microRNA Family Research Articles

Expression analyses of miRNA Up-MIR-843 and its target genes in Ulva prolifera

microRNAs (miRNA) families play a critical role in plant growth, development, and responses to abiotic stress. In this study, we characterized Up-miR-843 and its targets genes in Ulva prolifera responses to nitrogen depravation and heat stress. The data demonstrated that 184 target genes of Up-miR-843 could be successfully validated. N deficiency not heat stress stimulus induced increase in abundance of the Up-miR-843 while exhibited reverse expression of target genes, including cyclin A3 and cyclin L, which were strictly required for cell cycle progression. In addition, U. prolifera with highly expression of Up-miR-843 showed improved biomass, and photosynthesis compared with that under normal growth conditions. Thus, the N deprivation and heat responsive miRNAs might be a possible member mediating the expression of these target genes, which further regulated the growth of U. prolifera.

Transgenic expression of human C19MC miRNAs impacts placental morphogenesis

IntroductionThe chromosome 19 miRNA cluster (C19MC) encodes a large family of microRNAs (miRNAs) that are abundantly expressed in the placenta of higher primates and also in certain cancers. In the placenta, miRNAs from this cluster account for nearly 40% of all miRNAs present in trophoblasts. However, the function of these miRNAs in the placenta remains poorly understood. Recent observations reveal a role for these miRNAs in cell migration, and suggest that they are involved in the development and function of the human placenta. Here, we examine the placenta in transgenic mice expressing the human C19MC miRNAs. MethodsWe produced transgenic mice using pronuclear microinjection of a bacterial artificial chromosome plasmid carrying the entire human C19MC locus and derived a homozygous line using crossbreeding. We performed morphological characterization and profiled gene expression changes in the placentas of the transgenic mice. ResultsC19MC transgenic mice delivered on time with no gross malformations. The placentas of transgenic mice expressed C19MC miRNAs and were larger than wild type placentas. Histologically, we found that the transgenic placenta exhibited projections of spongiotrophoblasts that penetrated deep into the labyrinth. Gene expression analysis revealed alterations in the expression of several genes involved in cell migration, with evidence of enhanced cell proliferation. DiscussionMice that were humanized for transgenically overexpressed C19MC miRNAs exhibit enlarged placentas with aberrant delineation of cell layers. The observed phenotype and the related gene expression changes suggest disrupted migration of placental cell subpopulations.

Research Progress of MicroRNAs and Related Factors in the Pathogenesis of Lichen Planus

Abstract More evidences show that microRNAs play an important role in the pathogenesis of inflammation and autoimmune diseases including Lichen planus, and are an attractive therapeutic target. MicroRNA family plays an important role in the regulation of gene expression, which involves cell proliferation, apoptosis, growth, differentiation and metabolism, vascularization, immune response and other biological processes. The changes of expression profile and expression level are closely related to the occurrence and development of many diseases, such as tumor, inflammatory disease and autoimmune disease relevant. However, there are few reports on microRNA in the pathogenesis of LP. This review summarizes the research advance of microRNAs (microRNAs -137, microRNAs -125b, microRNAs -138, microRNAs -27b, microRNAs -203) and their downstream proteins in LP.

Abstract 5070: LIN28A-mediated repression of Let-7a is involved in resistin-induced STAT3 upregulation in breast cancer cells and supports growth and stemness properties

