Exosomal circ_0084043 derived from colorectal cancer-associated fibroblasts promotes in vitro endothelial cell angiogenesis by regulating the miR-140-3p/HIF-1α/VEGF signaling axis.

Background: Circular RNAs (circRNAs) hold potential as diagnostic markers for colorectal cancer (CRC); however, their functional mechanisms remain incompletely elucidated. This work investigates the clinical implications of a unique set comprising six circRNAs derived from serum in CRC. Furthermore, we delve into the role of exosomal circ_0084043, originating from colorectal cancer-associated fibroblasts (CAFs), with a specific focus on its contribution to endothelial cell angiogenesis. Methods: The study analyzed circRNA levels in serum samples obtained from both CRC and control groups using qRT-PCR. Additionally, exosomes originating from colorectal CAFs and normal fibroblasts (NFs) were purified and confirmed by electron microscopy and Western blotting techniques. The proangiogenic effects of CAF-derived exosomal circ_0084043 were assessed in endothelial cells through proliferation, migration, and in vitro capillary tube formation assays. Gain- and loss-of-function experiments were employed to clarify the role of the circ_0084043/miR-140-3p/HIF-1α axis in endothelial cell angiogenesis, utilizing luciferase reporter assay, Western blotting, and ELISA for mechanism elucidation. Results: The candidate circRNAs (circ_0060745, circ_001569, circ_007142, circ_0084043, Circ_BANP, and CiRS-7) exhibited notably elevated expression in CRC patient sera compared to the levels observed in healthy individuals. Except for CiRS-7, all circRNAs showed elevated expression in CRC patients with positive lymph node metastasis and advanced tumor stages. Exosomes released by colorectal CAFs augmented endothelial cell proliferation, migration, and angiogenesis by upregulating VEGF expression and secretion. Circ_0084043 was highly detected in endothelial cells treated with CAF-derived exosomes. Silencing circ_0084043 reduced VEGFA expression and diminished CAF exosome-induced endothelial cell processes, indicating its pivotal role in angiogenesis. Circ_0084043 sponges miR-140-3p, regulating HIF-1α, and a reverse relationship was also identified between miR-140-3p and VEGFA in endothelial cells. Inhibiting miR-140-3p mitigated circ_0084043 knockdown effects in CAF exosome-treated endothelial cells. Co-transfection of si-circ_0084043 and a miR-140-3p inhibitor reversed the inhibited migration and angiogenesis caused by circ_0084043 knockdown in CAF exosome-treated endothelial cells. Inhibiting miR-140-3p rescued reduced VEGFA expression due to circ_0084043 knockdown in endothelial cells exposed to CAF-derived exosomes, indicating modulation of the circ_0084043/miR-140-3p/VEGF signaling in CAF-derived exosome-induced angiogenesis. Conclusions: This study unveiled a distinctive signature of six serum-derived circular RNAs, indicating their potential as promising diagnostic biomarkers for CRC. Importantly, exosomal circ_0084043 originating from colorectal CAFs was identified as playing a crucial role in endothelial cell angiogenesis, exerting its influence through the modulation of the miR-140-3p/HIF-1α/VEGF signaling axis.

Cancer-associated fibroblasts drive colorectal cancer cell progression through exosomal miR-20a-5p-mediated targeting of PTEN and stimulating interleukin-6 production.

