Calumenin knockdown, by intronic artificial microRNA, to improve expression efficiency of the recombinant human coagulation factor IX.

OBJECTIVES: To improve the expression efficiency of recombinant hFIX, by enhancing its γ-carboxylation, which is inhibited by Calumenin (CALU), we used intronic artificial microRNAs (amiRNAs) for the CALU downregulation. METHODS: Two human CALU (hCALU)-specific amiRNAs were designed, validated and inserted within a truncated form of the hFIX intron 1, in either 3'- or 5'-untranslated regions of the hFIX cDNA, in an expression vector. After transfections of a human cell line with the recombinant constructs, processing of the miRNAs confirmed by RT-PCR, using stem-loop primers. The hFIX and hCALU expression assessments were done based on RT-PCR results. The Gamma(γ)-carboxylation of the expressed hFIX was examined by a barium citrate precipitation method, followed by Enzyme-Linked Immunosorbent Assay. RESULTS: Efficient CALU down regulations, with more than 30-fold decrease, occurred in the cells carrying either of the two examined the 3'-located amiRNAs. The CALU downregulation in the same cells doubled the FIX γ-carboxylation, although the transcription of the FIX decreased significantly. On the other hand, while the expression of the amiRNAs from the 5'-located intron had no decreasing effect on the expression level of CALU, the level of hFIX transcription in these cells increased almost twofold compared to the construct without amiRNA. CONCLUSION: The CALU downregulation, consistent with efficient hFIX γ-carboxylation, occurred in the cells carrying either of the two amiRNAs containing constructs, although it was affected by the locations of the amiRNA carrying introns, suggesting a possible need to optimize the conditions for the amiRNAs expression.

Functional mechanisms of miR-192 family in cancer.

By growing research on the mechanisms and functions of microRNAs (miRNAs, miRs), the role of these noncoding RNAs gained more attention in healthcare. Due to the remarkable regulatory role of miRNAs, any dysregulation in their expression causes cellular functional impairment. In recent years, it has become increasingly apparent that these small molecules contribute to development, cell differentiation, proliferation, apoptosis, and tumor growth. In many studies, the miR-192 family has been suggested as a potential prognostic and diagnostic biomarker and even as a possible therapeutic target for several cancers. However, the mechanistic effects of the miR-192 family on cancer cells are still controversial. Here, we have reviewed each family member of the miR-192 including miR-192, miR-194, and miR-215, and discussed their mechanistic roles in various cancers.

The expression of pluripotency and neuronal differentiation markers under the influence of electromagnetic field and nitric oxide.

Nitric oxide (NO) is a diatomic free radical compound that as a secondary messenger contributes to cell physiological functions and its variations influence proteins activity and triggering intracellular signaling cascades. Low frequency electromagnetic field (EMF) alters the cell biology such as cell differentiation by targeting the plasma membrane and entering force to the ions and small electrical ligands. The effect of these chemical (NO) and physical (EMF) factors on the expression of the stemness and neuronal differentiation markers in rat bone marrow mesenchymal stem cells (BMSC) was investigated. The cells were treated with low (50micromolar) and high (1mM) concentrations of Deta-NO as a NO donor molecule and 50Hz low frequency EMF. The expression of pluripotency and neuronal differentiation genes and proteins was investigated using real time qPCR and Immunocytochemistry techniques. The simultaneous treatment of EMF with NO (1mM) led to the down-regulation of stemness markers expression and up-regulation of neuronal differentiation markers expression. Cell proliferation decreased and cell morphology changed which caused the majority of cells obtains neuronal protein markers in their cytoplasm. The decrease in the expression of neuronal differentiation Nestin and DCX markers without any change in the expression of pluripotency Oct4 marker (treated with low concentration of NO) indicates protection of stemness state in these cells. Treatment with NO demonstrated a double behavior. NO low concentration helped the cells protect the stemness state but NO high concentration plus EMF pushed cells into differentiation pathway.

Overcoming the non-kinetic activity of EGFR1 using multi-functionalized mesoporous silica nanocarrier for in vitro delivery of siRNA.

Triple-negative breast cancer (TNBC) does not respond to HER2-targeted and hormone-based medicines. Epidermal growth factor receptor 1 (EGFR1) is commonly overexpressed in up to 70% of TNBC cases, so targeting cancer cells via this receptor could emerge as a favored modality for TNBC therapy due to its target specificity. The development of mesoporous silica nanoparticles (MSNs) as carriers for siRNAs remains a rapidly growing area of research. For this purpose, a multi-functionalized KIT-6 containing the guanidinium ionic liquid (GuIL), PEI and PEGylated folic acid (FA-PEG) was designed. Accordingly, KIT-6 was fabricated and modified with FA-PEG and PEI polymers attached on the surface and the GuIL placed in the mesopores. Subsequent to confirming the structure of this multi-functionalized KIT-6- based nanocarrier using TEM, SEM, AFM, BET, BJH, DLS and Zeta Potential, it was investigated for uploading and transferring the anti-EGFR1 siRNAs to the MD-MBA-231 cell line. The rate of cellular uptake, cellular localization and endolysosomal escape was evaluated based on the fluorescent intensity of FAM-labelled siRNA using flowcytometry analysis and confocal laser scanning microscopy (CLSM). The 64% cellular uptake after 4 h incubation, clearly suggested the successful delivery of siRNA into the cells and, CLSM demonstrated that siRNA@[FA-PEGylated/PEI@GuIL@KIT-6] may escape endosomal entrapment after 6 h incubation. Using qPCR, quantitative evaluation of EGFR1 gene expression, a knockdown of 82% was found, which resulted in a functional change in the expression of EGFR1 targets. Co-treatment of chemotherapy drug "carboplatin" in combination with siRNA@[FA-PEGylated/PEI@GuIL@KIT-6] exhibited a remarkable cytotoxic effect in comparison to carboplatin alone.