Abstract Breast cancer (BC) is the most frequently diagnosed malignancy and continues to be the second leading cause of cancer-related death in women in the United States. Dysregulation of Let-7 family microRNAs is observed in BC and associated with disease pathogenesis. Previously, we reported a significant increase in serum levels of resistin in BC patients as compared to the healthy individuals. Moreover, this increase was significantly greater in BC patients of African American background as compared to those of European descent. We also demonstrated that treatment of BC cells with resistin promoted their growth, aggressiveness, stemness and chemoresistance by inducing the expression and activation of STAT3. Here we present data to show that resistin induces STAT3 expression in BC cells in a LIN28A and Let-7a-dependent manner. Treatment of BC cells with resistin led to the most significant decrease in the expression of Let-7a among other Let-7 family members. Furthermore, resistin treatment also induced the expression of LIN28A at both transcript and protein levels and its silencing abrogated resistin-mediated Let-7a suppression. Let-7a restoration or LIN28A silencing abolished the resistin-induced growth, clonogenicity and sphere-forming ability of BC cells. Restoration of Let-7a suppressed the expression of several genes associated with growth, survival and stemness. Ingenuity Pathway Analysis suggested STAT3 as a putative central node associated with Let-7a-mediated gene regulation. Immunoblot analysis confirmed that Let-7a restoration dramatically suppressed basal as well as resistin-induced STAT3 expression. STAT3 regulation by Let-7a was further confirmed 3'UTR-reporter assay. Taken together, our findings demonstrate a novel resistin/LIN28A/Let-7a/STAT3 signaling axis regulating the growth and stemness phenotypes of BC cells. Citation Format: Sachin Kumar Deshmukh, Sanjeev K. Srivastava, Haseeb Zubair, Ajay P. Singh, Seema Singh. LIN28A-mediated repression of Let-7a is involved in resistin-induced STAT3 upregulation in breast cancer cells and supports growth and stemness properties [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5070.

MicroRNA-dependent inhibition of PFN2 orchestrates ERK activation and pluripotent state transitions by regulating endocytosis

Profilin2 (PFN2) is a target of the embryonic stem cell (ESC)-enriched miR-290 family of microRNAs (miRNAs) and an actin/dynamin-binding protein implicated in endocytosis. Here we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of miRNAs, PFN2 is up-regulated in ESCs, with a resulting decrease in endocytosis. Reintroduction of miR-290, knockout of Pfn2, or disruption of the PFN2-dynamin interaction domain in miRNA-deficient cells reverses the endocytosis defect. The reduced endocytosis is associated with impaired extracellular signal-regulated kinase (ERK) signaling, delayed ESC cell cycle progression, and repressed ESC differentiation. Mutagenesis of the single canonical conserved 3' UTR miR-290-binding site of Pfn2 or overexpression of the Pfn2 open reading frame alone in otherwise wild-type cells largely recapitulates these phenotypes. Taken together, these findings define an axis of posttranscriptional control, endocytosis, and signal transduction that is important for ESC proliferation and differentiation.

MicroRNA-708 prevents ethanol-induced hepatic lipid accumulation and inflammatory reaction via direct targeting ZEB1

Alcoholic liver disease (ALD) was a global liver disease which divided into liver inflammation, fatty liver, alcoholic hepatitis or cirrhosis. Abnormal expression levels of some microRNAs (miRNA) family members often lead to ALD and other liver diseases. MicroRNA-708 (miR-708) was known to suppress the proliferation and metastasis of hepatocellular carcinoma (HCC), but its role in the progression of ALD was not clear. In this study, the expression level of miR-708 was down-regulated in ethanol-induced L0-2 cells. ZEB1 could decrease the PPAR-α expression while increase the SREBP-1 expression. Meanwhile, the expression levels of TNF-α and IL-6 were up-regulated by ZEB1. Of note, ZEB1 aggravated the apoptotic rate of L0-2 cells induced by ethanol via inhibiting p-AKT and p-mTOR of AKT/mTOR signaling pathway. What's more, it was demonstrated that miR-708 family members particularly target ZEB1 3′-UTR regions and can down-regulate the expression level of ZEB1 in L0-2 cells. Sum up, these results indicated that miR-708 might inhibit the liver inflammation and lipid accumulation by targeting ZEB1 via regulating AKT/mTOR signaling pathway.

