S ilencing of SCD and SREBP1 Genes by Short Hairpin RNAs in Chicken Hepatic Cells in Vitro

RNA interference by short hairpin RNAs (shRNAs) is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each against SCD and SREBP1 genes involved in denovo lipid biosynthesis were designed upon considering parameters such as secondary structures of shRNAs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target), synthesized and cloned in pENTR/U6 entry vector to knockdown the expression of SCD and SREBP1 genes. After transfection of these shRNA constructs into the chicken embryonic hepatocytes, expressions of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression of SCD and SREBP1 genes was observed in hepatocytes. The shRNAs against SCD gene showed the knock down efficiency ranged from 20.4% (shRNA5) to 74.2% (shRNA2). In case of SREBP1 gene, the shRNAs showed knock-down efficiency ranging from 26.8% (shRNA4) to 95.85% (shRNA1). The shRNAs against both the genes introduced in chicken hepatocyte cells did not show any significant impact on expression of immune response genes (IFNA and IFNB) in those cells. These results clearly demonstrated the successful down regulation of the expression of SCD and SREBP1 genes by the shRNA molecules against both the target genes under in vitro condition. It is concluded that the shRNA molecules against SCD and SREBP1 genes showed great potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cells.

NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing Autophagy through GABARAP

A long noncoding RNA (lncRNA), nuclear enriched abundant transcript 1 (NEAT1) variant 1 (NEAT1v1), is involved in the maintenance of cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). CSCs are suggested to play important roles in therapeutic resistance. Therefore, we investigated whether NEAT1v1 is involved in the sensitivity to radiation therapy in HCC. Gene knockdown was performed using short hairpin RNAs, and NEAT1v1-overexpressing HCC cell lines were generated by stable transfection with a NEAT1v1-expressing plasmid DNA. Cells were irradiated using an X-ray generator. We found that NEAT1 knockdown enhanced the radiosensitivity of HCC cell lines and concomitantly inhibited autophagy. NEAT1v1 overexpression enhanced autophagy in the irradiated cells and conferred radioresistance. Gamma-aminobutyric acid receptor-associated protein (GABARAP) expression was downregulated by NEAT1 knockdown, whereas it was upregulated in NEAT1v1-overexpressing cells. Moreover, GABARAP was required for NEAT1v1-induced autophagy and radioresistance as its knockdown significantly inhibited autophagy and sensitized the cells to radiation. Since GABARAP is a crucial protein for the autophagosome-lysosome fusion, our results suggest that NEAT1v1 confers radioresistance to HCC by promoting autophagy through GABARAP.

CircRNA Microarray Profiling Reveals hsa_circ_0058493 as a Novel Biomarker for Imatinib-Resistant CML

Background: CircRNA has appeared as a critical molecular in the development of various cancers. However, the cellular function of circRNAs and exosomal circRNAs has not been well explored in Chronic myeloid leukemia (CML). Methods: Differentially expressed circRNAs were identified by a human circRNA microarray analysis. The expression of hsa_circ_0058493 in peripheral blood mononuclear cells (PBMCs) and exosomes was verified using quantitative real-time PCR. Short hairpin RNAs against hsa_circ_0058493 were constructed to silence the expression of circ_0058493. CCK8, flow cytometry and EdU assay were performed to investigate the biological functions of circ_0058493. Results: Hsa_circ_0058493 was significantly overexpressed in the PBMCs of CML patients and high level of circ_0058493 was associated with the poor clinical efficacy of imatinib. Silencing the expression of circ_0058493 significantly inhibited the development of imatinib-resistant CML cells. miR-548b-3p was overexpressed in circ_0058493-downregulated CML cells. Bioinformatic analysis revealed that circ_0058493 might exert its regulatory function acting as a “sponge” of miR-548b-3p. Moreover, hsa_circ_0058493 was significantly enriched in the exosomes derived from imatinib-resistant CML cells. Conclusion: Hsa_circ_0058493 in PBMCs could be a promising prognostic biomarker and might provide a therapeutic target for CML treatment.

Artificial miRNAs are potential gene therapy tools, especially for incurable monogenic disorders

Well-designed artificial miRNAs (amiRNAs) are as effective as short hairpin RNAs (shRNAs) but produce 10–80 times less siRNA. They enable long-term silencing and are safer than other RNAi triggers. They are suitable instruments for gene therapy techniques, especially for incurable monogenic diseases. In clinical studies, stereotactic injection of AAV5 directly into the striatum is the most effective approach. Intravenous injections would not only make patients more comfortable, but would also reduce the cost of complex brain surgery. In terms of structure, biogenesis, and expression levels, Ami RNAs are more "natural" than other gene therapy methods. They also utilise the cell's native protein machinery and do not produce irreversible alterations, unlike genome editing technologies. The amount of time spent on a technology determines its level of progression. ASOs have an edge in this regard, as seen by the number of authorized medicines. Perhaps RNAi is just around the corner.

