Inhibition Of Target Gene Expression Research Articles

Sugar-Baited Delivery of Small Interfering RNA for Gene Silencing in Adult Mosquitoes.

RNA interference (RNAi), an innate regulatory mechanism that is conserved across many eukaryotic species, has been harnessed for experimental gene silencing in many organisms, including mosquitoes. This protocol describes an optimized method for inducing RNAi in adult Aedes aegypti and Anopheles gambiae mosquitoes that involves feeding them a red-colored sugar bait containing small interfering RNA (siRNA). This oral delivery method is less physically disruptive than delivery by subcutaneous injection, and the use of siRNAs (in contrast to long dsRNAs) for RNAi enables the design of molecules that target conserved sites so that gene function can be studied in multiple species. After feeding, the behavioral and morbidity phenotypes that result from the suppression of target gene expression can then be analyzed.

Structure-Guided Approach to Relieving Transcriptional Repression in Resistance to Thyroid Hormone α.

ABSTRACTMutations in thyroid hormone receptor α (TRα), a ligand-inducible transcription factor, cause resistance to thyroid hormone α (RTHα). This disorder is characterized by tissue-specific hormone refractoriness and hypothyroidism due to the inhibition of target gene expression by mutant TRα-corepressor complexes. Using biophysical approaches, we show that RTHα-associated TRα mutants devoid of ligand-dependent transcription activation function unexpectedly retain the ability to bind thyroid hormone. Visualization of the ligand T3 within the crystal structure of a prototypic TRα mutant validates this notion. This finding prompted the synthesis of different thyroid hormone analogues, identifying a lead compound, ES08, which dissociates corepressor from mutant human TRα more efficaciously than T3. ES08 rescues developmental anomalies in a zebrafish model of RTHα and induces target gene expression in TRα mutation-containing cells from an RTHα patient more effectively than T3. Our observations provide proof of principle for developing synthetic ligands that can relieve transcriptional repression by the mutant TRα-corepressor complex for treatment of RTHα.

Identification of Pax protein inhibitors that suppress target gene expression and cancer cell proliferation

The Pax family of developmental control genes are frequently deregulated in human disease. In the kidney, Pax2 is expressed in developing nephrons but not in adult proximal and distal tubules, whereas polycystic kidney epithelia or renal cell carcinoma continues to express high levels. Pax2 reduction in mice or cell culture can slow proliferation of cystic epithelial cells or renal cancer cells. Thus, inhibition of Pax activity may be a viable, cell-type-specific therapy. We designed an unbiased, cell-based, high-throughput screen that identified triazolo pyrimidine derivatives that attenuate Pax transactivation ability. We show that BG-1 inhibits Pax2-positive cancer cell growth and target gene expression but has little effect on Pax2-negative cells. Chromatin immunoprecipitation suggests that these inhibitors prevent Pax protein interactions with the histone H3K4 methylation complex at Pax target genes in renal cells. Thus, these compounds may provide structural scaffolds for kidney-specific inhibitors with therapeutic potential.

SARS-CoV-2 Spike Targets USP33-IRF9 Axis via Exosomal miR-148a to Activate Human Microglia.

SARS-CoV-2, the novel coronavirus infection has consistently shown an association with neurological anomalies in patients, in addition to its usual respiratory distress syndrome. Multi-organ dysfunctions including neurological sequelae during COVID-19 persist even after declining viral load. We propose that SARS-CoV-2 gene product, Spike, is able to modify the host exosomal cargo, which gets transported to distant uninfected tissues and organs and can initiate a catastrophic immune cascade within Central Nervous System (CNS). SARS-CoV-2 Spike transfected cells release a significant amount of exosomes loaded with microRNAs such as miR-148a and miR-590. microRNAs gets internalized by human microglia and suppress target gene expression of USP33 (Ubiquitin Specific peptidase 33) and downstream IRF9 levels. Cellular levels of USP33 regulate the turnover time of IRF9 via deubiquitylation. Our results also demonstrate that absorption of modified exosomes effectively regulate the major pro-inflammatory gene expression profile of TNFα, NF-κB and IFN-β. These results uncover a bystander pathway of SARS-CoV-2 mediated CNS damage through hyperactivation of human microglia. Our results also attempt to explain the extra-pulmonary dysfunctions observed in COVID-19 cases when active replication of virus is not supported. Since Spike gene and mRNAs have been extensively picked up for vaccine development; the knowledge of host immune response against spike gene and protein holds a great significance. Our study therefore provides novel and relevant insights regarding the impact of Spike gene on shuttling of host microRNAs via exosomes to trigger the neuroinflammation.

PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors.

ABSTRACTRadial glia (RG) in the neocortex sequentially generate distinct subtypes of projection neurons, accounting for the diversity and complex assembly of cortical neural circuits. Mechanisms that drive the rapid and precise temporal progression of RG are beginning to be elucidated. Here, we reveal that the RG-specific transcriptional regulator PRDM16 promotes the transition of early to late phase of neurogenesis in the mouse neocortex. Loss of Prdm16 delays the timely progression of RG, leading to defective cortical laminar organization. Our genomic analyses demonstrate that PRDM16 regulates a subset of genes that are dynamically expressed between early and late neurogenesis. We show that PRDM16 suppresses target gene expression through limiting chromatin accessibility of permissive enhancers. We further confirm that crucial target genes regulated by PRDM16 are neuronal specification genes, cell cycle regulators and molecules required for neuronal migration. These findings provide evidence to support the finding that neural progenitors temporally shift the gene expression program to achieve neural cell diversity.

Association between microRNA-527 and glypican-3 in hepatocellular carcinoma.

The present study aimed to identify the specific microRNAs (miRNAs/miRs) and their corresponding target genes involved in hepatocellular carcinomas (HCCs). Microarray analysis was performed to examine the miRNA expression profiles of four paired HCC and corresponding non-cancerous (N) liver tissues using 985 miRNA probes. The Human miRNA Target database was used to identify the target genes of differentially expressed miRNAs between the HCC and N tissues. The protein expression levels of target genes in the HCC tissues and cell lines were evaluated using western blotting. miRNA-mediated suppression of target gene expression was evaluated by transiently transfecting the miRNA into the HCC cell lines. Of the 985 miRNAs evaluated, four miRNAs were differentially expressed (three upregulated and one downregulated miRNAs). Of these four miRNAs, miRNA-527 was highly downregulated in the HCC tissues. Glypican-3 (GPC-3) was predicted as a target gene of miRNA-527. Western blotting revealed that GPC-3 protein is highly expressed in the HCC tissues and HCC cell lines compared with N and normal cell lines. Transfection with miR-527 resulted in suppression of GPC-3 protein expression in the Cos7 cells. Furthermore, transfection with miR-527 also inhibited the intrinsic expression of GPC-3 in the Huh-7 cell line. This indicated that miR-527 in the HCC tissues may be an important novel miRNA that targets the GPC-3 gene expression. GPC-3, whose expression is regulated by miR-527, may be involved in the development and progression of HCC.

Double-stranded RNA confers resistance to pepper mottle virus in Nicotiana benthamiana

RNA interference (RNAi) is a regulatory mechanism of gene expression mediated by small RNAs. By using the RNAi technique, exogenous double-stranded RNA (dsRNA) designed to target mRNA, suppresses target gene expression levels in plants. In this study, we adopted the RNAi mechanism as a tool to protect plants from viruses. We designed and synthesized several dsRNAs targeting the pepper mottle virus (PepMoV) genes HC-Pro and NIb. When used on Nicotiana benthamiana plants, these dsRNAs protected the plant against viral infection over a specific period. By optimizing dsRNA and virus injection time, the protection efficiency of dsRNA by targeting virus genes could be maximized. It seems that exogenous dsRNA-derived RNA-induced silencing complex was able to defend the host against viral infection instantly. Furthermore, each dsRNA designed to target different regions within a transcript had varying levels of effects on virus survival in the host plants. When targeting the middle part of both the NIb and HC-Pro genes using the dsRNAs, the highest viral growth inhibitory effect was observed. An RLM-5′ RACE was performed using plant leaves infected with PepMoV after dsRNA treatment and it was observed that most of the mRNA cleavages occurred close to the 3′ part within the dsRNA target position on the mRNA. These results suggest that the dsRNA tool can be used as a plant vaccine platform for crop protection.