BACKGROUND: This study evaluated the clinical relevance of a set of five serum-derived circulating microRNAs (miRNAs) in colorectal cancer (CRC). Additionally, we investigated the role of miR-20a-5p released by exosomes derived from cancer-associated fibroblasts (CAFs) in the context of CRC. METHODS: The expression levels of five circulating serum-derived miRNAs (miR-20a-5p, miR-122-5p, miR-139-3p, miR-143-5p, and miR-193a-5p) were quantified by real-time quantitative PCR (RT-qPCR), and their associations with clinicopathological characteristics in CRC patients were assessed. The diagnostic accuracy of these miRNAs was determined through Receiver Operating Characteristic (ROC) curve analysis. CAFs and normal fibroblasts (NFs) were isolated from tissue samples, and subsequently, exosomes derived from these cells were isolated and meticulously characterized using electron microscopy and Western blotting. The cellular internalization of fluorescent-labeled exosomes was visualized by confocal microscopy. Gain- and loss-of-function experiments were conducted to elucidate the oncogenic role of miR-20a-5p transferred by exosomes derived from CAFs in CRC progression. The underlying mechanisms were uncovered through luciferase reporter assay, Western blotting, enzyme-linked immunosorbent assays, as well as proliferation and migration assays. RESULTS: The expression levels of serum-derived circulating miR-20a-5p and miR-122-5p were significantly higher in CRC and were positively correlated with advanced stages of tumorigenesis and lymph node metastasis (LNM). In contrast, circulating miR-139-3p, miR-143-5p, and miR-193a-5p were down-regulated in CRC and associated with early tumorigenesis. Except for miR-139-3p, they showed a negative correlation with LNM status. Among the candidate miRNAs, significantly elevated levels of miR-20a-5p were observed in both cellular and exosomal fractions of CAFs. Our findings indicated that miR-20a-5p induces the expression of EMT markers, partly by targeting PTEN. Exosomal miR-20a secreted by CAFs emerged as a key factor enhancing the proliferation and migration of CRC cells. The inhibition of miR-20a impaired the proliferative and migratory potential of CAF-derived exosomes in SW480 CRC cells, suggesting that the oncogenic effects of CAF-derived exosomes are mediated through the exosomal transfer of miR-20a. Furthermore, exosomes originating from CAFs induced increased nuclear translocation of the NF-kB p65 transcription factor in SW480 CRC cells, leading to increased interleukin-6 (IL-6) production. CONCLUSIONS: We established a set of five circulating miRNAs as a non-invasive biomarker for CRC diagnosis. Additionally, our findings shed light on the intricate mechanisms underpinning the oncogenic impacts of CAF-derived exosomes and underscore the pivotal role of miR-20a-5p in CRC progression.

The Expression of miR-34c-5p Induces G0/G1 Cell Cycle Arrest and Apoptosis in SW480 Colon Cancer Cell.

Background: Expression of the miR-34 family, including miR-34a/b/c, has been reported to inhibit the progression of several cancer types by inhibiting cell proliferation and inducing apoptosis. Objectives: We attempted to investigate the effect of SW480 cell transfection with miR-34c-5p mimics on cell proliferation. Methods: To do this, SW480 colon cancer cell line was transfected with miR-34c-5p mimics, scramble sequence, and the vehicle in PBS mock, and then cell proliferation was assessed by MTT assay. The population of cells in cell cycle phases, ROS generation, and apoptosis rate were evaluated by flow cytometry. Additionally, we determined the relative expression of apoptotic genes through real-time PCR technique. Results: We observed a reduced proliferation rate in cells transfected with miR-34c-5p compared to the control group (P <0.05). We also found that miR-34c-5p caused a significant increase in apoptosis rate (P < 0.001) and cell cycle arrest in the G0 and G1 phases (P < 0.05). Moreover, a significant increase was reported in the expression of pro-apoptotic genes, including BAK (P < 0.001), BAX and BAD (P < 0.0001), and Caspase 7/9 (P < 0.0001). Conclusions: However, no remarkable difference was seen in the expression of MCL1, BCL2, and CASPASE 3 genes. Our conclusion is that overexpression of miR-34c-5p could be considered a promising approach for colorectal cancer treatment.

Silymarin-Loaded Tin(IV) Nanoparticles Exhibit Enhanced Bioavailability and Antiproliferative Effects on Colorectal Cancer Cells.

Silymarin (SM) exhibits potential therapeutic effects due to having antioxidant activity. However, the low solubility and bioavailability of SM restrict its biological performance. To overcome this limitation, this study aimed to develop a nanoformulation composed of SM and dimethyltindichloride and investigate the effect of SM-loaded Sn nanoparticles on cancer cell growth and survival. An SM-Sn complex was synthesized and then characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR), EDS-MAP, dynamic light scattering (DLS), and ζ-potential analysis. After that, the SW480 colorectal cancer cell line was treated with different concentrations of SM and the SM-Sn complex. Cell viability was examined through the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, analyzing apoptosis, and live-dead assay. The lipid peroxidation rate was assessed through the measurement of thiobarbituric acid (TBA). Intracellular reactive oxygen species (ROS) level and cell population in the cell cycle were measured using a flow cytometry instrument. To evaluate the colonization ability of SW480 cells, a colony formation assay was performed. Gene expression analysis was also conducted using a real-time polymerase chain reaction (PCR) technique. The findings of this study revealed the effectiveness of the SM-Sn complex in decreasing SW480 cell viability by inducing cell death-associated mechanisms. We found that the SM-Sn complex increases intracellular ROS level and malondialdehyde (MDA) content. It was also revealed that the SM-Sn complex induces cell cycle arrest and the expression of apoptotic genes. In addition, the SM-Sn complex could effectively hinder SW480 cells from constituting colonies. We conclude that the use of tin(IV) as a scaffold for enhanced delivery of SM could be considered an efficient option for inhibiting cancer cell proliferation and survival.

