Increased expression of H19/miR-675 is associated with a low fat free mass index in patients with COPD

BackgroundLoss of muscle mass and strength is a significant comorbidity in patients with chronic obstructive pulmonary disease (COPD) that limits their quality of life and has prognostic implications but does not affect everyone equally. To identify mechanisms that may contribute to the susceptibility to a low muscle mass, we investigated microRNA (miRNA) expression, methylation status, and regeneration in quadriceps muscle from COPD patients and the effect of miRNAs on myoblast proliferation in vitro. The relationships of miRNA expression with muscle mass and strength was also determined in a group of healthy older men.MethodsWe identified miRNAs associated with a low fat-free mass (FFM) phenotype in a small group of patients with COPD using a PCR screen of 750 miRNAs. The expression of two differentially expressed miRNAs (miR-675 and miR-519a) was determined in an expanded group of COPD patients and their associations with FFM and strength identified. The association of these miRNAs with FFM and strength was also explored in a group of healthy community-dwelling older men. As the expression of the miRNAs associated with FFM could be regulated by methylation, the relative methylation of the H19 ICR was determined. Furthermore, the proportion of myofibres with centralized nuclei, as a marker of muscle regeneration, in the muscle of COPD patients was identified by immunofluorescence.ResultsImprinted miRNAs (miR-675 and from a cluster, C19MC which includes miR-519a) were differentially expressed in the quadriceps of patients with a low fat-free mass index (FFMI) compared to those with a normal FFMI. In larger cohorts, miR-675 and its host gene (H19) were higher in patients with a low FFMI and strength. The association of miR-519a expression with FFMI was present in male patients with severe COPD. Similar associations of miR expression with lean mass and strength were not observed in healthy community dwelling older men participating in the Hertfordshire Sarcopenia Study. Relative methylation of the H19 ICR was reduced in COPD patients with muscle weakness but was not associated with FFM. In vitro, miR-675 inhibited myoblast proliferation and patients with a low FFMI had fewer centralized nuclei suggesting miR-675 represses regeneration.ConclusionsThe data suggest that increased expression of miR-675/H19 and altered methylation of the H19 imprinting control region are associated with a low FFMI in patients with COPD but not in healthy community dwelling older men suggesting that epigenetic control of this loci may contribute to a susceptibility to a low FFMI.<br/

Simultaneous targeted delivery of doxorubicin and KRAS suppression by a hybrid molecule containing miR-143 and AS1411 aptamer

Abstract Hybrid molecules can be engineered to target tumors by merging drugs with the same or distinct mechanisms of action. The coexistence of multiple pharmacologically active entities within the cancer cell enhances the therapeutic efficacy of the hybrid molecule compared to single-target inhibitors. KRAS is considered the most common oncogenic gene in human cancers and is targeted by tumor suppressor miR-143. Therefore, an increase in miR-143 expression is a promising way to inhibit CRC cell growth. This research aims to develop a hybrid anticancer drug carrier by combining miR-143 and AS1411 aptamers through a hybridization strand (MAH) and loading doxorubicin (Dox), a chemotherapy drug. The uptake capability of MAH into the SW480 CRC cells was confirmed by detecting fluorescence intensity with a fluorescence microscope. After treatment of MAH in SW480 cells, the level of miR-143 was increased, but KRAS expression was decreased for both mRNA and protein. KRAS downstream target proteins, ERK and AKT, were downregulated as well. Furthermore, it was confirmed that DOX could be gradually released from MAH, with approximately 95% released over 72 h. Treating cells with Dox-MAH resulted in the inhibition of cell proliferation and induction of apoptosis. The protein expression of procaspase-3 and Bcl-2 was decreased, while Bax was increased, confirming that Dox-MAH triggered the cell apoptosis. The success of this research proposed a new strategy for a drug delivery system, which has multiple functions simultaneously; CRC cell-specificity, Dox carrier, and miR-143 delivery

Multifunctional molecular hybrid for targeted colorectal cancer cells: Integrating doxorubicin, AS1411 aptamer, and T9/U4 ASO.

Colorectal cancer (CRC) poses a global health challenge, with current treatments often harming both cancerous and normal cells. To improve efficacy, a multifunctional drug delivery platform has been developed, integrating bioactive materials, anticancer agents, and targeted recognition ligands into a single molecule. This study aimed to create a molecular hybrid (MH) containing doxorubicin, AS1411 aptamer, and T9/U4 ASO to regulate SW480 cell proliferation. The AS1411 aptamer targets nucleolin, overexpressed on cancer cell membranes, while T9/U4 ASO inhibits human telomerase RNA activity, further hindering cancer cell proliferation. AS-T9/U4_MH was synthesized via oligonucleotide hybridization, followed by doxorubicin loading and evaluation of its impact on cell proliferation. Binding capability of this MH was verified using fluorescence microscopy and flow cytometry, demonstrating specific recognition of SW480 cells due to nucleolin availability on the cell surface. These findings were corroborated by both microscopy and flow cytometry. AS-T9/U4_MH exhibited anti-proliferative effects, with the doxorubicin-loaded system demonstrating encapsulation and reduced toxicity. Moreover, the presence of Dox within AS-T9/U4_MH led to a notable reduction in hTERT and vimentin expression in SW480 cells. Additionally, examination of apoptotic pathways unveiled a marked decrease in Bcl-2 expression and a simultaneous increase in Bax expression in SW480 cells treated with Dox-loaded AS-T9/U4_MH, indicating its impact on promoting apoptosis. This molecular hybrid shows promise as a platform for integrating chemotherapeutic drugs with bioactive materials for cancer therapy

Interaction study of Dox<b>-</b>incorporated AS1411 aptamer and nucleolin by molecular dynamics simulation

AS1411 aptamer is able to recognise the nucleolin overexpressed on cancer cell membranes and has shown promise as a carrier of doxorubicin (Dox) to the cells. This study aimed to study the interaction between nucleolin and aptamers, in either the absence or presence of Dox, using molecular dynamics simulation. AS22nt aptamer was constructed by joining AS1411 aptamer with an additional 22 nucleotide (nt) sequence. NPT simulations were performed from initial docked configuration predicted by HDOCK. The binding of Dox to AS22nt aptamer occurred at the minor groove and the intercalation site in the duplex region. Nucleolin exhibited less flexibility upon binding to AS22nt. The dominant interaction between nucleolin and AS22nt was the electrostatic interaction. The presence of Dox in AS22nt affected the AS22nt-nucleolin interaction contributed by hydrogen bond, hydrophobic contact and ionic interaction. However, the presence of Dox in AS22nt had no impact on the interaction between nucleolin and AS22nt because the magnitudes of binding energy of nucleolin and aptamer with Dox or without Dox were comparable and they were within their calculated deviation. This understanding of nucleolin, AS1411 aptamer, and Dox interactions could provide us a way to prepare an effective targeted anticancer agent for cancer-suffering patients.</p