Transgenic rice overexpressing insect endogenous microRNA csu-novel-260 is resistant to striped stem borer under field conditions.

RNA interference (RNAi) is a promising approach for developing insect-resistant crops. In the first two proof-of-concept studies, DNA fragments derived from essential insect genes were constructed into plant expression cassettes as inverted repeats, enabling long double-stranded RNAs (dsRNAs) to be transcribed in host plants (Baum et al., 2007; Mao et al., 2007). After ingestion, dsRNAs overexpressed in planta suppressed target gene expression via small interference RNA (siRNA)-mediated RNAi in western corn rootworm (Diabrotica virgifera virgifera) (Baum et al., 2007) and cotton bollworm (Helicoverpa armigera) (Mao et al., 2007), and thereby reduced their viability. Subsequent studies have mostly employed a similar approach and attempted to develop RNAi crops against various insect species (Liu et al., 2020). However, except for coleopterans that are generally susceptible to RNAi, most insects (e.g., lepidopterans, dipterans, hymenopterans, and hemipterans) exhibit unpredictable responses to dsRNA-induced RNAi, and this has become a hurdle for ubiquitous adaption of this strategy (Cooper et al., 2019). Potential factors influencing RNAi efficacy in insects involve dsRNA stability, cellular dsRNA absorption, core RNAi machinery integrity, systemic RNAi spread, and target gene amenability (Cooper et al., 2019).

MicroRNAs in the Blood-Brain Barrier in Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic (HI) brain injury is a leading cause of acute mortality and chronic disability in newborns. Current evidence shows that cerebral microvascular response and compromised blood-brain barrier (BBB) integrity occur rapidly and could primarily be responsible for the brain injury observed in many infants with HI brain injury. MicroRNAs (miRNAs) are a type of highly conserved non-coding RNAs (ncRNAs), which consist of 21-25 nucleotides in length and usually lead to suppression of target gene expression. Growing evidence has revealed that brain-enriched miRNAs act as versatile regulators of BBB dysfunctions in various neurological disorders including neonatal HI brain injury. In the present review, we summarize the current findings regarding the role of miRNAs in BBB impairment after hypoxia/ischemia brain injury. Specifically, we focus on the recent progress of miRNAs in the pathologies of neonatal HI brain injury. These findings can not only deepen our understanding of the role of miRNAs in BBB impairment in HI brain injury, but also provide insight into the development of new therapeutic strategies for preservation of BBB integrity under pathological conditions.

Understanding Abnormal SMO-SHH Signaling in Autism Spectrum Disorder: Potential Drug Target and Therapeutic Goals.

Autism is a multifactorial neurodevelopmental condition; it demonstrates some main characteristics, such as impaired social relationships and increased repetitive behavior. The initiation of autism spectrum disorder is mostly triggered during brain development by the deregulation of signaling pathways. Sonic hedgehog (SHH) signaling is one such mechanism that influences neurogenesis and neural processes during the development of the central nervous system. SMO-SHH signaling is also an important part of a broad variety of neurological processes, including neuronal cell differentiation, proliferation, and survival. Dysregulation of SMO-SHH signaling leads to many physiological changes that lead to neurological disorders such as ASD and contribute to cognitive decline. The aberrant downregulation of SMO-SHH signals contributes to the proteolytic cleavage of GLI (glioma-associated homolog) into GLI3 (repressor), which increases oxidative stress, neuronal excitotoxicity, neuroinflammation, and apoptosis by suppressing target gene expression. We outlined in this review that SMO-SHH deregulation plays a crucial role in the pathogenesis of autism and addresses the current status of SMO-SHH pathway modulators. Additionally, a greater understanding of the SHH signaling pathway is an effort to improve successful treatment for autism and other neurological disorders.

Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses.

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.

Differential inhibition of target gene expression by human microRNAs

microRNA (miRNA) target genes are commonly computationally predicted, yet how dependable predictions are remains unclear, as most predicted target genes have not been rigorously tested experimentally. In addition, the degree to which a miRNA differentially inhibits the expression of its targets is underappreciated. We selected human miR‐1, miR‐122, and miR‐124 as representatives to investigate the reliability of miRNA target predictions and to examine how miRNAs suppress their target genes. We first constructed target gene reporter libraries for the miRNAs based on three prediction programs: TargetScan, miRanda, and PicTar, and used reporter assays in cell cultures to directly evaluate whether as well as how strongly a predicted target gene is repressed by its cognate miRNA. We found that all three programs have approximately 72–85% success rates in predicting genuine miRNA targets, and that miRNAs inhibit different targets to various degrees. We then performed statistical and correlation analyses to examine how well parameters offered by the online programs and features in target mRNAs might predict the degrees of repression by miRNAs. To show that endogenous miRNAs act through the same mechanism, we correlated our reporter assay results with publically available mRNA expression data in vivo. Overexpression of miR‐1, miR‐122, or miR‐124 further led to differential reduction of target mRNA levels in cell cultures. Our studies systematically investigated hundreds of miRNA target genes, shed light onto the performance of miRNA target gene prediction programs, and suggested a new mechanism by which differential target repression by miRNAs regulates gene expression in vivo.Support or Funding InformationThis work was supported in part by the National Natural Science Foundation of China [grant number 31570843].

Length-dependent accumulation of double-stranded RNAs in plastids affects RNA interference efficiency in the Colorado potato beetle

Transplastomic potato plants expressing double-stranded RNA (dsRNA) targeted against essential genes of the Colorado potato beetle (CPB) can be lethal to larvae by triggering an RNA interference (RNAi) response. High accumulation levels of dsRNAs in plastids are crucial to confer an efficient RNAi response in the insects. However, whether length and sequence of the dsRNA determine the efficacy of RNAi and/or influence the level of dsRNA accumulation in plastids is not known. We compared the RNAi efficacy of different lengths of dsRNA targeted against the CPB β-Actin gene (ACT) by feeding in vitro-synthesized dsRNAs to larvae. We showed that, while the 60 bp dsRNA induced only a relatively low RNAi response in CPB, dsRNAs of 200 bp and longer caused high mortality and similar larval growth retardation. When the dsRNAs were expressed from the plastid (chloroplast) genome of potato plants, we found that their accumulation were negatively correlated with length. The level of dsRNA accumulation was positively associated with the observed mortality, suppression of larval growth, and suppression of target gene expression. Importantly, transplastomic potato plants expressing the 200 bp dsRNA were better protected from CPB than plants expressing the 297 bp dsRNA, the best-performing line in our previous study. Our results suggest that the length of dsRNAs is an important factor that influences their accumulation in plastids and thus determines the strength of the insecticidal RNAi effect. Our findings will aid the design of optimized dsRNA expression constructs for plant protection by plastid-mediated RNAi.

Development of CRISPR technology and its application in bone and cartilage tissue engineering

The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.

New Potent DOT1L Inhibitors for in Vivo Evaluation in Mouse.

In MLL-rearranged cancer cells, disruptor of telomeric silencing 1-like protein (DOT1L) is aberrantly recruited to ectopic loci leading to local hypermethylation of H3K79 and consequently misexpression of leukemogenic genes. A structure-guided optimization of a HTS hit led to the discovery of DOT1L inhibitors with subnanomolar potency, allowing testing of the therapeutic principle of DOT1L inhibition in a preclinical mouse tumor xenograft model. Compounds displaying good exposure in mouse and nanomolar inhibition of target gene expression in cells were obtained and tested in vivo.

