written from the paper’s full text.
TL;DR
This study generated small RNA and mRNA profiles from mouse E8.5 foregut, E14.5 Dlk1+ hepatoblasts, and adult liver, identifying 13 temporally clustered miRNA groups. It implicates endoderm-enriched mir302b and mir20a in repressing TGF-beta signaling by targeting Tgfbr2, with mir302b also targeting Kat2b and repressing liver markers in ESC differentiation.
Key findings
- Small RNA sequencing identified 592 miRNA/miRNA*; more than 60% were present in all three libraries, and 59%, 64%, and 91% of reads aligned to 430, 384, and 315 known miRNA genes in foregut, hepatoblast, and adult liver, respectively. K-means clustering yielded 13 temporal groups, including foregut-enriched Cluster A (mir302b, mir17-92), hepatoblast-enriched Cluster H (mir379), and adult-enriched Cluster L (let7 family).
- Integrating RNA-Seq, 5,227 transcripts were endoderm-enriched and 1,599 were hepatoblast-enriched. mir302b was predicted to target 575 of 1,599 (35%) hepatoblast-enriched genes versus 22% for a random set; 16 of 575 were predicted by all six algorithms, and six of these are implicated in TGF-beta signaling, including Tgfbr2, NF1A/B, Bcl6, Kat2b, and Camk2n1. mir20a, the most highly expressed miRNA in the foregut library, was also predicted to target Tgfbr2, Kat2b, and Camk2n1.
- Luciferase and western assays showed both mir302b and mir20a regulate Tgfbr2; only mir302b targeted Kat2b, and neither significantly affected the Camk2n1 reporter. Both suppressed TGF-beta-responsive 3TP-lux activity in a Tgfbr2-dependent manner.
- In ESC differentiation, forced mir302b decreased Tgfbr2 and liver markers Alb, Afp, Hnf4a, Ttr, Hpx, and Serpina1a; the TGF-beta inhibitor SB505124 similarly reduced liver markers but not Tgfbr2. WISH showed mir302b in epiblast/mesoderm at E6.5–E7.5, definitive endoderm at the 3-somite stage (~E8.25), foregut and hindgut but not midgut, and later ubiquitous expression absent from heart.
Methods
- C57BL/6J mice were used: E8.5 foregut endoderm, E14.5 Dlk1+ hepatoblasts isolated by FACS to >90% purity, and adult liver from a 6-month-old female. Small RNA libraries were sequenced on Illumina GAIIx; two foregut, two hepatoblast, and one adult liver replicates were generated, and one replicate was used for further analysis. miRNAs expressed at >10 RPM were used for K-means clustering; qRT-PCR and whole-mount in situ hybridization validated expression.
- RNA-Seq libraries were generated from E8.5 endoderm and E14.5 Dlk1+ liver cells. Alexa-Seq normalized average coverage values were used; transcripts were considered differentially expressed if NAC values differed by a factor of two with corrected P < 0.05 by Fisher’s exact test. miRNA target prediction used DIANA mirExTra and mirWalk, followed by luciferase 3′UTR reporter assays, western blotting, 3TP-lux TGF-beta reporter assays, and mouse ESC differentiation.
Limitations
- The paper states that Dlk1 expression may mark only a subset of hepatoblasts at E14.5; not all Dlk1+ cells express HNF4a, so different subpopulations may exist, and earlier Dlk1+ cells can be subdivided by EpCAM.
- The paper notes that gender differences likely exist, although there was substantial overlap between the adult female liver library and an adult male liver library; the adult liver sample was from a single 6-month-old female and only one adult liver library was generated.
- Evident from the text: foregut and hepatoblast analyses used one of two generated replicates; miRNA target predictions are computational and require validation; and the ESC differentiation model is not an in vivo liver development system, with no direct in vivo loss- or gain-of-function for mir302b/mir20a reported.
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Claims the paper makes
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- Findingown data
mir20a was predicted to target Tgfbr2, Kat2b, and Camk2n1 using six algorithms.
“we noted that mir20a, the most highly expressed miRNA in the foregut library, was also predicted to target Tgfbr2, Kat2b, and Camk2n1, using the above six algorithms.”
Quote in the paperSubstantial claim - Findingown data
miRNAs were abundantly expressed at all three stages studied: E8.5 endoderm, E14.5 hepatoblasts, and adult liver.
“We found that miRNAs were abundantly expressed at all three stages.”
- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
“Most of the annotated miR-NAs had an expression level ranging from 10 RPM to 1,000 RPM; only a few were expressed at more than 10 4 RPM (Fig. 1B, Table S2).”
Quote in the paperNot a substantial claim - Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
- Findingown data
mir302b expression in the endoderm was validated by whole-mount in situ hybridization.
“We validated the expression of mir302b in the endoderm by whole-mount in situ hybridization.”
Quote in the paperNot a substantial claim - Findingown data
Sixteen of 575 Hepatoblast-enriched genes were predicted to be mir302b targets by all six algorithms.
“Sixteen of 575 Hepatoblast-enriched genes were predicted to be mir302b tar-gets by all six algorithms (Table S5).”
Quote in the paperSubstantial claim - Findingown data
Only mir302b can target Kat2b.
“Together, these data demonstrate that both mir302b and mir20a are able to regulate Tgfbr2 expression, while only mir302b can target Kat2b.”
Quote in the paperSubstantial claim
- Findingown data
mir302b and mir20a are able to regulate Tgfbr2 expression.
“We found that through targeting Tgfbr2, mir302b and mir20a are able to regulate transforming growth factor beta (TGFb) signal transduction.”
- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · boosted
“Together, these data demonstrate that both mir302b and mir20a are able to regulate Tgfbr2 expression, while only mir302b can target Kat2b.”
Quote in the paperSubstantial claim - Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · boosted
- Findingown data
mir302b and mir20a suppress TGF-beta signal transduction by targeting Tgfbr2.
“demonstrating that both mir302b and mir20a are able to suppress TGFb signal transduction by targeting Tgfbr2.”
Quote in the paperSubstantial claim - Findingown data
In hepatoblasts, mir379, mir434-3p, and mir127 were highly enriched compared to adult liver and foregut.
“In hepatoblasts, mir379, mir434-3p, and mir127 were highly enriched compared to adult liver and foregut (Fig. S1C, Table S6).”
Quote in the paperNot a substantial claim - Findingnone given
mir302b can repress liver markers in an embryonic stem cell differentiation model.
“Moreover, mir302b can repress liver markers in an embryonic stem cell differentiation model.”
Quote in the paperSubstantial claim - Findingown data
K-means clustering identified 13 miRNA clusters with distinct temporal expression patterns.
“Using K-means clustering analysis, 13 miRNA clusters with distinct temporal expression patterns were identified.”
- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
“Thirteen temporally related groups (designated Clusters A-M) of miRNAs were identified, including three clusters in which the miRNAs were highly and specifically enriched in one library (Fig. S1D).”
Quote in the paperNot a substantial claim - Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
- Findingown data
mir29a and mir29b are the most specifically enriched miRNAs in adult liver, being virtually absent in foregut and hepatoblasts.
“Of note, the most specifically enriched miRNAs in the adult liver are mir29a and mir29b, being virtually absent in foregut and hepatoblasts.”
Quote in the paperNot a substantial claim - Findingown data
Six of these sixteen genes have been implicated in TGF-beta signaling, including Tgfbr2, NF1A/B, Bcl6, Kat2b, and Camk2n1.
“Of note, six of these have been implicated in TGFb signaling, including Tgfbr2, Nuclear Factor 1A and 1B (NF1A/B), Bcl6, Kat2b (also known as P/CAF), and Camk2n1.”
Quote in the paperSubstantial claim - Findingown data
mir302b expression was low or absent in newly formed endoderm at E7.5.
“We also found that mir302b expression was low or absent in newly formed endoderm at E7.5 (Fig. 3D).”
Quote in the paperNot a substantial claim - Findingown data
Later mir302b expression was observed in the hindgut.
“Later expression was also observed in the hindgut (Fig. 4).”
Quote in the paperNot a substantial claim - Findingown data
Our data illustrate dynamic patterns of miRNA expression during liver development.
“Our data illustrate the dynamic patterns of miRNA expression that occur during liver development.”
Quote in the paperSubstantial claim - Findingown data
Our microRNAome profiling provides detailed information on miRNA expression during liver development in vivo.
“Together, our microRNAome profiling provides detailed information on miRNA expression during liver development in vivo.”
Quote in the paperSubstantial claim - Findingown data
By cluster analysis, we identified three groups of highly expressed miRNAs enriched in either foregut, hepatoblasts, or adult liver.
