MicroRNA Let-7a Inhibits Proliferation of Human Prostate Cancer Cells In Vitro and In Vivo by Targeting E2F2 and CCND2

Previous work has shown reduced expression levels of let-7 in lung tumors. But little is known about the expression or mechanisms of let-7a in prostate cancer. In this study, we used in vitro and in vivo approaches to investigate whether E2F2 and CCND2 are direct targets of let-7a, and if let-7a acts as a tumor suppressor in prostate cancer by down-regulating E2F2 and CCND2.Findings Real-time RT-PCR demonstrated that decreased levels of let-7a are present in resected prostate cancer samples and prostate cancer cell lines. Cellular proliferation was inhibited in PC3 cells and LNCaP cells after transfection with let-7a. Cell cycle analysis showed that let-7a induced cell cycle arrest at the G1/S phase. A dual-luciferase reporter assay demonstrated that the 3′UTR of E2F2 and CCND2 were directly bound to let-7a and western blotting analysis further indicated that let-7a down-regulated the expression of E2F2 and CCND2. Our xenograft models of prostate cancer confirmed the capability of let-7a to inhibit prostate tumor development in vivo.These findings help to unravel the anti-proliferative mechanisms of let-7a in prostate cancer. Let-7a may also be novel therapeutic candidate for prostate cancer given its ability to induce cell-cycle arrest and inhibit cell growth, especially in hormone-refractory prostate cancer

Identifying the Effects of Unprocessed let-7a-1 and let-7a-3 in Non-Small Cell Lung Cancer

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate protein levels typically by interacting with the 3′ untranslated region (3′-UTR) of target messenger RNA (mRNAs) and are often aberrantly expressed in cancer. The let-7 miRNA family members are commonly regarded as cancer suppressors, by down-regulating the expression of oncoproteins such as RAS, HMGA2, and MYC. However, prior work indicates that unprocessed let-7 RNAs may be positively correlated with cancer phenotypes in lung cancer cell lines. Our study aims to identify the effects of unprocessed let-7a-1 and let-7a-3 in non-small cell lung cancer, by transfecting plasmids that express unprocessed let-7a-1 and let-7a-3 into 3 different lung cancer cell lines. We then proceeded to conduct functional assays to measure the differences in anchorage independent growth, cell proliferation, and cell migration in all cell lines transfected with unprocessed let-7, in contrast to cells transfected with a control vector and thus far determined that unprocessed let-7a-1 can enhance anchorage independent growth. Thus, we created truncations of the let-7a-1 miRNA to identify the cis regions of this miRNA that is responsible for the change in phenotype. Our results suggest that cells transfected with truncated, yet unprocessed let-7a-1 have increased anchorage independent growth, a major hallmark of cancer cell. There is still a need to replicate the functional assays that were conducted while continuing to create constructs of both let-7a-1 and let-7a-3 in order to further identify the sequence of the miRNAs responsible for the enhanced cancer phenotypes

Involvement of microRNA Lethal-7a in the Regulation of Embryo Implantation in Mice

MicroRNAs interact with multiple mRNAs resulting in their degradation and/or translational repression. This report used the delayed implantation model to determine the role of miRNAs in blastocysts. Dormant blastocysts in delayed implanting mice were activated by estradiol. Differential expression of 45 out of 238 miRNAs examined was found between the dormant and the activated blastocysts. Five of the nine members of the microRNA lethal-7 (let-7) family were down-regulated after activation. Human blastocysts also had a low expression of let-7 family. Forced-expression of a family member, let-7a in mouse blastocysts decreased the number of implantation sites (let-7a: 1.1±0.4; control: 3.8±0.4) in vivo, and reduced the percentages of blastocyst that attached (let-7a: 42.0±8.3%; control: 79.0±5.1%) and spreaded (let-7a: 33.5±2.9%; control: 67.3±3.8%) on fibronectin in vitro. Integrin-β3, a known implantation-related molecule, was demonstrated to be a target of let-7a by 3′-untranslated region reporter assay in cervical cancer cells HeLa, and Western blotting in mouse blastocysts. The inhibitory effect of forced-expression of let-7a on blastocyst attachment and outgrowth was partially nullified in vitro and in vivo by forced-expression of integrin-β3. This study provides the first direct evidence that let-7a is involved in regulating the implantation process partly via modulation of the expression of integrin-β3. (200 words)

Cloning and characterization of the 5' flanking region of microRNA let-7a-1/let-7f-1 gene cluster in human lung cancer cell

In order to elucidate the molecular basis of microRNA let-7a-1/let-7f-1 gene cluster, the transcription initiation site which was determined by 5’ rapid amplification of cDNA ends (5’RACE) and 2.1 kb of the 5' flanking region proximal to the pre-let-7a-1 was isolated and characterized. The promoter activity of the 2.1 kb fragment was analyzed by a firefly luciferase-encoding gene expression vector (pGL3) transiently transfected into lung cancer cell line A549. The 2.1 kb promoter of let-7a-1/let-7f-1 displayed a lower activity and was significantly enhanced by ectopic expression of c/EBPα or p53 and treatment with dexamethasone. Despite the induction of other let-7 family members such as let-7a-3, let-7c and let-7d, all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cRA) display little enhancement effect on 2.1 kb promoter of let-7a-1/let-7f-1, as well as 1,25-(OH)2D3.Key words: let-7a-1; let-7f-1, 5’ rapid amplification of cDNA ends (5’RACE), promoter, lung cancer

MicroRNA let-7a suppresses breast cancer cell migration and invasion through downregulation of C-C chemokine receptor type 7.