Extracellular vesicles from Heligmosomoides bakeri and Trichuris muris contain distinct microRNA families and small RNAs that could underpin different functions in the host

Extracellular vesicles (EVs) have emerged as a ubiquitous component of helminth excretory-secretory products that can deliver parasite molecules to host cells to elicit immunomodulatory effects. RNAs are one type of cargo molecule that can underpin EV functions, hence there is extensive interest in characterising the RNAs that are present in EVs from different helminth species. Here we outline methods for identifying all of the small RNAs (sRNA) in helminth EVs and address how different methodologies may influence the sRNAs detected. We show that different EV purification methods introduce relatively little variation in the sRNAs that are detected, and that different RNA library preparation methods yielded larger differences. We compared the EV sRNAs in the gastrointestinal nematode Heligmosomoides bakeri with those in EVs from the distantly related gastrointestinal nematode Trichuris muris, and found that many of the sRNAs in both organisms derive from repetitive elements or intergenic regions. However, only in H. bakeri do these RNAs contain a 5′ triphosphate, and Guanine (G) starting nucleotide, consistent with their biogenesis by RNA-dependent RNA polymerases (RdRPs). Distinct microRNA (miRNA) families are carried in EVs from each parasite, with H. bakeri EVs specific for miR-71, miR-49, miR-63, miR-259 and miR-240 gene families, and T. muris EVs specific for miR-1, miR-1822 and miR-252, and enriched for miR-59, miR-72 and miR-44 families, with the miR-9, miR-10, miR-80 and let-7 families abundant in both. We found a larger proportion of miRNA reads derive from the mouse host in T. muris EVs, compared with H. bakeri EVs. Our report underscores potential biases in the sRNAs sequenced based on library preparation methods, suggests specific nematode lineages have evolved distinct sRNA synthesis/export pathways, and highlights specific differences in EV miRNAs from H. bakeri and T. muris that may underpin functional adaptation to their host niches.

Expansion of MIR482/2118 by a class-II transposable element in cotton.

Some plant microRNA (miRNA) families contain multiple members generating identical or highly similar mature miRNA variants. Mechanisms underlying the expansion of miRNA families remain elusive, although tandem and/or segmental duplications have been proposed. In this study of two tetraploid cottons, Gossypium hirsutum and Gossypium barbadense, and their extant diploid progenitors, Gossypium arboreum and Gossypium raimondii, we investigated the gain and loss of members of the miR482/2118 superfamily, which modulates the expression of nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance genes. We found significant expansion of MIR482/2118d in G.barbadense, G.hirsutum and G.raimondii, but not in G.arboreum. Several newly expanded MIR482/2118d loci have mutated to produce different miR482/2118 variants with altered target-gene specificity. Based on detailed analysis of sequences flanking these MIR482/2118 loci, we found that this expansion of MIR482/2118d and its derivatives resulted from an initial capture of an MIR482/2118d by a class-II DNA transposable element (TE) in G.raimondii prior to the tetraploidization event, followed by transposition to new genomic locations in G.barbadense, G.hirsutum and G.raimondii. The 'GosTE' involved in the capture and proliferation of MIR482/2118d and its derivatives belongs to the PIF/Harbinger superfamily, generating a 3-bp target site duplication upon insertion at new locations. All orthologous MIR482/2118 loci in the two diploids were retained in the two tetraploids, but mutation(s) in miR482/2118 were observed across all four species as well as in different cultivars of both G.barbadense and G.hirsutum, suggesting a dynamic co-evolution of miR482/2118 and its NBS-LRR targets. Our results provide fresh insights into the mechanisms contributing to MIRNA proliferation and enrich our knowledge on TEs.

