Let-7d Expression Research Articles

Down-regulation of microRNA let-7d inhibits the proliferation and invasion of trophoblast cells in preeclampsia.

Preeclampsia (PE) is a serious pregnancy complication that continues to adverse effects on mother and the fetus. The study investigated the effect of microRNA let-7d (miR-let-7d) on the proliferation and invasion of trophoblast cells (TC) in PE. In situ hybridization techniques were employed to evaluate the miR-let-7d expressions in the placental tissues from 63 PE patients as well as 65 normal placental tissues. Transfection of pre-let-7d, anti-let-7d, and their corresponding control sequences was performed in TCs obtained from PE. The blank group was composed of TCs no transfection and normal group consisted of normal TCs. Expressions of proliferation and invasion markers in TCs were detected by qRT-PCR. Proliferation, apoptosis, and the invasion ability of the TCs were determined using a CCK-8 assay, flow cytometry, and Transwell assay. In comparison to the normal placental tissues, PE placental tissues exhibited increased levels of let-7d expression. Following transfection of anti-let-7d, when compared with the blank group, the anti-let-7d group displayed increased levels of proliferation ability, expression of proliferation labeling proteins PCNA and Ki67, number of TCs as well as expressions of the invasion related proteins MMP-2, MMP-9, and TIMP-1. In contrast declines in cell apoptosis rate were observed. Compared with the blank group, the changes of the indexes were reversed in the pre-let-7d group. The study provided evidence suggesting that low expression levels of miR-let-7d plays a central role in suppressing apoptosis in addition to promoting the proliferation and invasion of PE TCs.

Different levels of let-7d expression modulate response of FaDu cells to irradiation and chemotherapeutics.

The implication of the let-7 family in cancer development is multifaceted. The family acts as tumor suppressor miRNA although overexpression of let-7 has also been described in many types of cancer, including head and neck squamous cell carcinoma (HNSCC). The aim of this study includes whether different expression levels of let-7d has an influence on chemo- and radiosensitivity. FaDu cell line models with a gradually increased level of let-7d (models from A to E) were generated with the lentiviral system. Expression levels of pluripotency, chemo-radioresistance/apoptosis, and targets of mRNAs were analyzed by real-time reverse transcription-PCR (qRT-PCR). Radiosensitivity was analyzed using a clonogenic assay after irradiation. Response to cisplatin, 5-FU, doxorubicin, and paclitaxel was done with MTT assay. Statistically significant decrease of K-RAS (p = 0.0369) and CASPASE3 (p = 0.0342) were observed with the growing expression level of let-7d. Cisplatin, 5-FU and doxorubicin caused similar decreased of cell survival with the increase of let-7d level (p = 0.004, post-trend p = 0.046; p = 0.004, post trend p = 0.0005 and p<0.0001, post trend p = 0.0001, respectively). All models were resistant to paclitaxel, irrespective of let-7d expression levels. Only two of the generated models (A and C) were radiosensitive (p = 0.0002). Conclusion: the above results indicated that the level of let-7d expression is an important factor for cell response to irradiation and chemotherapeutics.

Let-7d miRNA prevents TGF-β1-induced EMT and renal fibrogenesis through regulation of HMGA2 expression