Abstract 408: Mir148a Family Regulates Cardiac Differentiation of Human Embryonic Stem Cells by Inhibiting The Dll1 -mediated Notch Signaling Pathway

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate into spontaneously beating cardiomyocytes in vitro, and hold great promise for cardiovascular disease modeling, therapy and drug discovery. However, these applications were hampered by low yield and purity of the in vitro differentiated cardiomyocytes. Although miRNAs represent a type of potential candidates to promote cardiac differentiation, most of cardiomyocyte-enriched miRNAs have not been functionally investigated until now. This study investigated the roles of MIR148A family in cardiac differentiation from hESCs. The MIR148A family is composed of MIR148A , MIR148B and MIR152 , three highly conserved miRNAs sharing same seed sequences. The expression levels of all MIR148A family members were progressively increased during cardiac differentiation in vitro. By using CRISPR-Cas9-mediated knockout, we demonstrated that triple knockout of MIR148A family ( MIR148A -TKO), rather than individual knockout of each members, could grossly inhibited TNNT2 + cardiomyocyte generation. Ectopic expression of MIR152 significantly restored the cardiac differentiation efficiency of MIR148A -TKO hESCs. The transcriptome analysis identified a total of 1071 upregulated genes and 766 down-regulated genes in the MIR148A -TKO hESC-derived cells compared to wild-type hESC-derived cells at day 4 of cardiac differentiation. Gene Ontology analysis revealed that most genes down-regulated in MIR148A -TKO hESC-derived cells were involved in the events of lateral/cardiac mesoderm development, while the up-regulated genes were mainly involved in the events of paraxial/somitic development. Furthermore, the NOTCH ligand Delta-like1 ( DLL1 ) was validated as the target gene of MIR148A family, and the knockdown of DLL1 could inhibit target gene expression of Notch signaling pathway, and promote cardiac differentiation of MIR148A -TKO hESCs. Our findings demonstrate a new function of MIR148A family during cardiac differentiation. Synergistic inhibition of DLL1 -mediated Notch signaling represents a major mechanism for the MIR148A family to inhibit undesired lineage formation and promote hESC differentiation into cardiomyocytes.

Differential Inhibition of Target Gene Expression by Human microRNAs

microRNAs (miRNAs) exert their functions by repressing the expression of their target genes, but most miRNA target genes are unknown, and the degree to which a miRNA differentially inhibits the expression of its targets is underappreciated. We selected human miR-1, miR-122, and miR-124 as representatives to investigate the reliability of miRNA target predictions and examine how miRNAs suppress their targets. We constructed miRNA target gene reporter libraries based on prediction programs TargetScan, miRanda, and PicTar, and performed large-scale reporter assays to directly evaluate whether and how strongly a predicted target gene is repressed by its miRNA. We then performed statistical analyses to examine parameters that contributed to the miRNA inhibition of target genes. We found that the three programs have approximately 72–85% success rates in predicting genuine targets and that the miRNA inhibition of different targets varies in extent. We also identified parameters that could predict the degrees of miRNA repression, and further showed that differential miR-124 repression might contribute to differential gene expression in vivo. Our studies systematically investigated hundreds of miRNA target genes, shed light on factors influencing miRNA functions, and suggested a new mechanism by which differential target repression by miRNAs regulates endogenous gene expression.

Silencing RRM2 inhibits multiple myeloma bytargeting the Wnt/β‑catenin signaling pathway.