“By way of cluster analysis, we identified three groups of highly expressed miRNAs that are enriched in either foregut, hepatoblasts, or adult liver (Fig. 2A; Fig. S1D).”
Quote in the paperNot a substantial claim - Findingown data
Forced expression of mir302b during hepatoblast expansion resulted in decreased expression of Tgfbr2 and liver markers compared to control cells.
“Forced expression of mir302b through lentiviral vector during hepatoblast expansion resulted in decreased expression of Tgfbr2 and liver markers, compared to control cells (Figs. 7, 8).”
Quote in the paperSubstantial claim - Findingown data
A similar reduction of liver markers, but not Tgfbr2, was observed with the TGF-beta inhibitor SB505124.
“A similar reduction of liver markers, but not Tgfbr2, was observed with the TGFb inhibitor, SB505124 (Fig. S9D).”
Quote in the paperSubstantial claim - Findingown data
Sections of E8.75 embryos showed mir302b expression expanded to the entire gut.
“Sections of these embryos showed that mir302b expression expanded to the entire gut (Fig. 4A-D, black arrowhead).”
Quote in the paperNot a substantial claim - Interpretationown data
De-repressing TGF-beta signaling by down-regulation of mir302b provides a favorable environment for hepatoblast development.
“These results demonstrate that mir302b represses liver development during ESC differentiation and suggests that de-repressing TGFb signaling by down-regulation of mir302b provides a favorable environment for hepatoblast development.”
Quote in the paperSubstantial claim - Interpretationown data
mir302b represses liver development during ESC differentiation.
“These results demonstrate that mir302b represses liver development during ESC differentiation and suggests that de-repressing TGFb signaling by down-regulation of mir302b provides a favorable environment for hepatoblast development.”
Quote in the paperSubstantial claim - Interpretationreasoning
Down-regulation of mir302b and mir20a during early liver development may relieve suppression of TGF-beta signaling to promote hepatoblast proliferation.
“Therefore, down-regulation of mir302b and mir20a during early liver development may relieve the suppression of TGFb signaling to promote hepatoblast proliferation.”
Quote in the paperSubstantial claim - Interpretationown data
mir302b is highly expressed at the time of endoderm patterning.
“Together, our data show that mir302b is highly expressed at the time of endoderm patterning.”
Quote in the paperSubstantial claim - Interpretationreasoning
mir302b may function to compensate for loss of mir20a in the endoderm.
“In the endoderm, mir302b may function to compensate for loss of mir20a.”
Quote in the paperNot a substantial claim
- May combine two claims. 20
- Classified as probably not a substantial claim. 19
- The quote only partly states this claim. 2
May combine two claims.
Findingown datamir302b is an endoderm-enriched miRNA whose predicted targets are expressed highly in E14.5 hepatoblasts but low in the endoderm.
“mir302b, an endoderm-enriched miRNA, was identified as an miRNA whose predicted targets are expressed highly in E14.5 hepatoblasts but low in the endoderm.”
- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · hedged
“Analysis showed that mir302b potentially targets 575 out of the total 1,599 (35%) Hepatoblasts-enriched genes (compared to 22% for a random set of genes).”
Quote in the paperSubstantial claim- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · hedged
Probably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataMore than 60% of miRNAs were present in all three libraries (foregut, hepatoblasts, adult liver).
“Of note, more than 60% of miRNAs were present in all three libraries (Fig. 1C).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn foregut and hepatoblast libraries, 59% and 64% of reads aligned to 430 and 384 known miRNA genes, respectively, while 91% aligned to 315 miRNA genes in adult liver.
“In the foregut and hepatoblast libraries, 59% and 64% of reads aligned to 430 and 384 known miRNA genes, respectively, while 91% aligned to 315 miRNA genes in adult liver (Fig. 1A).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown datamir183-182 comprises 13% of total miRNA reads in foregut and is highly enriched compared to hepatoblasts (>35-fold) and liver (>250-fold).
“A second locus, mir183-182, comprises 13% of the total miRNA reads in the foregut, and is also highly enriched compared to hepatoblasts (>35-fold) and liver (>250-fold).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataA total of 5,227 transcripts were enriched in endoderm compared to hepatoblasts, while 1,599 genes were more highly expressed in fetal liver (hepatoblasts).
