Cationic Liposomes Carrying HPV16 E6-siRNA Inhibit the Proliferation, Migration, and Invasion of Cervical Cancer Cells.

The E6 and E7 oncoproteins of high-risk types of human papillomavirus (HR-HPV) are crucial for the development of cervical cancer (CC). Small interfering RNAs (siRNAs) are explored as novel therapies that silence these oncogenes, but their clinical use is hampered by inefficient delivery systems. Modification (pegylation) with polyethylene glycol (PEG) of liposomal siRNA complexes (siRNA lipoplexes) may improve systemic stability. We studied the effect of siRNA targeting HPV16 E6, delivered via cationic liposomes (lipoplexes), on cellular processes in a cervical carcinoma cell line (CaSki) and its potential therapeutic use. Lipoplexes-PEG-HPV16 E6, composed of DOTAP, Chol, DOPE, and DSPE-PEG2000 were prepared. The results showed that pegylation (5% DSPE-PEG2000) provided stable siRNA protection, with a particle size of 86.42 ± 3.19 nm and a complexation efficiency of over 80%; the siRNA remained stable for 30 days. These lipoplexes significantly reduced HPV16 E6 protein levels and restored p53 protein expression, inhibiting carcinogenic processes such as proliferation by 25.74%, migration (95.7%), and cell invasion (97.8%) at concentrations of 20 nM, 200 nM, and 80 nM, respectively. In conclusion, cationic lipoplexes-PEG-HPV16 E6 show promise as siRNA carriers for silencing HPV16 E6 in CC.

MiR-21 Regulates Growth and Migration of Cervical Cancer Cells by RECK Signaling Pathway.

Expression of miR-21 has been found to be altered in almost all types of cancers, and it has been classified as an oncogenic microRNA. In addition, the expression of tumor suppressor gene RECK is associated with miR-21 overexpression in high-grade cervical lesions. In the present study, we analyze the role of miR-21 in RECK gene regulation in cervical cancer cells. To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression using siRNAs. We analyzed the expression of miR-21 and RECK, as well as functional effects on cell proliferation and migration. We found that in cervical cancer cells, there was an inverse correlation between miR-21 expression and RECK mRNA and protein expression. SiRNAs to miR-21 increased luciferase reporter activity in construct plasmids containing the RECK-3'-UTR microRNA response elements MRE21-1, MRE21-2, and MRE21-3. The role of miR-21 in cell proliferation was also analyzed, and cancer cells transfected with siRNAs exhibited a markedly reduced cell proliferation and migration. Our findings indicate that miR-21 post-transcriptionally down-regulates the expression of RECK to promote cell proliferation and cell migration inhibition in cervical cancer cell survival. Therefore, miR-21 and RECK may be potential therapeutic targets in gene therapy for cervical cancer.

Noncoding RNAs and Colorectal Cancer: A General Overview.

Colorectal cancer (CRC) is the second most prevalent cancer in the world in which nonmelanoma skin cases are not considered. Different epigenetic mechanisms play a role in the development of cancer. Noncoding RNAs (ncRNAs) are RNA molecules transcribed from noncoding regions of the genome. These are divided into sncRNAs (small noncoding RNAs: <200 nucleotides - including miRNAs [microRNAs], siRNAs [small interfering RNAs], piRNAs [piwi-interacting RNAs], snoRNAs [small nucleolar RNAs]) and lncRNAs (long noncoding RNAs: >200 nucleotides - includingcircular RNAs [circRNAs]). NcRNAs can act as oncogenes or as tumor suppressor genes in CRC and are potential biomarkers of diagnosis, with possible clinical implications. This article aims to conduct a general review of all types of non-coding RNAs and their influence in colorectal cancer, focus on biomarkers of CRC and their possible applications in clinical practice, as well as review their biogenesis and functions. Furthermore, we seek to summarize possible databases available for new searches and studies that require sequence annotation, comparison sequences and target prediction for ncRNAs with the hope of gathering information that can aid in the process of understanding and translating the use of ncRNAs into clinical practice.