INTRODUCTION: C-C chemokine receptor type 7 (CCR7) plays an important role in chemotactic and metastatic responses in various cancers, including breast cancer. In the present study, the authors demonstrated that microRNA (miRNA) let-7a downregulates CCR7 expression and directly influences the migration and invasion of breast cancer cells. METHODS: The expression of CCR7, its ligand CCL21, and let-7a was detected in breast cancer cell lines and in breast cancer patient tissues. Synthetic let-7a and an inhibitor of let-7a were transfected into MDA-MB-231 and MCF-7 breast cancer cells, respectively, and cell proliferation, cell migration, and invasion assays were performed. To confirm the fact that 3'UTR of CCR7 is a direct target of let-7a, a luciferase assay for the reporter gene expressing the let-7a binding sites of CCR7 3'UTR was used. An in vivo invasion animal model system using transparent zebrafish embryos was also established to determine the let-7a effect on breast cancer cell invasion. RESULTS: First, a higher expression of both CCR7 and CCL21 in malignant tissues than in their normal counterparts from breast cancer patients was observed. In addition, a reverse correlation in the expression of CCR7 and let-7a in breast cancer cell lines and breast cancer patient tissues was detected. Synthetic let-7a decreased breast cancer cell proliferation, migration, and invasion, as well as CCR7 protein expression in MDA-MB-231 cells. The let-7a inhibitor reversed the let-7a effects on the MCF-7 cells. The 3'UTR of CCR7 was confirmed as a direct target of let-7a by using the luciferase assay for the reporter gene expressing let-7a CCR7 3'UTR binding sites. Notably, when analyzing in vivo invasion, MDA-MB 231 cells after synthetic let-7a transfection were unable to invade the vessels in zebrafish embryos. CONCLUSIONS: The results from the present study suggest that targeting of CCL21-CCR7 signaling is a valid approach for breast cancer therapy and that let-7a directly binds to the 3'UTR of CCR7 and blocks its protein expression, thereby suppressing migration and invasion of human breast cancer cells. Furthermore, the present study underscores the therapeutic potential of let-7a as an antitumor and antimetastatic manager in breast cancer patients.ope

Regulation of the let-7a-3 Promoter by NF-κB

Changes in microRNA expression have been linked to a wide array of pathological states. However, little is known about the regulation of microRNA expression. The let-7 microRNA is a tumor suppressor that inhibits cellular proliferation and promotes differentiation, and is frequently lost in tumors. We investigated the transcriptional regulation of two let-7 family members, let-7a-3 and let-7b, which form a microRNA cluster and are located 864 bp apart on chromosome 22q13.31. Previous reports present conflicting data on the role of the NF-κB transcription factor in regulating let-7. We cloned three fragments upstream of the let-7a-3/let-7b miRNA genomic region into a plasmid containing a luciferase reporter gene. Ectopic expression of subunits of NF-κB (p50 or p65/RelA) significantly increased luciferase activity in HeLa, 293, 293T and 3T3 cells, indicating that the let-7a-3/let-7b promoter is highly responsive to NF-κB. Mutation of a putative NF-κB binding site at bp −833 reduced basal promoter activity and decreased promoter activity in the presence of p50 or p65 overexpression. Mutation of a second putative binding site, at bp −947 also decreased promoter activity basally and in response to p65 induction, indicating that both sites contribute to NF-κB responsiveness. While the levels of the endogenous primary let-7a and let-7b transcript were induced in response to NF-κB overexpression in 293T cells, the levels of fully processed, mature let-7a and let-7b miRNAs did not increase. Instead, levels of Lin-28B, a protein that blocks let-7 maturation, were induced by NF-κB. Increased Lin-28B levels could contribute to the lack of an increase in mature let-7a and let-7b. Our results suggest that the final biological outcome of NF-κB activation on let-7 expression may vary depending upon the cellular context. We discuss our results in the context of NF-κB activity in repressing self-renewal and promoting differentiation