“A total of 5,227 transcripts were enriched in endoderm compared to hepatoblasts (Endoderm-enriched), while 1,599 genes were found to be more highly expressed in the fetal liver (Hepatoblasts-enriched).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataOnly three sequences at unannotated regions had features resembling novel miRNAs, all present in embryonic tissues but low in adult.
“Surprisingly, among the sequences that mapped at unannotated regions, only three had features resembling novel miRNAs, all of which were present in embryonic tissues but were low in adult (Supporting Table S1).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn foregut, 28% of miRNA reads belong to the mir17-92 locus, which includes mir20a and mir17, both expressed over 19-fold higher in foregut than hepatoblasts or liver.
“In the foregut, 28% of the miRNA reads belong to the mir17-92 locus of miRNAs, which includes mir20a and mir17, both of which are expressed over 19-fold higher in the foregut than hepatoblasts or liver (Fig. S1C, Table S3).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe Dlk1-Dio3 locus contains over 60 miRNAs, comprising 41% of total miRNA reads in the hepatoblast library.
“The Dlk1-Dio3 locus contains over 60 miRNAs, comprising 41% of the total miRNA reads obtained in the hepatoblast library.”
Quote in the paperNot a substantial claimThe quote only partly states this claim.
Findingown datamir20a is the most highly expressed miRNA in the endoderm/foregut library.
“mir20a, the most highly expressed miRNA in the endoderm library”
- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
“mir20a, the most highly expressed miRNA in the foregut library”
Quote in the paperNot a substantial claim- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
May combine two claims.
Findingown datamir302b expression reduced Tgfbr2 protein expression, while mir20a knockdown increased Tgfbr2 expression.
“Moreover, expression of mir302b in HEK293T cells reduced Tgfbr2 protein expression, while knockdown of mir20a increased Tgfbr2 expression (Fig. 5C).”
Quote in the paperSubstantial claimMay combine two claims. The quote only partly states this claim.
Findingown datamir302b is expressed in the endoderm at the onset of organogenesis, with expression first detected at approximately the 3-somite stage (E8.25) and throughout the foregut.
“The expression of mir302b in definitive endoderm was first detected at approximately the 3-somite stage (E8.25) (Fig. 3E,F; Fig. S5G-L).”
- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
“mir302b expression was evident throughout the foregut (Fig. 3F, black arrowhead), encompassing the region that contains liver progenitors, and in the hindgut region but was excluded from the midgut (Fig. S5J-L).”
Quote in the paperNot a substantial claim- Also stated in: § MicroRNAome of Foregut, Hepatoblast, and Adult Liver. · plain
May combine two claims.
Findingown data3TP-lux promoter activity was reduced in cells expressing mir302b but increased in cells with mir20a knockdown, irrespective of TGF-beta addition.
“Irrespective of addition of TGFb, the promoter activity was reduced in cells expressing mir302b but increased in cells with mir20a knockdown (Fig. 6).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataOf 592 miRNA/miRNA* identified, more than 60% were expressed in both male and female liver libraries, with expression level correlation 0.5807.
“Of the 592 miRNA/miRNA* identified, more than 60% were expressed in both libraries, and the expression level correlation was 0.5807 (Fig. S1B).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown datamir302b is the most highly enriched miRNA in the foregut, with essentially no expression in hepatoblasts or adult liver.
“Of note, mir302b was the most highly enriched miRNA in the foregut, with essentially no expression in hepatoblasts or adult liver.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown datamir20a can target Tgfbr2 expression and repress TGF-beta signaling.
“In addition to mir302b, we show that mir20a can target Tgfbr2 expression and repress TGFb signaling.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown datamir302b expression was evident throughout the foregut encompassing the region that contains liver progenitors, and in the hindgut region but excluded from the midgut.
“mir302b expression was evident throughout the foregut (Fig. 3F, black arrowhead), encompassing the region that contains liver progenitors, and in the hindgut region but was excluded from the midgut (Fig. S5J-L).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataRepressing TGF-beta signaling by SB505124 or mir302b reduced expression of liver markers during mouse ESC differentiation, partially resembling liver hypoplasia in smad2 and smad3 heterozygous mouse.
“We observed that repressing TGFb signaling by SB505124 or mir302b reduced the expression of liver markers during mouse ESC differentiation, partially resembling the phenomena of liver hypoplasia in smad2 and smad3 heterozygous mouse.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationComplete depletion of the mir17 family, including mir20a, causes embryonic lethality around E14.5 with increased apoptosis in the liver.