Downregulation of the let‑7 family of microRNAs may promote insulin receptor/insulin‑like growth factor signalling pathways in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type characterized by dysregulated cell signalling pathways and resistance to treatment. The insulin-like growth factor (IGF) signalling pathway has been identified to have a role in tumour progression and therapy resistance. However, its regulatory roles in PDAC have remained to be fully elucidated. In the present study, dysregulated microRNAs (miRNAs) in PDAC were explored with a focus on those that may be involved in regulating the insulin/IGF signalling pathway. A total of 208 patients were recruited, comprising 112 patients with PDAC, 50 patients with chronic pancreatitis (CP) and 46 subjects as a control group (CG). miRNA-specific quantitative PCR assays were used to measure 300 candidate miRNAs. The Student's t-test was applied to compare miRNA regulation between cancer patients and controls with a false discovery rate correction using Bonferroni-type comparison procedures. The DIANA-mirPath v.3 tool and HMDD v3.0 were used to identify miRNA-mRNA interactions within specific pathways. In patients with PDAC, 42 miRNAs were significantly upregulated and 42 were downregulated compared to the CG (P<0.01). In the PDAC vs. CP analysis, 16 significantly (P<0.01) upregulated and 16 downregulated miRNAs were identified. Of note, members of the let-7 family of miRNAs were downregulated and were indicated to target several components of the insulin receptor (INSR)/IGF pathway, including receptors and binding proteins, for upregulation and thus, may enable the activation of the pathway. Downregulation of the let-7 family may help promote the INSR/IGF pathway in PDAC. It may thus be an effective target for the development of INSR/IGF pathway-specific treatment strategies.

Association of the serum microRNA-29 family with cognitive impairment in Parkinson's disease.

We aimed to examine whether miRNA-29s (miR-29s) in serum are associated with cognitive impairment in Parkinson’s disease (PD). Thirty-nine PD patients with normal cognition (PD-NC), 37 PD patients with mild cognitive impairment (PD-MCI), 22 PD patients with dementia (PDD) and 40 healthy controls were recruited. Detailed clinical evaluations and a schedule of neuropsychological tests were administered to all patients. MiR-29s expression in serum samples was assessed using reverse-transcription quantitative real-time PCR. We found that the levels of all three miR-29s in the PDD group were significantly lower than those in the PD-NC group (p < 0.05). In addition, the miR-29b level was downregulated in the PD-MCI group with respect to that in the PD-NC group (p < 0.05). After adjusting for years of education and the UPDRS-III subscore using a multivariate model, miR-29s showed significant associations with PDD. MiR-29b levels were shown to be associated with different subsets of PD cognition and could accurately discriminate PDD from non-PDD (area under the curve (AUC) = 0.859; 95% CI, 0.7817-0.9372). Further analysis of the cognitive domains found that the miR-29s levels were all associated with memory performance in PD patients. In summary, miR-29s are associated with cognitive impairment in PD.

Loss of miR-132/212 Has No Long-Term Beneficial Effect on Cardiac Function After Permanent Coronary Occlusion in Mice.

Background: Myocardial infarction (MI) is caused by occlusion of the coronary artery and induces ischemia in the myocardium and eventually a massive loss in cardiomyocytes. Studies have shown many factors or treatments that can affect the healing and remodeling of the heart upon infarction, leading to better cardiac performance and clinical outcome. Previously, miR-132/212 has been shown to play an important role in arteriogenesis in a mouse model of hindlimb ischemia and in the regulation of cardiac contractility in hypertrophic cardiomyopathy in mice. In this study, we explored the role of miR-132/212 during ischemia in a murine MI model.Methods and Results: miR-132/212 knockout mice show enhanced cardiac contractile function at baseline compared to wild-type controls, as assessed by echocardiography. One day after induction of MI by permanent occlusion, miR-132/212 knockout mice display similar levels of cardiac damage as wild-type controls, as demonstrated by infarction size quantification and LDH release, although a trend toward more cardiomyocyte cell death was observed in the knockout mice as shown by TUNEL staining. Four weeks after MI, miR-132/212 knockout mice show no differences in terms of cardiac function, expression of cardiac stress markers, and fibrotic remodeling, although vascularization was reduced. In line with these in vivo observation, overexpression of miR-132 or miR-212 in neonatal rat cardiomyocyte suppress hypoxia induced cardiomyocyte cell death.Conclusion: Although we previously observed a role in collateral formation and myocardial contractility, the absence of miR-132/212 did not affect the overall myocardial performance upon a permanent occlusion of the coronary artery. This suggests an interplay of different roles of this miR-132/212 before and during MI, including an inhibitory effect on cell death and angiogenesis, and a positive effect on cardiac contractility and autophagic response. Thus, spatial or tissue specific manipulation of this microRNA family may be essential to fully understand the roles and to develop interventions to reduce infarct size.