Methylation in MIRLET7A3 Gene Induces the Expression of IGF-II and Its mRNA Binding Proteins IGF2BP-2 and 3 in Hepatocellular Carcinoma

miR-let-7a is a tumor suppressor miRNA with reduced expression in most cancers. Methylation of MIRLET7A3 gene was reported to be the cause of this suppression in several cancers; however, it was not explicitly investigated in hepatocellular carcinoma (HCC). We aimed at investigating miR-let-7a expression and molecular mode in HCC, identifying drug-targetable networks, which might be affected by its abundance. Our results illustrated a significant repression of miR-let-7a, which correlated with hypermethylation of its gene of origin MIRLRT7A3. This was further supported by the induction of miR-let-7a expression upon treatment of HCC cells with a DNA-methyltransferase inhibitor. Using a computational approach, insulin-like growth factor (IGF)-II and IGF-2 mRNA binding proteins (IGF2BP)-2/-3 were identified as potential targets for miR-let-7a that was further confirmed experimentally. Indeed, miR-let-7a mimics diminished IGF-II as well as IGF2BP-2/-3 expression. Direct binding of miR-let-7a to each respective transcript was confirmed using a luciferase reporter assay. In conclusion, this study suggests that DNA hypermethylation leads to epigenetic repression of miR-let-7a in HCC cells, which induces the oncogenic IGF-signaling pathway

Investigation and identification of let-7a related functional proteins in gastric carcinoma by proteomics

Abstract. MicroRNAs are small noncoding RNA molecules that control expression of target genes. Our previous studies show that let-7a decreased in gastric carcinoma and that up-regulation of let-7a by gene augmentation inhibited gastric carcinoma cell growth both in vitro and in vivo, whereas it remains largely unclear as to how let-7a affects tumor growth. In this study, proteins associated with the function of let-7a were detected by high throughout screening. The cell line of SGC-7901 stablely overexpressing let-7a was successfully established by gene cloning. Two-dimensional gel electrophoresis (2-DEy was used to separate the total proteins of SGC-7901/let-7a, SGC-7901/EV and SGC-7901, and PDQuest software was applied to analyze 2-DE images. Ten different protein spots were identified by MALDI-TOF-MS, and they may be the proteins associated with let-7a function. The overexpressed proteins included Antioxidant protein 2, Insulin-like growth factor binding protein 2, Protein disulfide isomerase A2, C-1-tetrahydrofolate synthase, Cyclin-dependent kinase inhibitor1 (CDKN1) and Rho-GTPase activating protein 4. The underexpressed proteins consisted of S-phase kinase-associated protein 2 (Spk2), Platelet membrane glycoprotein, Fibronectin and Cks1 protein. Furthermore, the different expression levels of the partial proteins (CDKN1, Spk2 and Fibronectin) were confirmed by western blot analysis. The data suggest that these differential proteins are involved in a novel let-7a signal pathway and these findings provide the basis to investigate the functional mechanisms of let-7a in gastric carcinoma

TGF-ß induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression.

Abstract TGF-ß/Activin induces epithelial-to-mesenchymal transition (EMT) and stemness in pancreatic ductal adenocarcinoma (PDAC). However, the microRNAs (miRNAs) regulated during this response have remained yet undetermined. Here, we show that TGF-ß transcriptionally induces MIR100HG lncRNA, containing miR-100, miR-125b and let-7a in its intron, via SMAD2/3. Interestingly, we find that although the pro-tumourigenic miR-100 and miR-125b accordingly increase, the amount of anti-tumourigenic let-7a is unchanged, as TGF-ß also induces LIN28B inhibiting its maturation. Notably, we demonstrate that inactivation of miR-125b or miR-100 affects the TGF-ß-mediated response indicating that these miRNAs are important TGF-ß effectors. We integrated AGO2-RIP-seq with RNA-seq to identify the global regulation exerted by these miRNAs in PDAC cells. Transcripts targeted by miR-125b and miR-100 significantly overlap and mainly inhibit p53 and cell-cell junctions’ pathways. Together, we uncover that TGF-ß induces an lncRNA, whose encoded miRNAs, miR-100, let-7a and miR-125b, play opposing roles in controlling PDAC tumourigenesis

MicroRNA-Let-7a regulates the function of microglia in inflammation

Microglia have multiple functions in cerebrovascular and neurodegenerative diseases. Regulation of microglial function during inflammatory stress is important for treatment of central nervous system (CNS) diseases because microglia secrete various substances that affect neurons and glia. MicroRNA-Let-7a (miR-Let-7a) is a tumor suppressor miRNA that has been reported to target transcripts that encode proteins involved in apoptosis. In the present study, we examined the essential role of miR-Let-7a in inflammatory stress by over-expressing miR-Let-7a to investigate its role in determining the BV2 microglial phenotype, a cell line often used as a model of activated microglia. We found that inflammatory factors and Reactive Oxygen Species (ROS) production levels were altered according to miR-Let-7a expression level as measured by Western blot analysis, reverse transcription PCR, quantitative real time PCR, the measurement of nitrite (indicative of the nitric oxide (NO) pathway), and immunocytochemistry (ICC). Our results suggest that miR-Let-7a is involved in the function of microglia in the setting of inflammatory injury. In response to inflammation, miR-Let-7a participates in the reduction of nitrite production and the expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-6 and is involved in increased expression of brain derived neurotrophic factor (BDNF), interleukin (IL)-10, and IL-4 in microglia. Thus, miRNA-Let-7a could act as a regulator of the function of microglia in inflammation.ope