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Abstract Non-small-cell lung carcinoma (NSCLC) is a type of epithelial lung cancer accounting for about 85% of all lung cancers. In our research, a novel lupene derivative namely acetoxy-lup-5(6), 20(29)-diene (ALUP), as well as two known triterpenes; lupeol (LUP) and betulinic acid (BA) were isolated through the chromatographic purification of the 95% ethanolic extract of Thymus capitatus. Identification of the compounds was carried out by physicochemical properties as well as spectral 1D and 2D NMR analysis. The anti-cancer activity of the three triterpenes was assessed on non-small cell lung cancer cell line; A549 using MTT assay and cell cycle analysis using annexin V/propidium iodide. The molecular mechanism underlying anti-apoptotic effects was determined by analyzing Let-7 miRNA and miRNA-21 expression, the mRNA gene expression level of Bax, CASP-8, CD95, Bcl2, KRAS, VEGF, Cyclin D1 using qRT-PCR. Our results revealed that the three isolated compounds ALUP, LUP, and BA caused cell cycle arrest at the G2/M phase with an increase in the apoptosis which may be attributed to their significant effect on raising Bax, CASP-8, and CD95 and reducing the mRNA expression levels of Bcl-2, KRAS, VEGF, and Cyclin D1 compared to control cells. RT-PCR results showed that the ALUP, LUP, and BA significantly downregulated miRNA-21 expression. Meanwhile, the three compounds caused significant overexpression of Let-7 miRNA. This is the first report on the anti-cancer activity of acetoxy-lup-5(6), 20(29)-diene (ALUP) in reducing the proliferation and differentiation of the A549 cell line through inducing apoptosis. Finally, by targeting the Let-7 miRNA/Cyclin D1/VEGF cascade, acetoxy-lup-5(6), 20(29)-diene could be a potential therapeutic agent for lung cancer.

miRNA biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryo-EM and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "fUN" motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a noncanonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC-motif and Drosha's PAZ-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP., Competing Interests: Declaration of interest Authors declare no competing financial interest.

Recently, breast cancer stem cells (BCSCs) have rapidly emerged as a novel target for the therapy of breast cancer as they play critical roles in tumor growth, maintenance, metastasis, and recurrence. Let-7 miRNA is known to be downregulated in a variety of cancers, especially BCSCs, whereas CDK4 being overexpressed in human epidermal growth factor receptor 2 (HER-2) overexpressing tumor cells. In this study, let-7 miRNA and CDK4-specific siRNA were chosen as therapeutic agents and co-encapsulated in Herceptin-conjugated cationic liposomes for breast cancer therapy. Particle size, zeta potential, and encapsulation efficacy of mi/siRNA-loaded PEGylated liposome conjugated with Herceptin (Her-PEG-Lipo-mi/siRNA) were 176 nm, 28.1 mV, and 99.7% ± 0.1%, respectively. Enhanced cellular uptake (86%) was observed by fluorescence microscopy when SK-BR-3 cells were treated with Her-PEG-Lipo-mi/siRNA. Also, the increased amount of let-7a mRNA and decreased amount of cellular CDK4 mRNA were observed by qRT-PCR when SK-BR-3 cells were treated with Her-PEG-Lipo-mi/siRNA, which was even more so when SK-BR-3 stem cells were used (197 vs 768 times increase for let-7a, 62% vs 68% decrease for CDK4). Growth inhibition (65%) and migration arrest (0.5%) of the cells were achieved by the treatment of the cells with Her-PEG-Lipo-mi/siRNA, but not with mi/siRNA complex or other formulations. In conclusion, an efficient liposomal delivery system for the combination of miRNA and siRNA to target the BCSCs was developed and could be used as an efficacious therapeutic modality for breast cancer.

Abstract Background In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19–40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts. Methods The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins. Results The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged. Conclusions Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.