Suppressing nNOS Enzyme by Small-Interfering RNAs Protects SH-SY5Y Cells and Nigral Dopaminergic Neurons from 6-OHDA Injury.

Nitric oxide (NO) has chemical properties that make it uniquely suitable as an intracellular and intercellular messenger. NO is produced by the activity of the enzyme nitric oxide synthases (NOS). There is substantial and mounting evidence that slight abnormalities of NO may underlie a wide range of neurodegenerative disorders. NO participates of the oxidative stress and inflammatory processes that contribute to the progressive dopaminergic loss in Parkinson's disease (PD). The present study aimed to evaluate in vitro and in vivo the effects of neuronal NOS-targeted siRNAs on the injury caused in dopaminergic neurons by the toxin 6-hidroxydopamine (6-OHDA). First, we confirmed (immunohistochemistry and Western blotting) that SH-SY5Y cell lineage expresses the dopaminergic marker tyrosine hydroxylase (TH) and the protein under analysis, neuronal NOS (nNOS). We designed four siRNAs by using the BIOPREDsi algorithm choosing the one providing the highest knockdown of nNOS mRNA in SH-SY5Y cells, as determined by qPCR. siRNA 4400 carried by liposomes was internalized into cells, caused a concentration-dependent knockdown on nNOS, and reduced the toxicity induced by 6-OHDA (p < 0.05). Regarding in vivo action in the dopamine-depleted animals, intra-striatal injection of siRNA 4400 at 4 days prior 6-OHDA produced a decrease in the rotational behavior induced by apomorphine. Finally, siRNA 4400 mitigated the loss of TH(+) cells in substantia nigra dorsal and ventral part. In conclusion, the suppression of nNOS enzyme by targeted siRNAs modified the progressive death of dopaminergic cells induced by 6-OHDA and merits further pre-clinical investigations as a neuroprotective approach for PD.

Small RNA differential expression and regulation in Tuxpeño maize embryogenic callus induction and establishment.

Somatic embryogenesis represents an alternative developmental process used to achieve genetic transformation and to approach key questions in maize development. It is known that embryogenic callus induction and plant regeneration are accompanied by microRNA expression changes. However, small RNA (sRNA) populations have not been explored during the proliferative callus subculture establishment and their impact on maintaining the dedifferentiated status and embryogenic potential is far from being completely understood. Here we globally tested the sRNA populations in explants (immature embryos), induced and established maize embryogenic callus from the Mexican cultivar VS-535, Tuxpeño landrace. We detected readjustments in 24 nt and 21-22 nt sRNAs during the embryogenic callus (EC) establishment and maintenance. A follow up on specific microRNAs (miRNAs) indicated that miRNAs related to stress response substantially increase upon the callus proliferation establishment, correlating with a reduction in some of their target levels. On the other hand, while 24 nt-long heterochromatic small interfering RNAs (hc-siRNAs) derived from transposable retroelements transiently decreased in abundance during the EC establishment, a population of 22 nt-hc-siRNAs increased. This was accompanied by reduction in transposon expression in the established callus subcultures. We conclude that stress- and development-related miRNAs are highly expressed upon maize EC callus induction and during maintenance of the subcultures, while miRNAs involved in hormone response only transiently increase during induction. In addition, the establishment of a proliferative status in embryogenic callus is accompanied by important readjustments in hc-siRNAs mapping to long tandem repeat (LTR) retrotransposons, and their expression regulation.

The legume miR1514a modulates a NAC transcription factor transcript to trigger phasiRNA formation in response to drought.