Identification, characterization, and expression analysis of cowpea (Vigna unguiculata [L.] Walp.) miRNAs in response to cowpea severe mosaic virus (CPSMV) challenge.

Cowpea miRNAs and Argonaute genes showed differential expression patterns in response to CPSMV challenge Several biotic stresses affect cowpea production and yield. CPSMV stands out for causing severe negative impacts on cowpea. Plants have two main induced immune systems. In the basal system (PTI, PAMP-triggered immunity), plants recognize and respond to conserved molecular patterns associated with pathogens (PAMPs). The second type (ETI, Effector-triggered immunity) is induced after plant recognition of specific factors from pathogens. RNA silencing is another important defense mechanism in plants. Our research group has been using biochemical and proteomic approaches to learn which proteins and pathways are involved and could explain why some cowpea genotypes are resistant whereas others are susceptible to CPSMV. This current study was conducted to determine the role of cowpea miRNA in the interaction between a resistant cowpea genotype (BRS-Marataoã) and CPSMV. Previously identified and deposited plant microRNA sequences were used to find out all possible microRNAs in the cowpea genome. This search detected 617 mature microRNAs, which were distributed in 89 microRNA families. Next, 4 out of these 617 miRNAs and their possible target genes that encode the proteins Kat-p80, DEAD-Box, GST, and SPB9, all involved in the defense response of cowpea to CPSMV, had their expression compared between cowpea leaves uninoculated and inoculated with CPSMV. Additionally, the differential expression of genes that encode the Argonaute (AGO) proteins 1, 2, 4, 6, and 10 is reported. In summary, the studied miRNAs and AGO 2 and AGO4 associated genes showed differential expression patterns in response to CPSMV challenge, which indicate their role in cowpea defense.

High Mobility Group AT-Hook 2 (HMGA2) Oncogenicity in Mesenchymal and Epithelial Neoplasia.

High mobility group AT-hook 2 (HMGA2) has been associated with increased cell proliferation and cell cycle dysregulation, leading to the ontogeny of varied tumor types and their metastatic potentials, a frequently used index of disease prognosis. In this review, we deepen our understanding of HMGA2 pathogenicity by exploring the mechanisms by which HMGA2 misexpression and ectopic expression induces mesenchymal and epithelial tumorigenesis respectively and distinguish the pathogenesis of benign from malignant mesenchymal tumors. Importantly, we highlight the regulatory role of let-7 microRNA family of tumor suppressors in determining HMGA2 misexpression events leading to tumor pathogenesis and focused on possible mechanisms by which HMGA2 could propagate lymphangioleiomyomatosis (LAM), benign mesenchymal tumors of the lungs. Lastly, we discuss potential therapeutic strategies for epithelial and mesenchymal tumorigenesis based on targeting the HMGA2 signaling pathway.

MiR-103/107 regulates left-right asymmetry in zebrafish by modulating Kupffer’s vesicle development and ciliogenesis

In zebrafish, cilia movement within the Kupffer’s vesicle (KV) generates a fluid flow responsible for accumulating nodal signals exclusively in the left lateral plate mesoderm, thereby initiating left-right patterning (LRP). Defects in LRP cause devastating congenital disorders including congenital heart malformations due to organ mis-positioning. We identified the miR-103/107 family to be involved in regulating LRP. Depletion of miR-103/107 in zebrafish embryos resulted in malpositioned and malformed visceral organs and hearts due to disturbed LRP gene expression, indicating early defects in LRP. Additionally, loss of miR-103/107 affected KV morphogenesis and cilia formation without disturbing endoderm development. Human fibroblasts depleted of miR-103a/107 often failed to extend cilia or developed shorter cilia, indicating functional conservation between species. We identified arl6, araf and foxH1 as direct targets of miR-103/107 providing a mechanistic link to cilia development and nodal signal titration. We describe a new microRNA family controlling KV development and hence influencing establishment of internal organ asymmetry.