Single-strand selective uracil-DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A number of long noncoding RNAs (lncRNAs) have been identified in silkworm, but little is known about their functions. Recent study showed that the let-7 miRNA cluster (contains let-7, miR-2795, and miR-100) was transcribed from the last exon of lncRNA lncR17454 in silkworm. To investigate the functional role of lncR17454, dsRNAs of lncR17454 were injected into the hemolymph of 1-d-old third-instar larvae of Bombyx mori , repression of lncR17454 led to molting arrestment during the larval–larval and larval–pupal transition of silkworm, which was consistent to the result as let-7 knockdown in other studies. The expression level of mature let-7, miR-100, and miR-2795 decreased 40%, 36%, and 40%, respectively, while the mRNA level of two predicted target genes of let-7, the Broad Complex isoform 2 (BR-C-Z2) and the BTB-Zinc finger transcription repression factor gene Abrupt (Ab), increased significantly after lncR17454 knockdown. In contrast, when adding the 20-Hydroxyecdysone (20E) to silkworm BmN4 cell lines, the expression level of lncR17454 and let-7 cluster all increased significantly, but the expression of Abrupt , the predicted target gene of let-7, was repressed. Dual-luciferase reporter assays confirmed Abrupt was the real target of let-7. Here we found that the lncRNA lncR17454 can play regulator roles in the metamorphosis of silkworm through let-7 miRNA cluster and the ecdysone signaling pathway, which will provide new clues for lepidopteran pest control. [ABSTRACT FROM AUTHOR]

miRNA is a promising class of biomarkers whose levels can be assayed to detect various forms of cancer and other serious diseases. These short, noncoding nucleic acids are difficult to detect due to their low abundance and the marginal stability of their duplexes with DNA probes. In addition, miRNAs within the same family have high sequence homology, and often, related miRNA differ in sequence by only a single base. In this report, we demonstrate an independent detection seven members of the let-7 family of miRNA in a single run. Key to success is the use of mini-PEG-substituted PNA amphiphiles (γPNAA) and highly fluorescent DNA nanotags in micelle tagging electrophoresis (MTE). Multiplexed detection is accomplished in capillary electrophoresis (CE) using oligomeric nanotags of pre-programmed lengths where the presence of a specific miRNA links its nanotag to a micelle drag-tag, which shifts the nanotag elution time to a defined region for detection. We further demonstrate that the peak shape and elution time are unaffected by the presence of up to 10 mg/ml of serum protein in the sample, with a total runtime of less than 4 min and a LOD of 10-100 pM. We discuss efforts to substantially decrease the detection limit using nanotags that are >1000 bp in length., (© 2021 Wiley Periodicals LLC.)

Medulloblastoma (MB) is the most common pediatric embryonal brain tumor. The current consensus classifies MB into four molecular subgroups: sonic hedgehog-activated (SHH), wingless-activated (WNT), Group 3, and Group 4. MYCN and let-7 play a critical role in MB. Thus, we inferred the activity of miRNAs in MB by using the ActMiR procedure. SHH-MB has higher MYCN expression than the other subgroups. We showed that high MYCN expression with high let-7 activity is significantly associated with worse overall survival, and this association was validated in an independent MB dataset. Altogether, our results suggest that let-7 activity and MYCN can further categorize heterogeneous SHH tumors into more and less-favorable prognostic subtypes, which provide critical information for personalizing treatment options for SHH-MB. Comparing the expression differences between the two SHH-MB prognostic subtypes with compound perturbation profiles, we identified FGFR inhibitors as one potential treatment option for SHH-MB patients with the less-favorable prognostic subtype.