Recent studies have identified microRNAs as post-transcriptional regulators involved in stress responses. miR1514a is a legume microRNA that is induced in response to drought stress in Phaseolus vulgaris (common bean) and shows differential accumulation levels in roots during water deficit in two cultivars with different drought tolerance phenotypes. A recent degradome analysis revealed that miR1514a targets the transcripts of two NAC transcription factors (TFs), Phvul.010g121000 and Phvul.010g120700. Furthermore, expression studies and small RNA-seq data indicate that only Phvul.010g120700 generates phasiRNAs, which also accumulate under water deficit conditions. To confirm these results, we over-expressed miR1514a in transgenic hairy roots, and observed a reduced accumulation of Phvul.010g120700 and an increase in NAC-derived phasiRNAs; inhibition of miR1514a activity resulted in the opposite effect. Moreover, we determined that a NAC-derived phasiRNA associates with ARGONAUTE 1 (AGO1), suggesting that it is functional. In addition, a transcriptome analysis of transgenic hairy roots with reduced miR1514a levels revealed several differentially expressed transcripts, mainly involved in metabolism and stress responses, suggesting they are regulated by the NAC TF and/or by phasiRNAs. This work therefore demonstrates the participation of miR1514 in the regulation of a NAC transcription factor transcript through phasiRNA production during the plant response to water deficit.

Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells.

BACKGROUND: Expression of the microRNA miR-21 has been found to be altered in almost all types of cancers and it has been classified as an oncogenic microRNA or oncomir. Due to the critical functions of its target proteins in various signaling pathways, miR-21 is an attractive target for genetic and pharmacological modulation in various cancers. Cervical cancer is the second most common cause of death from cancer in women worldwide and persistent HPV infection is the main etiologic agent. This malignancy merits special attention for the development of new treatment strategies. In the present study we analyze the role of miR-21 in cervical cancer cells. METHODS: To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression in a cervical intraepithelial neoplasia-derived cell lines using siRNAs. The effect of miR-21 on gene expression was assessed in cervical cancer cells transfected with the siRNA expression plasmid pSIMIR21. We identified the tumor suppressor gene PTEN as a target of miR-21 and determined the mechanism of its regulation throughout reporter construct plasmids. Using this model, we analyzed the expression of miR-21 and PTEN as well as functional effects such as autophagy and apoptosis induction. RESULTS: In SiHa cells, there was an inverse correlation between miR-21 expression and PTEN mRNA level as well as PTEN protein expression in cervical cancer cells. Transfection with the pSIMIR21 plasmid increased luciferase reporter activity in construct plasmids containing the PTEN-3'-UTR microRNA response elements MRE21-1 and MRE21-2. The role of miR-21 in cell proliferation was also analyzed in SiHa and HeLa cells transfected with the pSIMIR21 plasmid, and tumor cells exhibited markedly reduced cell proliferation along with autophagy and apoptosis induction. CONCLUSIONS: We conclude that miR-21 post-transcriptionally down-regulates the expression of PTEN to promote cell proliferation and cervical cancer cell survival. Therefore, it may be a potential therapeutic target in gene therapy for cervical cancer.

In vitro and in vivo inhibition of rabies virus replication by RNA interference

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.

In vitro and in vivo inhibition of rabies virus replication by RNA interference

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.(AU)

In vitro and in vivo inhibition of rabies virus replication by RNA interference.

Rabies is a zoonotic disease that affects all mammals and leads to more than 55,000 human deaths every year, caused by rabies virus (RABV) (Mononegavirales: Rhabdoviridae: Lyssavirus). Currently, human rabies treatment is based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. The aim of this study was to assess the decrease in the titer of rabies virus both in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs were used with antisense strands complementary to rabies virus nucleoprotein (N) mRNA. BHK-21 cells monolayers were infected with 1000 to 0.1 TCID50 of PV and after 2 hours the cells were transfected with each of tree RNAs in separate using Lipofectamine-2000. All three siRNAs reduced the titer of PV strain in a least 0.72 logTCID50/mL and no cytotoxic effect was observed in the monolayers treated with Lipofectamine-2000. Swiss albino mice infected with 10.000 to 1 LD of PV strain by the intracerebral route were also transfected after two hours of infection with a pool 3 siRNAs with Lipofectamine-2000 by the intracerebral route, resulting in a survival rate of 30% in mice inoculated with 100 LD50, while the same dose led to 100% mortality in untreated animals. Lipofectamine-2000 showed no toxic effect in control mice. These results suggest that intracerebral administration of siRNAs might be an effective antiviral strategy for rabies.