Abstract A29: Differential regulation of apoptosis and senescence by the miR-449 family microRNAs and its implications in tumor microenvironments of NSCLC

Abstract MicroRNAs (miRNAs) play important roles in various biologic processes, and aberrant miRNA expression is one of the mechanisms of tumorigenesis. Previous studies indicate that oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a and contributes to the development of cervical cancer. In addition, the traditional Chinese medicine Gynostemma pentaphyllum (Thunb.) Makino may exert its pharmacologic effects through modifying the gut microbiota by regulating hepatic miR-34a expression. MiR-449 miRs are members of the tumor suppressor miR-34 family and are evolutionarily conserved. In this study, we demonstrated that, in lung cancer cells, ectopic expression of miR-449 family, including miR-449a, miR-449b, and miR-449c, decreased the levels of several G1 phase proteins including cyclin D1, cyclin D3, CDK4, and CDK6 via directly targeting their mRNA’s 3’-UTR. We also observed a differential induction of apoptosis vs. senescence by the miR-449 cluster. While miR-449a and miR-449c induced senescence, miR-449b induced apoptosis and sensitized cancer cells to apoptotic stimuli, such as serum deprivation. Interestingly, among this cluster miRNAs, only miR-449a could induce significant senescence-associated secretory phenotype (SASP), e.g., expression of IL-8, IL-6, IL-1b, and TNF-alpha. Moreover, we also found that miR-449a increased NF-kB phosphorylation at S276, but not phosphorylation site of S536, and promoted the expression of IL-8 mRNA. In this regard, the gut microbiota has been indicated to induce proinflammatory cytokines such as IL-8 to influence the outcomes of cancer patients. In addition to MRX34 (a miR-34a mimic) used in clinical trials, miR-449 family may also potentially become a new therapeutic candidate for NSCLC and other solid tumors’ treatment. Citation Format: Chun-Yu Cho, Shih-Ying Chung, Shuang-En Chuang. Differential regulation of apoptosis and senescence by the miR-449 family microRNAs and its implications in tumor microenvironments of NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A29.

MicroRNA-200 family governs ovarian inclusion cyst formation and mode of ovarian cancer spread.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-β expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

Characterization and functional analysis of microRNA399 in Cunninghamia lanceolata

The miR399 is a conserved microRNA (miRNA) family, and it has been characterized as an essential regulator of phosphorus transport in plants. However, the biological function of miR399 in Cunninghamia lanceolata is still largely unclear. In this study, the comparison of mature miR399 sequence revealed a high similarity between Arabidopsis thaliana and C. lanceolate, and the pre-miR399 was capable of forming a typical stem-loop hairpin structure. A gene PHOSPHATE 2 (PHO2) was identified as a target of cln-miR399 using 5' rapid amplification of cDNA ends. Furthermore, the relationship between cln-miR399 and PHO2 was further confirmed through a transient co-expression of both genes in Nicotiana benthamiana. To examine the function of miR399 in Arabidopsis, miR399-overexpressing transgenic Arabidopsis thaliana was acquired using Agrobacterium-mediated approach. Real-time PCR showed that the amount of cln-MIR399 transcripts was higher in miR399-overexpressing plants than in wild-type plants, which was accompanied with down-regulation of expression of its target gene AtPHO2. The P content was 1.40 to 1.56-fold higher in the leaves of three transgenic lines than in wild type plants. However, the P content in the roots of the three transgenic lines was 24.5 - 37.2 % less than that in wild type plants. Moreover, the transcriptions of three phosphate transporter genes (PHT1, PHT2, and PHT3) were up-regulated in roots of miR399-overexpressing Arabidopsis plants. Interestingly, the transgenic lines exhibited retarded growth under normal P conditions compared with the wild type. Our findings demonstrate that cln-miR399 may play crucial roles in P transport and plant growth via regulation of its target gene PHO2.

