Target Of Let-7c Research Articles

CircRNA Profiling Reveals an Abundant circFUT10 that Promotes Adipocyte Proliferation and Inhibits Adipocyte Differentiation via Sponging let-7

Adipose development is regulated by a series of complex processes, and non-coding RNAs (ncRNAs), including circular RNAs (circRNAs), play important roles in regulating proliferation and differentiation of adipocytes. In this study, we profiled circRNA expression in cattle fat tissue during calf and adult developmental stages and detected 14,274 circRNA candidates. Some circRNAs are differentially expressed between two developmental stages. We characterized circFUT10, named for its host gene FUT10, a highly expressed and abundant circRNA. Luciferase screening, an RNA-binding protein immunoprecipitation (RIP) assay, quantitative real-time PCR, and western blotting assays indicated that circFUT10 directly binds let-7c/let-e, and PPARGC1B (peroxisome proliferator-activated receptor γ coactivator 1-β) is identified as a target of let-7c. Flow cytometry, EdU (5-ethynyl-2′-deoxyuridine) incorporation, a CCK-8 (cell counting kit-8) assay, oil red O staining, and western blotting assays demonstrated that circFUT10 promotes adipocyte proliferation and inhibits cell differentiation by sponging let-7c. The results demonstrate that circFUT10 binding of let-7c promotes cell proliferation and inhibits cell differentiation by targeting PPARGC1B in cattle adipocytes.

The let-7c/HoxB7 axis regulates the cell proliferation, migration and apoptosis in hepatocellular carcinoma.

Ectopic expression of HOX-containing genes is closely related to carcinogenesis, acting as either tumor suppressors or oncogenes. A preliminary bioinformatics analysis showed that HoxB7 is a possible target of let-7c. In this study, we aimed to investigate the relationship between HoxB7 and let-7c in liver carcinogenesis. We found that HoxB7 was upregulated in hepatocellular carcinoma (HCC) tissues and cells and negatively correlated with survival time, whereas let-7c was downregulated and positively correlated with survival time in patients with HCC. Let-7c overexpression suppressed proliferation, migration but promoted cell apoptosis in HCC cells. We validated that HoxB7 is a target of let-7c. Consistently, let-7c overexpression reversed the promotional effects of HoxB7 on proliferation and migration in HCC cells, and increased the cell apoptotic rate reduced by HoxB7. Furthermore, let-7c overexpression reversed the promotional effect of HoxB7 on tumor growth in subcutaneous HCC tumor model. Our data suggest that the let-7c/HoxB7 axis regulates HCC development, which may provide a novel therapeutic strategy for the treatment of HCC.

Let-7c regulates proliferation and osteodifferentiation of human adipose-derived mesenchymal stem cells under oxidative stress by targeting SCD-1.

Osteoporosis is a progressive bone disease characterized by decreased bone mass and density, which usually parallels a reduced antioxidative capacity and increased reactive oxygen species formation. Adipose-derived mesenchymal stem cells (ADMSCs), a population of self-renewing multipotent cells, are a well-recognized source of potential bone precursors with significant clinical potential for tissue regeneration. We previously showed that overexpressing stearoyl-CoA desaturase 1 (SCD-1) promotes osteogenic differentiation of mesenchymal stem cells. Micro-RNAs (miRNAs) are noncoding RNAs recently recognized to play key roles in many developmental processes, and miRNA let-7c is downregulated during osteoinduction. We found that let-7c was upregulated in the serum of patients with postmenopausal osteoporosis compared with healthy controls. Levels of let-7c during osteogenic differentiation of ADMSCs were examined under oxidative stress in vitro and found to be upregulated. Overexpression of let-7c inhibited osteogenic differentiation, whereas inhibition of let-7c function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase activity, and matrix mineralization. The luciferase reporter assay was used to validate SCD-1 as a target of let-7c. Further experiments showed that silencing of SCD-1 significantly attenuated the effect of let-7c inhibitor on osteoblast markers, providing strong evidence that let-7c modulates osteogenic differentiation by targeting SCD-1. Inhibition of let-7c promoted the translocation of β-catenin into nuclei, thus activating Wnt/β-catenin signaling. Collectively, these data suggest that let-7c is induced under oxidative stress conditions and in osteoporosis, reducing SCD-1 protein levels, switching off Wnt/β-catenin signaling, and inhibiting osteogenic differentiation. Thus, let-7c may be a potential therapeutic target in the treatment of osteoporosis and especially postmenopausal osteoporosis.

Let-7c restores radiosensitivity and chemosensitivity and impairs stemness in oral cancer cells through inhibiting interleukin-8.