Most human genes are associated with promoters embedded in non-methylated, G + C-rich CpG islands (CGIs). Not all CGIs are found at annotated promoters, however, raising the possibility that many serve as promoters for transcripts that do not code for proteins. To test this hypothesis, we searched for novel transcripts in embryonic stem cells (ESCs) that originate within orphan CGIs. Among several candidates, we detected a transcript that included three members of the let-7 micro-RNA family: Let-7a-1, let-7f-1, and let-7d. Deletion of the CGI prevented expression of the precursor RNA and depleted the included miRNAs. Mice homozygous for this mutation were sub-viable and showed growth and other defects. The results suggest that despite the identity of their seed sequences, members of the let-7 miRNA family exert distinct functions that cannot be complemented by other members.

miRNAs are small noncoding RNAs capable of regulating gene expression at the post-transcriptional level. A growing body of evidence demonstrated that let-7 family of miRNAs, as one of the highly conserved miRNAs, plays an important role in cell differentiation and development, as well as tumor suppressor function depending on their levels of expression. To explore the physiological significance of let-7 in regulating cell fate decisions, we present a coarse grained model of let-7 biogenesis network, in which let-7 and its regulator Lin28 inhibit mutually. The dynamics of this minimal network architecture indicates that, as the concentration of Lin28 increases, the system undergoes a transition from monostability to a bistability and then to a one-way switch with increasing strength of positive feedback of let-7, while in the absence of Lin28 inhibition, the system loses bistability. Moreover, the ratio of degradation rates of let-7 and Lin28 is critical for the switching sensitivity and resistance to stimulus fluctuations. These findings may highlight why let-7 is required for normal gene expression in the context of embryonic development and oncogenesis, which will facilitate the development of approaches to exploit this regulatory pathway by manipulating Lin28/let-7 axis for novel treatments of human diseases.

Maintenance of normal endothelial function is critical to various aspects of blood vessel function, but its regulation is poorly understood. In this study, we show that disruption of baseline fibroblast growth factor (FGF) signaling to the endothelium leads to a dramatic reduction in let-7 miRNA levels that, in turn, increases expression of transforming growth factor (TGF)-β ligands and receptors and activation of TGF-β signaling, leading to endothelial-to-mesenchymal transition (Endo-MT). We also find that Endo-MT is an important driver of neointima formation in a murine transplant arteriopathy model and in rejection of human transplant lesions. The decline in endothelial FGF signaling input is due to the appearance of an FGF resistance state that is characterized by inflammation-dependent reduction in expression and activation of key components of the FGF signaling cascade. These results establish FGF signaling as a critical factor in maintenance of endothelial homeostasis and point to an unexpected role of Endo-MT in vascular pathology.

Genetic robustness, the preservation of evolved phenotypes against genotypic mutations, is one of the central concepts in evolution. In recent years a large body of work has focused on the origins, mechanisms, and consequences of robustness in a wide range of biological systems. In particular, research on ncRNAs studied the ability of sequences to maintain folded structures against single-point mutations. In these studies, the structure is merely a reference. However, recent work revealed evidence that structure itself contributes to the genetic robustness of ncRNAs. We follow this line of thought and consider sequence-structure pairs as the unit of evolution and introduce the spectrum of extended mutational robustness (EMR spectrum) as a measurement of genetic robustness. Our analysis of the miRNA let-7 family captures key features of structure-modulated evolution and facilitates the study of robustness against multiple-point mutations., (© 2019 He et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

LINE-1 retrotransposons are insertional mutagens capable of altering the genomic landscape in many ways. Activation of the normally silent LINE-1 retrotransposon is associated with a high level of cancer-associated DNA damage and genomic instability. Studies of LINE-1 have so far focused mainly on changes in gene expression, and our knowledge of its impact on functional noncoding RNAs is in its infancy. However, current evidence suggests that a significant number of human miRNAs originate from retrotransposon sequences. Furthermore, LINE-1 is generally not expressed in normal tissues while its expression is widespread in epithelial cancers. Based on our recent studies, we demonstrate a functional link between aberrant LINE-1 expression and deregulation of let-7 miRNA expression. Since the expression of let-7 is modulated by LINE-1 activity, we discuss possible mechanisms for this effect and how the silencing of LINE-1 activation could provide new therapeutic options for cancer treatment. Based on the deep sequencing of small RNAs in parallel with gene expression profiling in breast cancer cells, we have identified potential pathways linking L1 activity to let-7 processing and maturation and ultimately to the control of stemness in