scholarly journals Viromers as carriers for mRNA-mediated expression of therapeutic molecules under inflammatory conditions

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Edith Jansig ◽  
Stefanie Geissler ◽  
Vera Rieckmann ◽  
Anja Kuenemund ◽  
Benjamin Hietel ◽  
...  
Keyword(s):  
Treatment Options ◽  
Biological Molecules ◽  
Luciferase Reporter ◽  
Active Components ◽  
Reporter Expression ◽  
Inflammatory Conditions ◽  
Therapeutic Molecules ◽  
In Vitro Protein Expression

Abstract Therapeutic mRNA delivery has been described for several treatment options, such as vaccination and cancer immunotherapy. However, mRNA delivery has to be accompanied by the development and testing of suitable carrier materials due to the instability of mRNAs in human body fluids. In the present study, we investigated the ability of recently developed Viromers to deliver mRNAs in a classical inflammatory setting. We tested mRNAs coding for active components of preclinical (7ND) and approved (sTNF-RII) biologics, in vitro and in vivo. 7ND is an established blocker of the CCR2 axis, whereas sTNF-RII is the active component of the approved drug Etanercept. Viromer/mRNA complexes were transfected into murine macrophages in vitro. Protein expression was analysed using Luciferase reporter expression and mainly identified in spleen, blood and bone marrow in vivo. 7ND-mRNA delivery led to efficient blockage of monocytes infiltration in thioglycolate-induced peritonitis in mice, underlining the ability of Viromers to deliver a therapeutic mRNA cargo without overt toxicity. Therefore, we propose Viromer-based mRNA delivery as a suitable option for the treatment of inflammatory disorders beyond infusion of biological molecules.

scholarly journals Hsa-miR-557 Inhibits Osteosarcoma Growth Through Targeting KRAS

2022 ◽  
Vol 12 ◽  
Author(s):  
Zhi Qiao ◽  
Jinfeng Li ◽  
Hongwei Kou ◽  
Xiangrong Chen ◽  
Deming Bao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nude Mice ◽  
Target Genes ◽  
Tumor Formation ◽  
Cell Cycle Analysis ◽  
Luciferase Reporter ◽  
Expression Of Genes ◽  
Therapeutic Molecules

Objective: Osteosarcoma is the most common malignancy in the skeletal system; studies showed an important role of miRNAs in tumorigenesis, indicating miRNAs as possible therapeutic molecules. This study found abnormal hsa-miR-557 expression levels in osteosarcoma and tried to explore the potential function and the mechanism.Methods: Differential expression genes of osteosarcoma were analyzed using GSE28423 from the GEO database. Survival analysis of miRNAs was conducted with data obtained from the TARGET-OS database. STRING and miRDIP were used to predict target genes of hsa-miR-557; KRAS was then verified using dual-luciferase reporter assay. Expression of genes was detected by qPCR, and levels of proteins were detected by Western blot. The proliferation ability of cells was detected by CCK-8 and cell cycle analysis. Tumor formation assay in nude mice was used to detect the influence of osteosarcoma by hsa-miR-557 in vivo.Results: Analysis from the GEO and TARGET databases found 12 miRNAs that are significantly related to the osteosarcoma prognosis, 7 downregulated (hsa-miR-140-3p, hsa-miR-564, hsa-miR-765, hsa-miR-1224-5p, hsa-miR-95, hsa-miR-940, and hsa-miR-557) and 5 upregulated (hsa-miR-362-3p, hsa-miR-149, hsa-miR-96, hsa-miR-744, and hsa-miR-769-5p). CCK-8 analysis and cell cycle analysis found that hsa-miR-557 could significantly inhibit the proliferation of osteosarcoma cells. The tumor formation assay in nude mice showed that tumor sizes and weights were inhibited by hsa-miR-557 transfection. Further studies also proved that hsa-miR-557 could target the 3′UTR of KRAS and modulate phosphorylation of downstream proteins.Conclusion: This study showed that hsa-miR-557 could inhibit osteosarcoma growth both in vivo and in vitro, by modulating KRAS expression.

scholarly journals Possibility for Transcriptional Targeting of Cancer-Associated Fibroblasts—Limitations and Opportunities