Osteogenic differentiation of human induced pluripotent stem cell in the presence of testosterone and 17 ß-estradiol in vitro.

Recently, numerous scientific approaches have been explored to treat various diseases using stem cells. In 2006, induced pluripotent stem cell (iPSC) were introduced by Takahashi and Yamanaka and showed the potential of self-renewing and differentiation into all types of targeted cells in vitro. In this investigation, we studied the effect of testosterone (T) individually or in the presence of 17 ß-estradiol (E2) on osteogenic differentiation of human iPSC (hiPSC) during 2 wk. The optimal concentrations of sex steroid hormones were examined by MTT assay and acridine orange (AO) staining. The impact of E2 and T either individually or together as a combination was examined by ALP activity; the content of total mineral calcium, by von Kossa and alizarin red staining. Additionally, the expression rate of osteogenic specific markers was studied via real-time RT-PCR and immunocytochemistry analyses at day 14 of differentiation. The obtained results illustrated that the differentiation medium supplemented with T-E2 increased not only the ALP enzyme activity and the content of calcium but also the osteogenic-related gene and protein expressions on the 14th day. Furthermore, the results were confirmed by mineralized matrix staining. In conclusion, these data suggest that T could be used as an effective factor for osteogenic induction of hiPSCs combined with the E2 in bone regeneration.

In vitro Delivery of HIV-1 Nef-Vpr DNA Construct Using the Human Antimicrobial Peptide LL-37.

BACKGROUND AND OBJECTIVES: DNA-based therapeutic vaccines have been proposed as a promising strategy for the treatment of established HIV infections. However, these vaccines are often associated with certain shortcomings, such as poor immunogenicity and low transfection efficiency. In this study, we investigated the ability of LL-37 to deliver a potential immunogenic fusion construct comprising HIV-1 nef and vpr genes into a mammalian cell line. METHODS: First, the pEGFP-N1 eukaryotic expression vector harboring the HIV-1 nef-vpr fusion was produced free of endotoxin on a large scale. Then, DNA/LL-37 complexes were prepared by coincubation of pEGFP-nef-vpr with LL-37 for 45 minutes at different nitrogen to phosphate (N/P) ratios. The formation of DNA/peptide complexes was investigated by gel retardation assay. Next, the stability and morphological characteristics of the nanoparticles were evaluated. The toxicity of LL-37 and the nanoparticles in HEK-293T cells were assessed by MTT assay. The transfection efficiency of the DNA/LL-37 complexes was studied by fluorescence microscopy, flow cytometry, and western blot analysis. RESULTS: LL-37 formed stable complexes with pEGFP-nef-vpr (diameter of 150-200 nm) while providing good protection against nucleolytic and proteolytic degradation. The peptide significantly affected cell viability even at low concentrations. However, the LL-37/DNA complexes had no significant cytotoxic effect. Treatment of cells with pEGFP-N1/LL-37 and pEGFP-nef-vpr/LL-37 resulted in transfection of 36.32% ± 1.13 and 25.55% ± 2.07 of cells, respectively. CONCLUSION: Given these findings and the important immunomodulatory and antiviral activities of LL- 37, the use of this peptide can be further exploited in the development of novel gene delivery strategies and vaccine design.

Biophysical, docking, and cellular studies on the effects of cerium oxide nanoparticles on blood components: in vitro.