Inactivation of Zeb1 in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure

ABSTRACTCleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (Grhl1-3) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, Grhl2, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. Grhl2-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of Grhl2-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors Ovol1 and Ovol2, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of Zeb1 expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in Zeb1−/− mice is evident, with rescue of both palatal and facial fusion seen in Grhl2−/−;Zeb1−/− embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.

Aberrant expression of miR‐29b‐3p influences heart development and cardiomyocyte proliferation by targeting NOTCH2

ObjectivesmicroRNA‐29 (miR‐29) family have shown different expression patterns in cardiovascular diseases. Our study aims to explore the effect and mechanism of miR‐29 family on cardiac development.Materials and methodsA total of 13 patients with congenital heart disease (CHD) and 7 controls were included in our study. Tissues were obtained from the right ventricular outflow tract (RVOT) after surgical resection or autopsy. The next‐generation sequencing was applied to screen the microRNA expression profiles of CHD. Quantitative RT‐PCR and Western blot were employed to measure genes expression. Tg Cmlc2: GFP reporter zebrafish embryos were injected with microRNA (miRNA) to explore its role in cardiac development in vivo. Dual‐luciferase reporter assay was designed to validate the target gene of miRNAs. CCK‐8 and EdU incorporation assays were performed to evaluate cardiomyocyte proliferation.ResultsOur study showed miR‐29b‐3p expression was significantly increased in the RVOT of the CHD patients. Injection of miR‐29b‐3p into zebrafish embryos induced higher mortality and malformation rates, developmental delay, cardiac malformation and dysfunction. miR‐29b‐3p inhibited cardiomyocyte proliferation, and its inhibitor promoted cardiomyocyte proliferation in vitro and in vivo. Furthermore, we identified that miR‐29b‐3p influenced cardiomyocyte proliferation by targeting NOTCH2, which was down‐regulated in the RVOT of the CHD patients.ConclusionThis study reveals that miR‐29b‐3p functions as a novel regulator of cardiac development and inhibits cardiomyocyte proliferation via NOTCH2, which provides novel insights into the aetiology and potential treatment of CHD.

Expression of the microRNA-200 Family, microRNA-205, and Markers of Epithelial–Mesenchymal Transition as Predictors for Endoscopic Submucosal Dissection over Esophagectomy in Esophageal Adenocarcinoma: A Single-Center Experience

Endoscopic submucosal dissection (ESD) is an effective treatment of early esophageal adenocarcinomas (EACs). The decision of ESD over esophagectomy is based on clinical evaluation of tumor depth and invasion. On a molecular level, tumor invasion is strongly associated with epithelial-to-mesenchymal transition (EMT). Here, we investigated whether localized ESD-resected and surgically resected EAC samples displayed different expression profiles of EMT protein and microRNA markers and whether these different expression profiles were able to retrospectively discriminate localized and surgically resected samples. By doing this, we aimed to evaluate whether preoperative measurement of EMT marker expression might support the decision regarding ESD over surgery. The results showed that ESD-resected samples displayed an epithelial expression profile, i.e., high expression of epithelial protein markers, whereas surgically resected samples displayed high expression of mesenchymal markers. In addition, the anti-EMT microRNA-205 was significantly more expressed in ESD-resected samples, whereas we found no significant differences in the expression levels of microRNA-200 family members. Furthermore, in our retrospective approach, we have demonstrated that measurement of selected EMT markers and microRNA-205 has significant discrimination power to distinguish ESD-resected and surgically resected samples. We suggest that the assessment of EMT status of EAC samples on a molecular level may support clinical evaluation regarding the applicability of ESD.