2021 ◽  
Vol 22 (7) ◽  
pp. 3298
Author(s):  
Dina V. Antonova ◽  
Marina V. Zinovyeva ◽  
Liya G. Kondratyeva ◽  
Alexander V. Sass ◽  
Irina V. Alekseenko ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Cancer Associated Fibroblasts ◽  
Transcriptional Targeting ◽  
Viral Promoters ◽  
Therapeutic Molecules ◽  
Selective Activity

Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting.

scholarly journals Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

2019 ◽  
Vol 20 (10) ◽  
pp. 2500 ◽  
Author(s):  
Vrathasha Vrathasha ◽  
Hilary Weidner ◽  
Anja Nohe
Keyword(s):  
Signaling Pathways ◽  
Treatment Options ◽  
Time Frame ◽  
Bmp Signaling ◽  
C2c12 Cells ◽  
Molecular Sequence ◽  
Sequence Of Events ◽  
Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

scholarly journals In vitro and in vivo assessment of the protective effect of sufentanil in acute lung injury

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098635
Author(s):  
Qi Gao ◽  
Ningqing Chang ◽  
Donglian Liu
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Protective Effect ◽  
High Mobility ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Hmgb1 Protein

Objectives To investigate the mechanisms underlying the protective effect of sufentanil against acute lung injury (ALI). Material and Methods Rats were administered lipopolysaccharide (LPS) by endotracheal instillation to establish a model of ALI. LPS was used to stimulate BEAS-2B cells. The targets and promoter activities of IκB were assessed using a luciferase reporter assay. Apoptosis of BEAS-2B cells was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Results Sufentanil treatment markedly reduced pathological changes in lung tissue, pulmonary edema and secretion of inflammatory factors associated with ALI in vivo and in vitro. In addition, sufentanil suppressed apoptosis induced by LPS and activated NF-κB both in vivo and in vitro. Furthermore, upregulation of high mobility group box protein 1 (HMGB1) protein levels and downregulation of miR-129-5p levels were observed in vivo and in vitro following sufentanil treatment. miR-129-5p targeted the 3ʹ untranslated region and its inhibition decreased promoter activities of IκB-α. miR-129-5p inhibition significantly weakened the protective effect of sufentanil on LPS-treated BEAS-2B cells. Conclusion Sufentanil regulated the miR-129-5p/HMGB1 axis to enhance IκB-α expression, suggesting that sufentanil represents a candidate drug for ALI protection and providing avenues for clinical treatment.

scholarly journals Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zhibin Liao ◽  
Hongwei Zhang ◽  
Chen Su ◽  
Furong Liu ◽  
Yachong Liu ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Animal Experiments ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Downstream Target ◽  
Protein Levels ◽  
Elevated Expression ◽  
Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

scholarly journals Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 308
Author(s):  
Ying-Ray Lee ◽  
Chia-Ming Chang ◽  
Yuan-Chieh Yeh ◽  
Chi-Ying F. Huang ◽  
Feng-Mao Lin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blotting ◽  
Expression Profiles ◽  
Plaque Assay ◽  
Virus Titer ◽  
Enterovirus 71 ◽  
Luciferase Reporter ◽  
Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

scholarly journals MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xixi Li ◽  
Shengli Hu ◽  
Haitao Yin ◽  
Hongbo Zhang ◽  
Dan Zhou ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Oral Feeding ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Culex Pipiens Pallens ◽  
Deltamethrin Resistance ◽  
Regulatory Functions ◽  
Pipiens Pallens

Abstract Background Culex pipiens (Cx. pipiens) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non-coding RNAs that have been demonstrated to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens. Methods We used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using dual-luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR-4448’s association with deltamethrin resistance by targeting CYP4H31in vivo. Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results MiR-4448 was downregulated in the deltamethrin-resistant strain (DR strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3′ untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR strain increased when CYP4H31 was knocked down in vivo. Conclusions In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

scholarly journals Molecular and Cellular Mechanisms of Metformin in Cervical Cancer

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2545
Author(s):  
Ya-Hui Chen ◽  
Po-Hui Wang ◽  
Pei-Ni Chen ◽  
Shun-Fa Yang ◽  
Yi-Hsuan Hsiao
Keyword(s):  
Cervical Cancer ◽  
Potential Role ◽  
Treatment Options ◽  
Cellular Mechanisms ◽  
Anticancer Effects ◽  
Clinical Benefits ◽  
Underlying Mechanisms