INTRODUCTION: The application of nanoparticles (NPs) in medicine and biology has received great interest due to their novel features. However, their adverse effects on the biological system are not well understood. MATERIALS AND METHODS: This study aims to evaluate the effect of cerium oxide nanoparticles (CNPs) on conformational changes of human hemoglobin (HHb) and lymphocytes by different spectroscopic (intrinsic and synchronous fluorescence spectroscopy and far and near circular dichroism [CD] spectroscopy), docking and cellular (MTT and flow cytometry) investigations. RESULTS AND DISCUSSION: Transmission electron microscopy (TEM) showed that CNP diameter is ~30 nm. The infrared spectrum demonstrated a strong band around 783 cm-1 corresponding to the CNP stretching bond. Fluorescence data revealed that the CNP is able to quench the intrinsic fluorescence of HHb through both dynamic and static quenching mechanisms. The binding constant (Kb ), number of binding sites (n), and thermodynamic parameters over three different temperatures indicated that hydrophobic interactions might play a considerable role in the interaction of CNPs with HHb. Synchronous fluorescence spectroscopy indicated that microenvironmental changes around Trp and Tyr residues remain almost unchanged. CD studies displayed that the regular secondary structure of HHb had no significant changes; however, the quaternary structure of protein is subjected to marginal structural changes. Docking studies showed the larger CNP cluster is more oriented toward experimental data, compared with smaller counterparts. Cellular assays revealed that CNP, at high concentrations (>50 µg/mL), initiated an antiproliferative response through apoptosis induction on lymphocytes. CONCLUSION: The findings may exhibit that, although CNPs did not significantly perturb the native conformation of HHb, they can stimulate some cellular adverse effects at high concentrations that may limit the medicinal and biological application of CNPs. In other words, CNP application in biological systems should be done at low concentrations.

Heme degradation and iron release of hemoglobin and oxidative stress of lymphocyte cells in the presence of silica nanoparticles.

Silica nanoparticles (SiO2 NPs) have been widely used in the medical and food sciences. However, their toxic effects against bio-macromolecules and cells are not well understood. The present study was aimed to investigate the adverse effects of fabricated SiO2 NPs on the human hemoglobin (Hb) by FTIR, CD, fluorescence, and UV-vis spectroscopic techniques. Moreover, the toxic effects of SiO2 NPs on the human lymphocyte cell was assessed by trypan blue, reactivate oxygen species (ROS), and apoptosis assays. It was shown that synthesized SiO2 NPs have an amorphous structure with dominant size of around 20-30 nm. FTIR results showed that SiO2 NPs bind to Hb and induce significant structural changes on the native structure of Hb. Near CD spectroscopy depicted that SiO2 NPs induced tertiary structural changes and heme displacement. Fluorescence spectroscopy demonstrated the production of heme degradation species in the Hb solution after interaction with SiO2 NPs. UV-vis spectroscopy experiment indicated the release of iron form Hb after interaction with SiO2 NPs in a concentration dependent manner. Live-dead staining, ROS detection and flow cytometry analysis revealed that human lymphocyte was sensitive towards the toxicity of SiO2 NPs in a ROS-mediated apoptosis mechanism. In conclusion, SiO2 NPs exhibited concentration-dependent toxicity.

Anti-Vascular Endothelial Growth Factor Effects of Sorafenib and Arsenic Trioxide in Acute Myeloid Leukemia Cell Lines

Acute myeloid leukemia (AML), is a clonal disorder caused by acquired somatic mutations and chromosomal rearrangements. According to some evidence, progression of hematolymphoid malignancies depends on the induction of new blood vessel formation under the influence of acute leukemia. Various factors are produced by cancer cells under hypoxic conditions to increase vascular formation. Among these, vascular endothelial growth factor (VEGF) plays a crucial role. Cytotoxicity and anticancer effects of arsenic trioxide (ATO) have been reported in many cancers. Sorafenib, known as an angiogenic inhibitor, decreases leukemic cell survival. The aim of this study was to indicate combination effects of ATO and sorafenib in two AML cell lines, KG-1 and U937. Effective doses was determined by MTT assay for both single and combination treatments. Percentages of apoptotic cells were evaluated by Annexin V FITC staining and mRNA levels of VEGF isoforms and receptor expression were investigated by Real-Time PCR. Our data show that sorafenib (5µM and 7µM in KG-1 and U937 cell lines respectively), ATO (1.618µM and 1µM in KG-1 and U937 cell lines respectively), and also their combination significantly increased the percentage of apoptotic cells. In addition the mRNA level of VEGF isoforms was downregulated in the U937 cell line while upregulated in KG-1 cells. Taken together, our results suggest that the VEGF autocrine loop may have an influence on AML development and progression and could be consider as a therapeutic target. The combination of sorafenib as a VEGF inhibitor with ATO synergistically inhibits cell proliferation and promotes apoptosis.