The let-7 family of microRNAs has been considered as tumor suppressors in various cancers; however, the role of let-7c in oral squamous cell carcinoma has not been determined yet. In this study, phenotypical behaviors and the radio/chemoresistance were examined subsequent to overexpression of let-7c. In addition, the expression of let-7c in cancer stem cells (CSCs) was evaluated and the effect of let-7c on stemness characteristics was assessed. Also, luciferase activity assays were performed to test whether interleukin (IL)-8 was a putative target of let-7c. Our results confirmed that the expression of let-7c in CSCs was reduced, while overexpression of let-7c attenuated the oncogenicity. Moreover, ectopic expression of let-7c in CSCs downregulated the stemness hallmarks and the radio/chemoresistance. Expression and secretion of IL-8 in oral CSCs were both reduced following overexpression of let-7c. Besides, the inhibitory effect of let-7c on various stemness phenotypes was reverted by IL-8, indicating that lower expression of let-7c may confer higher cancer stemness through a failure to downregulate IL-8. These findings revealed the significance of let-7c in the contribution of oral cancer stemness and radio/chemoresistance. Targeting let-7c and its downstream IL-8 may be beneficial to prevent cancer recurrence and metastasis of oral squamous cell carcinoma.

Let-7c miRNA Inhibits the Proliferation and Migration of Heat-Denatured Dermal Fibroblasts Through Down-Regulating HSP70.

Wound healing is a complex physiological process necessitating the coordinated action of various cell types, signals and microRNAs (miRNAs). However, little is known regarding the role of miRNAs in mediating this process. In the present study, we show that let-7c miRNA is decreased in heat-denatured fibroblasts and that inhibiting let-7c expression leads to the increased proliferation and migration of dermal fibroblasts, whereas the overexpression of let-7c exerts an opposite effect. Further investigation has identified heat shock protein 70 as a direct target of let-7c and has demonstrated that the expression of HSP70 in fibroblasts is negatively correlated with let-7c levels. Moreover, down-regulation of let-7c expression is accompanied by up-regulation of Bcl-2 expression and down-regulation of Bax expression, both of which are the downstream genes of HSP70. Notably, the knockdown of HSP70 by HSP70 siRNA apparently abrogates the stimulatory effect of let-7c inhibitor on heat-denatured fibroblasts proliferation and migration. Overall, we have identified let-7c as a key regulator that inhibits fibroblasts proliferation and migration during wound healing.

MicroRNA let-7c Inhibits Cell Proliferation and Induces Cell Cycle Arrest by Targeting CDC25A in Human Hepatocellular Carcinoma.

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3ʹ UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.

Let-7c inhibits metastatic ability of mouse hepatocarcinoma cells via targeting mannoside acetylglucosaminyltransferase 4 isoenzyme A

Aberrant glycosylation may promote tumor invasion and metastasis. To investigate whether microRNA (miRNA) is involved in glycosylation-related metastasis, we examined the role of let-7c, a well-known tumor-suppressor miRNA, in glycosylation in murine hepatocarcinoma cell lines Hca-F and Hca-P. We found that let-7c level was higher in Hca-P cells (with lower lymphatic metastasis potential) than in Hca-F cells (with higher lymphatic metastasis potential). Overexpression of let-7c decreased hyper-N-glycosylation of Hca-F cells and repressed their metastatic and invasive ability. Mannoside acetylglucosaminyltransferase 4, isoenzyme A (Mgat4a) is a key glycosyltransferase in the pathway of synthesizing complex N-glycans. Bioinformatics analysis indicates that Mgat4a may be a target of let-7c, which has been verified by dual-luciferase reporter gene assay. Furthermore, the anti-metastatic effect of overexpressed let-7c is similar to that of Mgat4a siRNAs transfection. Hence, our results suggest that let-7c may inhibit the metastatic ability of Hca-F cells, at least partially, via repressing Mgat4a activity.

Cardiomyogenesis is controlled by the miR-99a/let-7c cluster and epigenetic modifications