Cervical cancer is one of the major gynecologic malignancies worldwide. Treatment options include chemotherapy, surgical resection, radiotherapy, or a combination of these treatments; however, relapse and recurrence may occur, and the outcome may not be favorable. Metformin is an established, safe, well-tolerated drug used in the treatment of type 2 diabetes; it can be safely combined with other antidiabetic agents. Diabetes, possibly associated with an increased site-specific cancer risk, may relate to the progression or initiation of specific types of cancer. The potential effects of metformin in terms of cancer prevention and therapy have been widely studied, and a number of studies have indicated its potential role in cancer treatment. The most frequently proposed mechanism underlying the diabetes–cancer association is insulin resistance, which leads to secondary hyperinsulinemia; furthermore, insulin may exert mitogenic effects through the insulin-like growth factor 1 (IGF-1) receptor, and hyperglycemia may worsen carcinogenesis through the induction of oxidative stress. Evidence has suggested clinical benefits of metformin in the treatment of gynecologic cancers. Combining current anticancer drugs with metformin may increase their efficacy and diminish adverse drug reactions. Accumulating evidence is indicating that metformin exerts anticancer effects alone or in combination with other agents in cervical cancer in vitro and in vivo. Metformin might thus serve as an adjunct therapeutic agent for cervical cancer. Here, we reviewed the potential anticancer effects of metformin against cervical cancer and discussed possible underlying mechanisms.

scholarly journals Oncogenic lncRNA ZNF561-AS1 is essential for colorectal cancer proliferation and survival through regulation of miR-26a-3p/miR-128-5p-SRSF6 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zizhen Si ◽  
Lei Yu ◽  
Haoyu Jing ◽  
Lun Wu ◽  
Xidi Wang
Keyword(s):  
Colorectal Cancer ◽  
Rna Binding ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Cancer Proliferation ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model

Abstract Background Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. Methods ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. Results We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. Conclusion In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

scholarly journals miR-146a-5p Promotes Chondrocyte Apoptosis and Inhibits Autophagy of Osteoarthritis by Targeting NUMB

Cartilage ◽  
2021 ◽  
pp. 194760352110235
Author(s):  
Hongjun Zhang ◽  
Wendi Zheng ◽  
Du Li ◽  
Jia Zheng
Keyword(s):  
Caspase 3 ◽  
Cartilage Tissue ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Knee Cartilage ◽  
Before And After ◽  
Related Proteins ◽  
Human And Mouse

Objective miR-146a-5p was found to be significantly upregulated in cartilage tissue of patients with osteoarthritis (OA). NUMB was shown to be involved in the autophagy regulation process of cells. We aimed to learn whether NUMB was involved in the apoptosis or autophagy process of chondrocytes in OA and related with miR-146a-5p. Methods QRT-PCR was used to detect miR-146a-5p level in 22 OA cartilage tissues and 22 controls. The targets of miR-146a-5p were analyzed using software and the luciferase reporter experiment. The apoptosis and autophagy, and related proteins were detected in chondrocytes treated with miR-146a-5p mimic/inhibitor or pcDNA3.1-NUMB/si-NUMB and IL-1β, respectively. In vivo experiment, intra-articular injection of miR-146a-5p antagomir/NC was administered at the knee of OA male mice before and after model construction. Chondrocyte apoptosis and the expression of apoptosis and autophagy-related proteins were also detected. Results miR-146a-5p was highly expressed in knee cartilage tissue of patients with OA, while NUMB was lowly expressed and negatively regulated by miR-146a-5p. Upregulation of miR-146a-5p can promote cell apoptosis and reduce autophagy of human and mouse chondrocytes by modulating the levels of cleaved caspase-3, cleaved PARP, Bax, Beclin 1, ATG5, p62, LC3-I, and LC3-II. Increasing the low level of NUMB reversed the effects of miR-146a-5p on chondrocyte apoptosis and autophagy. Intra-articular injection of miR-146a-5p antagomir can also reverse the effects of miR-146a-5p on the apoptosis and autophagy of knee joint chondrocytes in OA mice. Conclusion Downregulation of miR-146a-5p suppresses the apoptosis and promotes autophagy of chondrocytes by targeting NUMB in vivo and in vitro.

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