Antiviral efficacy of short-hairpin RNAs and artificial microRNAs targeting foot-and-mouth disease virus

RNA interference (RNAi) is a well-conserved mechanism in eukaryotic cells that directs post-transcriptional gene silencing through small RNA molecules. RNAi has been proposed as an alternative approach for rapid and specific control of viruses including foot-and-mouth disease virus (FMDV), the causative agent of a devastating animal disease with high economic impact. The aim of this work was to assess the antiviral activity of different small RNA shuttles targeting the FMDV RNA-dependent RNA polymerase coding sequence (3D). Three target sequences were predicted within 3D considering RNA accessibility as a major criterion. The silencing efficacy of short-hairpin RNAs (shRNAs) and artificial microRNAs (amiRNAs) targeting the selected sequences was confirmed in fluorescent reporter assays. Furthermore, BHK-21 cells transiently expressing shRNAs or amiRNAs proved 70 to >95% inhibition of FMDV growth. Interestingly, dual expression of amiRNAs did not improve FMDV silencing. Lastly, stable cell lines constitutively expressing amiRNAs were established and characterized in terms of antiviral activity against FMDV. As expected, viral replication in these cell lines was delayed. These results show that the target RNA-accessibility-guided approach for RNAi design rendered efficient amiRNAs that constrain FMDV replication. The application of amiRNAs to complement FMDV vaccination in specific epidemiological scenarios shall be explored further.

Up-regulation of long non-coding RNA CYTOR induced by icariin promotes the viability and inhibits the apoptosis of chondrocytes

Background Icariin (ICAR) is the main effective component extracted from epimedium, and is reported to have the potential to treat osteoarthritis (OA). However, its pharmacological function on chondrocytes has not been fully clarified. Methods Different doses of ICAR were used to treat chondrocyte cell lines, including CHON-001 and ATDC5. Then the expressions of different lncRNAs were measured by qRT-PCR. Interleukin-1β (IL-1β) was used to simulate the inflammatory response environment of chondrocytes. Overexpression plasmids and short hairpin RNAs of lncRNA CYTOR were used to construct gain-of-function and loss of function models. CCK-8 was conducted to determine the cell viability. Flow cytometry was used to detect the apoptosis of chondrocytes. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the contents of inflammatory factors (IL-6, IL-8, TNF-α) in the supernatant of the chondrocytes. Results Compared with other lncRNAs, CYTOR was changed most significantly in both CHON-001 and ATDC5 cells after treatment with ICAR. ICAR promotes the viability and inhibits the apoptosis of CHON-001 and ATDC5 cells induced by IL-1β, accompanied with reduced levels of inflammatory factors. Overexpression of CYTOR facilitated the viability of chondrocytes, while repressed their apoptosis and inflammatory response. What’s more, knockdown of CYTOR reversed the protective effects of ICAR on chondrocytes. Conclusion CYTOR was a pivotal lncRNA involved in the protective function of ICAR on chondrocytes.

Plasmids Expressing shRNAs Specific to the Nucleocapsid Gene Inhibit the Replication of Porcine Deltacoronavirus In Vivo

Porcine deltacoronavirus (PDCoV) is a novel enteric coronavirus and is becoming one of the major causative agents of diarrhea in pig herds in recent years. To date, there are no commercial vaccines or antiviral pharmaceutical agents available to control PDCoV infection. Therefore, developing a reliable strategy against PDCoV is urgently needed. In this study, to observe the antiviral activity of RNA interference (RNAi), four short hairpin RNAs (shRNAs) specific to the nucleocapsid (N) gene of PDCoV were designed and tested in vitro. Of these, a double-shRNA-expression vector, designated as pSil-double-shRNA-N1, was the most effectively expressed, and the inhibition of PDCoV replication was then further evaluated in neonatal piglets. Our preliminary results reveal that plasmid-based double-shRNA-expression targeting the N gene of PDCoV can significantly protect LLC-PK1 cells and piglets from pathological lesions induced by PDCoV. Our study could benefit the investigation of the specific functions of viral genes related to PDCoV infection and offer a possible methodology of RNAi-based therapeutics for PDCoV infection.