TGF-β1-induced epithelial to mesenchymal transition (EMT) process of tubular epithelial cells plays a leading role in the occurrence and progression of renal fibrosis as seen in diabetic nephropathy (DN). High mobility group AT-hook 2 (HMGA2) is considered to be involved in TGF-β1-mediated EMT via multifactorial mechanisms. Specific microRNAs (miRNAs) are closely associated with EMT, and here we focused on let-7d miRNA as a regulator of HMGA2. This study aims to investigate the effects of HMGA2 on EMT process induced by TGF-β1 using small interfering RNA (siRNA) technique in vitro, and further explore the potential role of let-7d miRNA during renal fibrosis in DN. We demonstrated that siRNA targeting HMGA2 was sufficient to inhibit TGF-β1-induced EMT and fibrogenesis in rat kidney tubular epithelial cells (NRK52E). Furthermore, let-7d expression was significantly reduced by TGF-β1 stimulation, we focused on let-7d and found that overexpression of let-7d down-regulated the expression of HMGA2 and in turn suppressed TGF-β1-induced EMT and renal fibrogenesis. Inhibition of let-7d increased HMGA2 expression and enhanced the profibrogenic effects of TGF-β1 on NRK-52E cells. Consistent with the above observations in vitro, let-7d expression was also decreased in the kidneys of unilateral ureter obstruction model, accompanied by the correspondingly increased expression of HMGA2 and fibrotic genes in this model. Collectively, HMGA2 and let-7d miRNA significantly impact on the progression of TGF-β1-induced EMT and fibrogenesis both in vitro and in vivo, and they may represent novel targets for the prevention strategies of renal fibrosis in the context of DN.

MicroRNA Expression during Bovine Oocyte Maturation and Fertilization.

Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.

Ficus carica latex prevents invasion through induction of let-7d expression in GBM cell lines.

Glioblastoma multiforme (GBM) is one of the deadliest human malignancies. A cure for GBM remains elusive, and the overall survival time is less than 1 year. Thus, the development of more efficient therapeutic approaches for the treatment of these patients is required. Induction of tumor cell death by certain phytochemicals derived from medicinal herbs and dietary plants has become a new frontier for cancer therapy research. Although the cancer suppressive effect of Ficus carica (fig) latex (FCL) has been determined in a few cancer types, the effect of this latex on GBM tumors has not been investigated. Therefore, in the current study, the anti-proliferative activity of FCL and the effect of the FCL-temozolomide (TMZ) combination were tested in the T98G, U-138 MG, and U-87 MG GBM cell lines using the WST-1 assay. The mechanism of cell death was analyzed using Annexin-V/FITC and TUNEL assays, and the effect of FCL on invasion was tested using the chick chorioallantoic membrane assay. To determine the effect of FCL on GBM progression, the expression levels of 40 GBM associated miRNAs were analyzed in T98G cells using RT-qPCR. According to the obtained data, FCL causes cell death in GBM cells with different responses to TMZ, and this effect is synergistically increased in combination with TMZ. In addition, the current study is the first to demonstrate the effect of FCL on modulation of let-7d expression, which may be an important underlying mechanism of the anti-invasive effect of this extract.

Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7.

BackgroundMicroRNAs are endogenous small noncoding RNAs that are functionally involved in numerous critical cellular processes including tumorigenesis. Data mining using a microRNA array database suggested that let-7d microRNA may be associated with renal cell carcinoma (RCC) malignant progression. Here, we performed further analyses to determine whether let-7d is functionally linked to RCC malignancy.MethodsQuantitative real-time PCR was used to determine the level of mature let-7d in RCC clinical specimens and its correlation with clinicopathological data. Immunohistochemical staining was conducted to characterize the stroma of RCC. Let-7d overexpressing RCC cell lines combined with mouse models bearing cell-derived xenografts and patient-derived xenografts were used to assess the functional role of let-7d in vitro and in vivo.ResultsDownregulation of let-7d in clinical RCC samples was associated with advanced tumor grade and T stage and increased vascular invasion. An inverse relationship between let-7d expression and macrophage infiltration was found in clinical RCC samples. Functional studies indicated that ectopic expression of let-7d significantly inhibited RCC cell proliferation, migration, and peripheral blood monocyte (PBMC) recruitment in vitro, as well as tumor growth, metastasis, and tumor macrophage infiltration in vivo. In silico analysis and subsequent experimental validation confirmed collagen, type III, alpha 1 (COL3A1) and C-C subfamily chemokine member CCL7 as direct let-7d target genes. The addition of COL3A1 and CCL7 counteracted the inhibitory effects of let-7d on RCC cell proliferation, migration, and PBMC recruitment. The inhibition of let-7d increased cell proliferation, migration, and PBMC recruitment by the enhanced expression of COL3A1 and CCL7 genes in vitro. The mRNA levels of COL3A1 and CCL7 were inversely correlated with let-7d level in RCC clinical specimens.ConclusionsThese results suggest that let-7d may suppress RCC growth, metastasis, and tumor macrophage infiltration at least partially through targeting COL3A1 and CCL7.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-206) contains supplementary material, which is available to authorized users.