“Complete depletion of the mir17 family, including mir20a, causes embryonic lethality, with embryos dying around E14.5, exhibiting increased apoptosis in the liver.35”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [35]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Classified as probably not a substantial claim.
PremisecitationKat2b and Camk2n1 can modulate TGF-beta signaling.
“Kat2b and Camk2n1 can modulate TGFb signaling.25,26”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [25]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites [26]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMice heterozygous for both smad2 and smad3 die at midgestation with liver hypoplasia and anemia.
“Mice heterozygous for both smad2 and smad3 die at midgestation with liver hypoplasia and anemia.27”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [27]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisenone givenThe role for TGF-beta ligands themselves in endoderm organ formation is less well established.
“However, the role for TGFb ligands themselves in endoderm organ formation is less well established.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationmiRNAs expressed at greater than 10 RPM were used in K-means clustering analysis.
“miRNAs expressed at greater than 10 RPM were used in Kmeans clustering analysis.14”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisecitationmir20a expression promotes neuroblastoma development by regulating TGF-beta signaling.
“Expression of mir20a promotes neuroblastoma development by regulating TGFb signaling.34”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [34]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S4 (based).
Premisenone givenIt has been proposed that TGF-beta signaling must be inhibited during early organogenesis.
“It has been proposed that TGFb signaling must be inhibited during early organogenesis.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationLet7 functions by repressing self-renewal and promoting differentiation.
“Let7 functions by repressing self-renewal and promoting differentiation.28”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [28]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisecitationmir302b orthologs in Xenopus (mir427) and zebrafish (mir430) are critical for mesendodermal fate specification by balancing Nodal and Lefty activity.
“Studies of mir302b orthologs in Xenopus (mir427) and zebrafish (mir430) propose it is critical for mesendodermal fate specification by balancing Nodal and Lefty activity.32”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [32]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Classified as probably not a substantial claim. May combine two claims.
Study design and conductnone givenTranscripts were considered differentially expressed if NAC values differed by a factor of two and their corrected P-value was less than 0.05 by Fisher's exact test.
“Transcripts were considered differentially expressed if NAC values differed by a factor of two and their corrected P-value was less than 0.05 (by Fisher's exact test).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe TGFBR1 inhibitor SB431542 enhances hepatic lineage specification in hESC.
“Studies in hESC also showed that the TGFBR1 inhibitor, SB431542, enhances hepatic lineage specification.37”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [37]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisecitationmir302b was found to promote reprogramming of human fibroblasts into iPS cells in part by targeting human TGFBR2.
“Recently, mir302b was found to promote reprogramming of human fibroblasts into iPS cells in part by targeting human TGFBR2 and thus promoting a mesenchymal to epithelial transition.33”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [33]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisecitationmir379, mir541, mir434-3p, and mir127 are highly expressed in germline-competent iPS cells.
“In addition, mir379, mir541, mir434-3p, and mir127, are highly expressed in germline-competent iPS cells.30”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [30]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenStatistical significance was inferred at *P < 0.05 and **P < 0.01.
“Statistical significance was inferred at *P < 0.05 and **P < 0.01.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningDifferent subpopulations may exist among Dlk1+ cells.
“Thus, different subpopulations may exist.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationcitationInduction of mir302b in definitive endoderm initiates at the 3-somite stage, corresponding to the stage when liver and pancreatic progenitors are first specified.
“Thus, induction of mir302b in definitive endoderm initiates at the 3-somite stage, corresponding to the stage when liver and pancreatic progenitors are first specified.1”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [1]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Classified as probably not a substantial claim.
PremisereasoningHow miRNAs contribute to critical phases of early liver specification and progenitor cell proliferation remains unexplored.
“These studies demonstrate the importance of miRNAs during the later differentiation stages of liver development; however, how miRNAs contribute to critical phases of early liver specification and progenitor cell proliferation remains unexplored.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
Study design and conductnone givenAll data presented are representative of at least three independent experiments unless indicated otherwise.
“All data presented are representative of at least three independent experiments unless indicated otherwise.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationRecent findings suggested a positive correlation between activation of the imprinted Dlk1-Dio3 region and pluripotency in iPS cells.
“Recent findings suggested a positive correlation between the activation of the imprinted Dlk1-Dio3 region and pluripotency in induced pluripotent stem (iPS) cells.30”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [30]
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
The paper relies on this without support.