Understanding the molecular basis of cardiomyocyte development is critical for understanding the pathogenesis of pre- and post-natal cardiac disease. MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression that play an important role in many developmental processes. Here, we show that the miR-99a/let-7c cluster, mapping on human chromosome 21, is involved in the control of cardiomyogenesis by altering epigenetic factors. By perturbing miRNA expression in mouse embryonic stem cells, we find that let-7c promotes cardiomyogenesis by upregulating genes involved in mesoderm specification (T/Bra and Nodal) and cardiac differentiation (Mesp1, Nkx2.5 and Tbx5). The action of let-7c is restricted to the early phase of mesoderm formation at the expense of endoderm and its late activation redirects cells toward other mesodermal derivatives. The Polycomb complex group protein Ezh2 is a direct target of let-7c, which promotes cardiac differentiation by modifying the H3K27me3 marks from the promoters of crucial cardiac transcription factors (Nkx2.5, Mef2c, Tbx5). In contrast, miR-99a represses cardiac differentiation via the nucleosome-remodeling factor Smarca5, attenuating the Nodal/Smad2 signaling. We demonstrated that the identified targets are underexpressed in human Down syndrome fetal heart specimens. By perturbing the expression levels of these miRNAs in embryonic stem cells, we were able to demonstrate that these miRNAs control lineage- and stage-specific transcription factors, working in concert with chromatin modifiers to direct cardiomyogenesis.

Let-7c Inhibits NSCLC Cell Proliferation by Targeting HOXA1

The aim of the present study was to explore mechanisms by which let-7c suppresses NSCLC cell proliferation. The expression level of let-7c was quantified by qRT-PCR. A549 and H1299 cells were transfected with let-7c mimics to restore the expression of let-7c. The effects of let-7c were then assessed by cell proliferation, colony formation and cell cycle assay. Mouse experiments were used to confirm the effect of let-7c on tumorigenicity in vivo. Luciferase reporter assays and Western blotting were performed to identify target genes for let-7c. HOXA1 was identified as a novel target of let-7c. MTS, colony formation and flow cytometry assays demonstrated that forced expression of let-7c inhibited NSCLC cell proliferation by inducing G1 arrest in vitro, consistent with inhibitory effects induced by knockdown of HOXA1. Mouse experiments demonstrated that let-7c expression suppressed tumorigenesis. Furthermore, we found that let-7c could regulate the expression of HOXA1 downstream effectors CCND1, CDC25A and CDK2. Collectively, these results demonstrate let-7c inhibits NSCLC cell proliferation and tumorigenesis by partial direct targeting of the HOXA1 pathway, which suggests that restoration of let-7c expression may thus offer a potential therapeutic intervention strategy for NSCLC.

MicroRNA let-7c inhibits Bcl-xl expression and regulates ox-LDL-induced endothelial apoptosis

Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play a critical role in atherosclerosis. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. MiRNA let-7 family is known to be involved in the regulation of cell apoptosis. However, the function of let-7 in ox-LDL induced ECs apoptosis and atherosclerosis is still unknown. Here, we show that let-7c expression was markedly up-regulated in ox-LDL induced apoptotic human umbilical cord vein endothelial cells (HUVECs). Let-7c over-expression enhanced apoptosis in ECs whereas inhibition of let-7c could partly alleviate apoptotic cell death mediated by ox-LDL. Searching for how let-7c affected apoptosis, we discovered that antiapoptotic protein Bcl-xl was a direct target of let-7c in ECs. Our data suggest that let-7c contributes to endothelial apoptosis through suppression of Bcl-xl.

Abstract 5442: MicroRNAs may mediate multigenerational epigenetic inheritance of mammary cancer risk caused by maternal exposure to high-fat diet during pregnancy

Abstract Maternal dietary exposure to high-fat (HF) during pregnancy increases mammary cancer risk in daughters and granddaughters. It also causes persistent changes in mammary gland morphology in these two HF generations. Our results further show that an increase in mammary cancer risk can be transmitted to HF granddaughters either through the female or male germlines. Disease traits resulting from environmental in utero exposures can be transmitted through multiple generations by epigenetic means. Here, we investigated whether misregulation of expression of specific miRNAs may be involved in mediating the effects of maternal HF exposure during pregnancy on mammary cancer risk in multiple generations of offspring. Pregnant Sprague-Dawley rats were fed isocaloric control or HF diets (fat source: corn oil) throughout pregnancy. The effects on mammary cancer risk in daughters and granddaughters were examined and reported earlier. Total RNA was obtained from mammary glands on postnatal day (PND) 50 from control and HF daughters (F1 generation) and granddaughters (F2 generation). MicroRNA levels were measured using Applied Biosystems TaqManR Array Rodent MicroRNA Card Set v2.0. mRNA expression levels were assessed using q-PCR. Maternal HF exposure during pregnancy increased carcinogen-induced mammary tumorigenesis in both daughters and granddaughters (p=0.049 and p=0.028, respectively). Higher mammary tumor incidence was also observed in the F2 generation even if only one parent had been exposed to HF diet in utero. Fifty one miRNAs were significantly down-regulated on PND50 in mammary glands of HF daughters compared to controls. From these, five miRNAs were also down-regulated in mammary tissue of HF granddaughters compared to controls: let-7c, miR-7a, miR-17, miR-299 and miR-770. Down-regulation of miR-7a and miR-770 was transmitted only through the male germline; i.e., they were expressed at a significantly lower level in F2 daughters of in utero HF exposed fathers, but not HF mothers. Mammary mRNA levels of the polycomb gene, EZH2, a target of let-7c, were up-regulated in both HF daughters and granddaughters compared to controls (p=0.014). Our findings indicate that maternal exposure to HF diet during pregnancy causes a persistent suppression of some miRNAs in the offspring's mammary gland that is transmitted to next generation. The down-regulation of some of these miRNAs was transmitted through the male germline. Moreover, up-regulation of EZH2 transcript, a target gene for let-7c, in mammary tissue of HF daughters and granddaughters, confirms the functional consequence of suppression of this miRNA. Additional studies are needed to elucidate the consequences of suppression of other miRNAs uncovered in this study, and whether they are causally related to increased mammary cancer risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5442. doi:1538-7445.AM2012-5442

