GREEN ROADS 1291
Macabeo Eastman
Development of reliable and eco-accommodating methods for the synthesis of nanoparticles is a vital step in the field of nanotechnology. Silver nanoparticles are important because of their exceptional chemical, physical, and biological properties, and hence applications. In the last decade, numerous efforts were made to develop green methods of synthesis to avoid the hazardous byproducts. This review describes the methods of green synthesis for Ag-NPs and their numerous applications. It also describes the comparison of efficient synthesis methods via green routes over physical and chemical methods, which provide strong evidence for the selection of suitable method for the synthesis of Ag-NPs.
Fantastic use of Monza 1291 on this hospitality bar top. Used at the recent Dunhill Links golf in St Andrews and a pretty good place to escape the elements ! Huge shout out to www.wm-events.co.uk for this stunner !
Methods: A series of cell phenotype indicators, such as BrdU, colony formation, cell cycle, ATP production, ROS accumulation and cell ability to resist metabolic stress, were performed to clarify the effects of miR-1291 and ERRα expression on tumor cell proliferation and metabolism. A xenograft tumor model was used to evaluate cell tumorigenesis. Meta-analysis and bioinformatic prediction were applied in the search for the bridge-link between miR-1291 and CPT1C. RT-qPCR, western-blot and IHC analysis were used for the detection of mRNA and protein expression. Luciferase assays and ChIP assays were conducted for in-depth mechanism studies.
Results: The expression of miR-1291 inhibited growth and tumorigenesis as a result of modulation of metabolism. CPT1C expression was indirectly and negatively correlated with miR-1291 levels. ESRRA was identified as a prominent differentially expressed gene in both breast and pancreatic cancer samples, and estrogen-related receptor α (ERRα) was found to link miR-1291 and CPT1C. MiR-1291 targeted ERRα and CPT1C was identified as a newly described ERRα target gene. Moreover, ERRα was found to influence cancer cell metabolism and proliferation, consistent with the cellular changes caused by miR-1291.
Since both miR-1291 and CPT1C were identified as tumor biomarkers in pancreatic cancer 1,2,11 and both are closely related to tumor metabolism 4,5,12, the assumption was made that they may be mechanistically linked in their impact on cancer. Furthermore, through Meta-analysis and bioinformatic prediction, ERRα was found in the present study to be the conduit between miR-1291 and CPT1C.
miR-1291 inhibits cancer cell proliferation and metabolism. (A) A WST-8 assay was performed to examine the viability of PANC-1 and MDA-MB-231 cells after overexpression of miR-1291. BrdU activity was used to measure cell proliferation capacity after treated with miR-1291 plasmid. Data are mean SD (n = 5). (B) For colony formation assays, the cells were stained with Diff-Quik after being cultured for an additional 14 days. (C) The cell cycle was determined by flow cytometry after transfection with miR-1291 and immunoblot analysis of cell cycle-related proteins, such as cyclin A/D/E after transfection with miR-1291. Data are mean SD (n = 3). (D) ATP production in miR-1291-transfected cells. Data are mean SD (n = 5). (E) Intracellular accumulation of ROS in two cell lines. Data are mean SD (n = 5). (F) RT-qPCR analysis to determine the expression of the mitochondriogenesis-related NRF1, PGC-1A, TFAM, and CYBA mRNAs. Data are mean SD (n = 6). (G) Glycolysis inhibition test with 2-deoxyglucose and a glucose deprivation test with glucose to measure the anti-metabolic stress ability of cells. Data are mean SD (n = 5).
miR-1291 reduces the tumor cell tumorigenicity. (A) The images of xenograft tumor-bearing mice and tumors derived from PANC-1 and MDA-MB-231 cells infected with retroviruses expressing either miR-1291 or control vector (n = 8). (B) Subcutaneous xenograft tumors arising from PANC-1 and MDA-MB-231 cells were monitored for 4 weeks and 3 weeks, respectively. Tumor weights of mice in both groups. Tumor sizes are presented as mean SD over time (n = 8). (C) Comparison of the ratio of dissected tumor weights over body weights of mice. Data are mean SD (n = 8). (D) IHC analysis was used to determine Ki67 levels in tumor samples. (E) Western blot assay were used to measure ERRα and CPT1C protein in tumor samples.
miR-1291 indirectly regulates CPT1C. (A) Expression of CPT1C mRNA in PANC-1 and MDA-MB-231 cells after transfection with miR-1291. Data are mean SD (n = 6). (B) Western blot assay was used to detect expression of CPT1C after transfection with miR-1291. Band intensity was evaluated by Quantity one software. Data are mean SD (n = 3). (C) Luciferase reporter gene assays were conducted in HEK-293T cells treated with different CPT1C reporter plasmids to define the impact of miR-1291. ST-miR1291-PANC-1 and ST-miR1291-231 cell lines were also used to verify the effect of miR-1291 on luciferase activities of the CPTIC promoters. Data are mean SD (n = 5).