The E8.5 foregut contains liver progenitor cells.
“E8.5 foregut, containing liver progenitor cells”
The paper relies on this without support.
The miRNA target prediction algorithms used (mirWalk and its component algorithms) accurately predict functional miRNA-mRNA targeting pairs.
“we utilized a more stringent miRNA target prediction algorithm, mirWalk.24 mirWalk integrates five additional prediction algorithms, each using different approaches including TargetScan, DIANA-microT, miRDB, miRanda, and PITA.”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Percentage of hepatoblast-enriched genes targeted by mir302b is misstated
MinorThe reported 575 of 1,599 genes is 35.96%, which rounds to 36%, not 35%.
Reported 35 · recomputed 35.959975
“mir302b potentially targets 575 out of the total 1,599 (35%) Hepatoblasts-enriched genes”
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Self-citationOrganogenesis and development of the liver
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TGF-β Receptor Type II Deficiency Results in Defects of Yolk Sac Hematopoiesis and Vasculogenesis
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let-7 microRNAs in development, stem cells and cancer
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BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm
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Isolation of hepatoblasts based on the expression of Dlk/Pref-1
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The miR-17-92 MicroRNA Cluster Regulates Multiple Components of the TGF-β Pathway in Neuroblastoma
Mestdagh, P., Boström, A.-K., Impens, F., Fredlund, E., Van Peer, G., De Antonellis, P., von Stedingk, K., Ghesquière, B., Schulte, S., Dews, M., et al. (2010) · Molecular Cell · doi:10.1016/j.molcel.2010.11.038 · Cited by 314 · see citing works
Activation of the Imprinted Dlk1-Dio3 Region Correlates with Pluripotency Levels of Mouse Stem Cells
Liu, L., Luo, G., Yang, W., Zhao, X., Zheng, Q., Lv, Z., Li, W., Wu, H., Wang, L., Wang, X., et al. (2010) · Journal of Biological Chemistry · doi:10.1074/jbc.m110.131995 · Cited by 268 · see citing works
Dynamic Signaling Network for the Specification of Embryonic Pancreas and Liver Progenitors
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miR-29b is activated during neuronal maturation and targets BH3-only genes to restrict apoptosisPreprint
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Disruption of Dicer1 Induces Dysregulated Fetal Gene Expression and Promotes Hepatocarcinogenesis
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The microRNA-30 family is required for vertebrate hepatobiliary development
Hand, N., Master, Z. R., Eauclaire, S. F., Weinblatt, D., Matthews, R. P., & Friedman, J. R. (2008) · Gastroenterology · doi:10.1053/j.gastro.2008.12.006 · Cited by 137 · see citing works
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The transcriptional co-activator P/CAF potentiates TGF-beta/Smad signaling
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Molecular Mechanism of Liver Development and Regeneration
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Hepatic function is preserved in the absence of mature microRNAs #
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Long-term culture of hepatic progenitors derived from mouse Dlk+ hepatoblasts
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Dicer is required for proper liver zonation
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The DIANA-mirExTra Web Server: From Gene Expression Data to MicroRNA Function
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“Foregut endoderm was isolated by mechanical dissociation from embryos at somite stage 8-12.(12) Since nearly 70% cells of E14.”
Self-citationTissue-specific regulation of mouse MicroRNA genes in endoderm-derived tissues
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Genomic analysis distinguishes phases of early development of the mouse atrio-ventricular canal
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Self-citation
Flagged first, then by citations
2 of the 39 references couldn’t be matched to a known paper.
Research works printed but not linked: 1
- GouonEvans, 2006, BMP4 is required for hepatic specification of mouse embryonic stem cellderived definitive endoderm., Nat Biotechnol, 24, 14021411
Cited by
Sorted by citations
<scp>TGF</scp>‐β signalling and liver disease
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Hippo Signaling Influences HNF4A and FOXA2 Enhancer Switching during Hepatocyte Differentiation
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“We next investigated the impact of temporal TF-DNA binding on associated target gene transcription by using RNA sequencing (RNA-seq) to profile the transcriptomes of both DLK1 + hepatoblasts and adult liver (Table S2).”