Differential Expression of miRNAs during the Course of Chronic Myeloid Leukemia.

Despite the high efficiency of CML therapy with tyrosine kinase inhibitor imatinib (Gleevec®, STI571), there are 20–30% of patients with suboptimal response or therapy failure. There are several known mechanisms of resistance (e.g. mutation in BCR-ABL kinase domain, BCR-ABL or hOCT-1 overexpression), while others are still uncovered. Aberrant expression of specific miRNAs might contribute to the resistance and disease progression, or could be the consequence of the disease status. Our aim was to screen the expression of known miRNAs in patient samples of different stages of CML treated with imatinib: 1. Major molecular response (MMR), 2. Suboptimal response/failure (not achieved cytogenetic response after 12 months of STI); 3. Start of hematological relapse; 4. Blast crisis (BC); 5. Diagnosis. Groups were created according to defined parameters (ELN definition) and were precisely characterized by BCR-ABL transcript level, cytogenetic, hematological and clinical examinations. Each group was represented by pooled leukocytes from peripheral blood of 5 patients, who contributed to the pool with the same number of cells; total pool concentration was 10x106 leukocytes/ml. Altogether 25 patient samples were tested. A pool of total leukocytes from 13 healthy donors was used as a control. Total RNA was isolated from the homogenate (5x106 leukocytes) of pooled samples. The RNA from the diagnosis pool was isolated in duplicate to check the technical variability of the assay. The miRNA expression was measured using the PIQORTM miRXplore arrays (Miltenyi Biotech) with 780 probes. Mean signal and mean local background intensities were obtained for each spot of the microarray images using the ImaGene® (Biodiscovery). Raw data derived from ImaGene software were processed with the PIQOR Analyzer (Miltenyi Biotech). As expected, most differentially expressed miRNAs (25 miRNAs with more than 2-fold change to control) were found in blast crisis. In BC we found the overexpression of polycistronic and oncogenic miR-17-92 cluster (miR-17, miR-19a, miR- 20a, miR-19b, and miR-92), which has been described in CD34+ cells from CML patients (Venturini et al.2007, Blood, 109 (10): 4399). Other important miRNAs that play roles in hematopoiesis were overexpressed in BC: e.g. miR-181a (16 fold), miR-222 (6 fold), miR-106a (3.5 fold), miR-221 (3 fold). Interestingly, the quantity of specific miRNAs differed in the context of a disease status. MiR-150 was downregulated at diagnosis, which was not observed in MMR and suboptimal response/failure. The downregulation appeared again at the start of hematological relapse (3 fold) and BC (10 fold). The miR-150 target is Myb oncogene encoding transcription factor required for proliferation, differentiation and survival of normal and leukemic hematopoietic cells. C-Myb has a longer half-life in BCR-ABL expressing cells than in normal ones. At the start of the hematological relapse and blast crisis let7c was upregulated but in MMR and suboptimal response/failure let7c level was equal to the control sample. The known target of let7c is c-Myc that in CML contributes to tumorigenesis. MiR-126 was downregulated in MMR, suboptimal response/ failure and start of hematological relapse, but in blast crisis it was overexpressed. The predicted miR-126 target is the antiapoptotic heat shock protein hsp70. It was shown that BCR-ABL expression results in the upregulation of hsp70. Attenuation of hsp70 levels either by imatinib or hsp70 antisense sensitized K562 cells to e.g. cytarabine and etoposide (Ossenkoppele 2005, Blood, 105 (3): 917). In conclusion, it seems that miR-150, 126 and let7c reflect the disease status and could become new possible markers in CML. Supported by GACR GA301/08/P154.