miR-1291 targets the ERRα pathway. (A) The Venn diagram displayed the overlaps between four different gene sets. (B) Levels of ESRRA and PGC-1A mRNAs in PANC-1 and MDA-MB-231 cells after transfection with miR-1291. The data are the mean SD (n = 6). (C) Western blot analysis of ERRα after transfection with miR-1291. Data are mean SD (n = 3). (D) MDA-MB-231 cell lines were stained for ERRα. (E) ERRα target gene mRNA levels measured under the same conditions described above. Data are mean SD (n = 6). (F) Bioinformatics analysis revealing two putative MRE sites for miR-1291 within the 3'UTR of ESRRA transcript. The seed sequence of miR-1291 is underlined. ESRRA 3'UTR luciferase reporter activities were determined. Data are mean SD (n = 5). (G) The sequence of ESRRA 3'UTR bearing the mutated MRE sites is shown. The ESRRA 3'UTR luciferase reporter activities were detected with two ERRα-Reporter-MUT plasmids. Data are mean SD (n = 5).
Synergistic regulation of miR-1291-ERRα-CPT1C signaling on tumor. (A) WST-8 and BrdU assays were performed to examine the effect of low CPT1C expression on the viability and proliferation capacity of WT and ST-miR1291 tumor cells. Data are mean SD (n = 5). (B) Glycolysis inhibition tests with 2-deoxyglucose and glucose deprivation tests with glucose were performed to measure the depletion of CPT1C expression on the anti-metabolic stress ability of WT and ST-miR1291 tumor cells. Data are mean SD (n = 5). (C) WST-8 and BrdU assays were performed to examine the influence of ERRα inhibition on the viability and proliferation capacity of WT and ST-miR1291 tumor cells. Data are mean SD (n = 5). (D) Glycolysis inhibition tests with 2-deoxyglucose and glucose deprivation tests with glucose were performed to measure the impact of reduction of ERRα expression on the anti-metabolic stress ability of WT and ST-miR1291 tumor cells. Data are mean SD (n = 5).
miR-1291 has received increasing attention in the study of cancer. In previous studies, miR-1291 levels were significantly lower in pancreatic tumor tissues, esophageal squamous cell carcinoma and renal cell carcinoma specimens compared with their levels in control tissues or cell lines 4,31,32. Prior studies also revealed that the restoration of miR-1291 expression inhibits cancer cell proliferation, invasion, tumorigenesis and the cell metabolome 2,4. Recombinant fully-humanized bioengineered tRNA/miR-1291 was produced in large quantities, and the therapeutic potential of this miR-1291 prodrug as a monotherapy for pancreatic cancer has gained increasing attention 1,33. In addition, certain targets have been identified for miR-1291, including the efflux transporter MRP1, the glucose transporter GLUT1, the essential signaling pathway for cell differentiation and fatty acid oxidation, FOXA2-AGR2, the mucus maker MUC1, the endoplasmic reticulum stress sensor IRE1α and GPC3 3-5,32,34,35, all of which are critical for cell energy metabolism. The above information may indicate that the mechanism of action of miR-1291 as a potential antitumor factor is likely to be achieved by comprehensively inhibiting cellular metabolic pathways. Therefore, the present study illuminated a new downstream regulatory mechanism for miR-1291 at the metabolic level.
As a member of the CPT family, CPT1C has emerged as a potential therapeutic target in various types of cancer, such as breast cancer and neuroblastoma 8,10,36. Compared to normal tissue, clinical tissue samples from patients with multiple cancers, especially pancreatic cancer and breast cancer, showed high CPT1C expression (37,38, and unpublished data). These data provided clinically relevant support for the present study. CPTs participate in the first vital step of fatty acid metabolism, the reversible transesterification of acyl-CoA esters and carnitines to form acyl-carnitine esters in the mitochondrial membrane 39. Cancer cells can tolerate energy stress by increasing fatty acid synthesis 40,41. The majority of tumors maintain survival and viability by consuming fatty acids, and the FAO process is the primary energy source 42. This is due to the fact that CPT1C, which is closely related to lipid metabolism, is induced under the conditions of glucose deprivation or hypoxia 10, 36. Moreover, CPT1C may be a key element of mitochondrial dysfunction-associated tumor cellular proliferation and senescence and functionally differs from the other three subtypes CPT1A, CPT1B and CPT2 11,12. Referring to the upstream regulatory mechanisms of CPT1C, the AMPK-ACC-CPT1 signaling pathway is recognized as a key regulator of FAO, and the expression of CPT1C can be induced by AMPK activation in a p53-dependent manner 10,43. Moreover, CPT1C is an important PPARα target that may be involved in cellular proliferation and senescence 22. However, the upstream regulatory mechanisms that lead to a series of CPT1C-dependent cell events remain unclarified, and no studies have reported connections between miRNAs and CPT1C. The current work revealed the molecular mechanism by which miR-1291 and CPT1C synergistically regulate the proliferation and metabolism of tumor cells.
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