MethodInfluentialSelf-citationEpigenetics in hepatocellular carcinoma: An update and future therapy perspectives
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Micro RNA s: the fine modulators of liver development and function
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“Target analysis demonstrated that miR-302b reduces the expression of transforming growth factor beta receptor II (TGFBR2, a type II receptor required for TGFb ligand signalling) and K (Lysine) acetyltransferase 2B (KAT2B, a histone acetyltransferase interacting directly with the intracellular TGFb signalling component…”
BackgroundRegulation of microRNAs and their role in liver development, regeneration and disease
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“They were utilized to explore patterns of RNA expression, and hence intrinsic gene interactions [10-12,18-25].”
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“Previous reports have also revealed the ability of miR-20a to regulate several cellular processes and to promote malignant transformation (36,37).”
PtenRegulates Retinal Amacrine Cell Number by Modulating Akt, Tgfβ, and Erk Signaling
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“…activates membrane inositol phospholipids by converting PIP2 (phosphatidylinositol-4,5-bisphosphate) into PIP3 (phosphatidylinositol-3,4,5-triphosphate), which serves as a lipid second messenger, activating downstream Akt kinases (Whitman et al., 1988; Engelman et al., 2006; Kriplani et al., 2015).”
BackgroundSingle-Cell Non-coding RNA in Embryonic Development
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MiR-126 Regulates Properties of SOX9+ Liver Progenitor Cells during Liver Repair by Targeting Hoxb6
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“One previous study has shown that miR-302b and miR-20a repress hepatic functions by regulating transforming growth factor b (TGF-b) (Wei et al., 2013).”
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“Stem cell properties of hepatoblasts are maintained by an miR-125/Lin28/Let-7 axis,((31)) and miR-302b and miR-20a repress hepatic functions.((32)) Later, in DEMAREZ, G ERARD, ET AL.”
BackgroundWeirather, J. L., Duggal, P., do Nascimento, E. L. T., Monteiro, G. R., Martins, D. R., Lacerda, H. G., Fakiola, M., Blackwell, J. M., Jerônimo, S. M. B., & Wilson, M. E. (2017) · Annals of Human Genetics · doi:10.1111/ahg.12180 · Cited by 10 · see citing works
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Expression patterns of Yes-associated protein 1 in the developing mouse liver
Wei, W., Lotto, J., & Hoodless, P. A. (2018) · Gene Expression Patterns · doi:10.1016/j.gep.2018.04.003 · Cited by 3 · see citing works
Self-citationAntitarget profiling of mycotoxins via molecular docking
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The Role of microRNAs in the Gut-Liver Axis
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Noncoding RNAs in liver cancer patients
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- Authors
- Wei, W., Hou, J., Alder, O., Ye, X., Lee, S., Cullum, R., Chu, A., Zhao, Y., Warner, S. M., Knight, D. A., Yang, D., Jones, S. J. M., Marra, M. A., & Hoodless, P. A.
- Affiliations
- BC Cancer Agency (CA)
- University of British Columbia (CA)
- St. Paul's Hospital (CA)
- Corresponding author
- Hoodless, Pamela A.
- ORCID
- Venue
- Hepatology
- Published
- 2013-06
- Volume
- 57
- Issue
- 6
- Pages
- 2491-2501
- ISSN
- 0270-9139 (ISSN-L)
- OpenAlex
- W2095478157
- Semantic Scholar
- 1f1c4f0dfc97d137cb8419c0774e294fa1fbe81d
- Cited by
- 19
- References
- 39
- Citations by year
- 2026: 1 · 2022: 1 · 2021: 1 · 2020: 2 · 2018: 4 · 2017: 2 · 2016: 3 · 2015: 1 · 2014: 4
- FWCI
- 0.83
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Hepatology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Genome Canada
- Canadian Institutes of Health Research (89806_1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Keywords
- microRNA, liver development, TGF-beta signaling, TGFBR2, hepatoblasts, endoderm, miRNA expression profiling, miRNA network, embryonic stem cell differentiation, mouse embryo
- MeSH terms
- Receptor, Transforming Growth Factor-beta Type II, p300-CBP-Associated Factor, Animals, Cell Differentiation, Endoderm, Female, Imidazoles, Liver, Male, Pyridines, RNA, Messenger, Signal Transduction, Genome, Protein Serine-Threonine Kinases, Receptors, Transforming Growth Factor beta, Gene Expression Profiling, MicroRNAs, Organogenesis, Gastrointestinal Tract, p300-CBP Transcription Factors, Mice, Benzodioxoles, Embryonic Stem Cells, Transforming Growth Factor beta1
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.
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