759: Ficus carica latex inhibits GBM cell proliferation by modulating let-7d expression

759: Ficus carica latex inhibits GBM cell proliferation by modulating let-7d expression

Mir-16 and 21 Deregulation Are Linked to Anti-Apoptotic Bcl-2 Gene Expression in Polycythemia Vera

AbstractAbstract 5057 Background:Polycythaemia vera (PV) is a clonal disorder characterized by an accumulation of normal red and white cells, platelets, and their progenitors in absence of a definable stimulus. Despite the advances in PV diagnosis its physiopathology remains not well elucidated. Apoptosis deregulation seems to contribute to disease establishment. MicroRNAs are small noncoding RNAs, post transcriptional repressors of genes by deadenilation, which play relevant roles in regulation of apoptotic machinery. Here we investigate the relation of apoptosis deregulation and apoptomirs expression in PV patients. Aims:To quantify the apoptomirs −16, −21, −15a, −26a, 130b, 29c and let-7d expression in peripheral leukocytes and CD34+ hematopoietic stem cells (HSC) in Polycythemia Vera (PV) patients and to correlate with targets genes expression data. Subjects and Methods:30 PV patients, 13 men and 17 women with a mean age (ma) of 64. 7y. 30 healthy subjects, 13 males and 17 females, ma=60. 2y and 23 bone marrow donors, 14 males and 9 females, ma=32. 6y. Peripheral leukocytes were obtained by Haes-Steril method and CD34+ hematopoietic stem cells were enriched by using the magnetically activated cell sorting, total RNA was extracted according to Trizol® method and High Capacity® Kit was used to synthesize cDNA. The microRNAs and apoptosis-related genes expression quantification was performed by real time PCR. Results were given as 2-ΔΔCt and statistical analyses were carried out by Mann-Whitney and Spearman tests. Western Blot was applied to detect Bcl-2 protein expression. Results:miR-16 and miR21 levels were increased in PV leukocytes (median= 3. 45 and 6. 97 respectively)) and CD34+ HSC (median= 3. 86 and 4. 07 respectively) in comparison to controls (control leukocytes median= 0. 483 and 0. 989 respectively) (CD34+ HSC median= 0. 978 and 1. 15, respectively) (p= 0. 0001 and 0. 0001 in leukocytes) and (p= 0. 0001 and 0. 0005 in CD34+ HSC, respectively). The Bcl-2 gene expression was decreased in PV leukocytes (m= 0.18) when compared to controls (m= 1. 03; p= 0. 019). In addition we found in CD34+ HSC negative correlation between miR-21 and anti-apoptotic gene Bcl-2 (r= −0. 417; p= 0. 033) and between miR16 and anti-apoptotic gene Bcl-2(r= −0. 384; p=0. 046). Bcl-2 western blot showed half expression in PV leukocytes when compared to controls. Conclusions:The results indicate the apoptomirs expression is linked to apoptosis deregulation in Polycythemia Vera physiopathology. Disclosures:No relevant conflicts of interest to declare.