Ribonucleotide reductase M2 (RRM2) is one of the two subunits that comprise ribonucleotide reductase (RR), the enzyme that catalyzes the conversion of ribonucleotide 5′-diphosphates into 2′-deoxyribonucleotides, which are required for DNA synthesis. RRM2 is a stress response factor important for the development of several tumors. However, its role in multiple myeloma (MM) remains to be fully elucidated. The present study aimed to investigate the role of RRM2 in MM. The expression of RRM2 in patients with MM was analyzed using the Oncomine database. The results demonstrated that RRM2 expression was higher in MM compared with healthy subjects. Reverse transcription-quantitative polymerase chain reaction and western blot results revealed that RRM2 expression was decreased following transfection with a small interfering RNA targeting RRM2 into NCI-H929 cells. RR activity and Cell Counting Kit-8 assays demonstrated that RRM2 silencing reduced RR activity and inhibited cell proliferation. Annexin V-propidium iodide staining indicated that the percentage of apoptotic NCI-H929 cells was increased following RRM2 silencing compared with that in the control group. Increased phosphorylation of H2AX indicated that RRM2 silencing may activate the DNA-damage response pathway in NCI-H929 cells. Western blot analysis revealed that protein levels of the apoptosis-associated factor Bcl-2 were reduced, whereas Bax, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1 were upregulated following RRM2 silencing compared with the control group. In addition, the results demonstrated that RRM2 silencing may inhibit target gene expression in the Wnt/β-catenin signaling pathway by increasing the phosphorylation of glucose synthase kinase 3β. These findings indicated that RRM2 may be involved in the proliferation and apoptosis of MM cells via the Wnt/β-catenin signaling pathway, suggesting that RRM2 may represent a novel therapeutic target for MM.

Reduced Glutamine Synthetase Activity Alters the Fecundity of Female Bactrocera dorsalis (Hendel)

Glutamine synthetase (GS) is a key enzyme in glutamine synthesis and is associated with multiple physiological processes in insects, such as embryonic development, heat shock response, and fecundity regulation. However, little is known about the influence of GS on female fecundity in the oriental fruit fly, Bactrocera dorsalis. Based on the cloning of BdGSs, mitochondrial BdGSm and cytoplasmic BdGSc, we determined their expressions in the tissues of adult B. dorsalis. BdGSm was highly expressed in the fat body, while BdGSc was highly expressed in the head and midgut. Gene silencing by RNA interference against two BdGSs isoforms suppressed target gene expression at the transcriptional level, leading to a reduced ovarian size and lower egg production. The specific inhibitor L-methionine S-sulfoximine suppressed enzyme activity, but only the gene expression of BdGSm was suppressed. A similar phenotype of delayed ovarian development occurred in the inhibitor bioassay. Significantly lower expression of vitellogenin and vitellogenin receptor was observed when GS enzyme activity was suppressed. These data illustrate the effects of two GS genes on adult fecundity by regulating vitellogenin synthesis in different ways.

Antisense yycG Regulation of Antibiotic Sensitivity of Methicillin-Resistant Staphylococcus aureus in Chronic Osteomyelitis.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an urgent medical problem in osteomyelitis. The YycFG two-component regulatory system (TCS) allows bacteria to adapt rapidly to physical, chemical, and biological stresses. The recombinant plasmid shuttle vector was used to overexpress an antisense RNA (asRNA) to inhibit target gene expression by sequence-specific double-stranded RNA complex degradation. In the current study, antisense yycG RNA (ASyycG)-overexpression MRSA clinical isolates were constructed. Methods: Bacterial growth was monitored, and biofilm biomass was determined by crystal violet microtiter assay. Quantitative reverse transcription polymerase chain reaction analysis was used to identify expression of yycF/G/H and icaA/D in MRSA and ASyycG strains. The expression of YycG protein was quantified by Western blot assays. The antibiotic resistance of ASyycG strains was compared with that of the MRSA strains. Results: The ASyycG strains showed a decrease in growth rate compared with the MRSA strains. Of note, overexpression of ASyycG led to a reduction in biofilm formation and adhesion force. ASyycG strains had decreased expressions of the yycF/G/H and icaA/D. Furthermore, Western blot data showed that expression of the YycG protein decreased by 40% in ASyycG strains compared with MRSA strains. In addition, the effect of yycG asRNA improved the susceptibility of ASyycG strains to cefoxitin. Conclusions: The ASyycG strains inhibited biofilm organization and increased antibiotic sensitivity, which may be attributed to altered intracellular polysaccharide construction.