H. pylori eradication did not improve dysregulation of specific oncogenic miRNAs in intestinal metaplastic glands

Many microRNAs (miRNAs) are differentially expressed in Helicobacter pylori-infected gastric mucosa and in gastric cancer tissue. We aimed to compare the effect of H. pylori eradication on gastric mucosal miRNAs in subjects in a high-risk group for gastric cancer compared to controls. Patients with a recent history of endoscopic resection for early gastric cancer and sex- and age-matched non-cancer controls were enrolled. The expression of 21 miRNAs was examined using gastric mucosal biopsy specimens and microdissected gastric glands from the lesser and greater curvatures of the gastric corpus both before and one year after H. pylori eradication. Twenty patients and 14 controls were enrolled. The expression of oncogenic miRNAs (miR-17/92 and the miR-106b-93-25 cluster, miR-21, miR-194, and miR-196) was significantly higher in the gastric mucosa of the cancer group than in the controls. H. pylori eradication resulted in a significant fall in the expression of oncogenic miRNAs only in the controls, whereas miR-223 expression was decreased and let-7d expression was increased in both groups. miR-196 was expressed only in intestinal metaplastic glands. The expression of oncogenic miRNAs was significantly higher in the intestinal metaplastic glands than in the non-intestinal metaplastic glands irrespective of H. pylori eradication. In neither group did H. pylori eradication significantly change any miRNA expression in the intestinal metaplastic glands. Dysregulation of specific miRNAs is present in H. pylori-induced corpus gastritis. H. pylori eradication improved miRNA dysregulation, but not in intestinal metaplastic glands or in the gastric mucosa of patients in a high-risk group for gastric cancer.

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

MYC Protein Inhibits Transcription of the MicroRNA Cluster MC-let-7a-1∼let-7d via Noncanonical E-box

The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.

Let-7d functions as novel regulator of epithelial-mesenchymal transition and chemoresistant property in oral cancer

Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide. Let-7 family has been shown to function as a tumor suppressor through regulating multiple oncogenic signaling. Recent study reported that combined underexpression of miR-205 and let-7d showed negative correlation with the survival prognosis of head and neck cancer patients. However, the let-7d-involved mechanism in regulating OSCC is still unclear. In this study, we first demonstrated that let-7d expression was significantly decreased while Twist and Snail expression was increased in OSCC cancer cell lines and primary cultures as compared to normal human oral keratinocyte cells. To further investigate the role of let-7d in OSCC, we applied the SPONGE method to knock down let-7d in OECM-1 and two primary OSCC cell types. The results showed that knockdown of let-7d promote epithelial-mesenchymal transition (EMT) traits and migratory/invasive capabilities in OSCC cells. Furthermore, down-expression of let-7d significantly activated Twist and Snail expressions and chemo-resistant abilities of OSCC cells. Notably, overexpression of let-7d effectively reversed the EMT phenotype, blocked migratory/invasive abilities, and further increased the chemosensitivity in oral cancer tumor initiating ALDH1+ cells. In sum, these results show that let-7d negatively modulates EMT expression and also plays a role in regulating chemo-resistant ability in oral cancer.

Regulation of MiR-124, Let-7d, and MiR-181a in the Accumbens Affects the Expression, Extinction, and Reinstatement of Cocaine-Induced Conditioned Place Preference

Molecular adaptations underlying drug seeking and relapse remain largely unknown. Studies highlight post-transcriptional modifications mediated by microRNAs (miRNAs) in addiction and other neurological disorders. We have previously shown that chronic cocaine suppresses miR-124 and let-7d and induces the expression of miR-181a in mesolimbic pathway. To further address the role and target gene regulation network of these miRNAs in vivo in cocaine addiction, we developed lentiviral vector (LV)-expressing miRNAs and their corresponding silencers for stable and regulatable miRNA expression. We tested in vivo miRNA gain and loss of function on cocaine-induced conditioned place preference (CPP) by localized LV-miRNA regulation in the nucleus accumbens (NAc). LV-miR-124 and let-7d expression in the NAc attenuates cocaine CPP, whereas LV-miR-181a enhances it. Silencing miRNAs by corresponding LV-miRNA silencers expressing perfect miRNA target sequences inversed this effect on cocaine CPP. Doxycycline treatment for switching off silencer expression abolished the observed behavioral changes. Behavioral changes mediated by LV-miRNA regulation resulted in dynamic alterations in transcription factors, receptors, and other effector genes involved in cocaine-induced plasticity. Our results describe a complex regulatory pathway mediated by miRNAs in cocaine-mediated neuronal adaptations.

Inhibition and Role of let-7d in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. To determine changes in expression and role of microRNAs in IPF. RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).