Search Results (512)

Sestrin2 (SESN2) is a stress-inducible protein known for its cytoprotective functions, but its role in diabetic vascular complications remains unclear. This study investigated the impact of SESN2 on methylglyoxal (MGO)-induced endothelial–mesenchymal transition (EndMT). Human endothelial cells were transfected with SESN2 siRNA duplexes to silence SESN2 expression, followed by MGO treatment. SESN2 knockdown significantly exacerbated MGO-induced oxidative stress, as evidenced by the reduced expression of antioxidant markers. Furthermore, SESN2 silencing enhanced the inflammatory response to MGO, demonstrated by the increased levels of pro-inflammatory cytokines. Notably, SESN2 deficiency promoted EndMT, a key process in diabetes-induced cardiovascular complications, as shown by the increased expression of mesenchymal markers and the decreased expression of endothelial markers. These findings suggest that SESN2 plays a critical protective role in endothelial cells against MGO-induced damage. The study provides novel insights into the molecular mechanisms underlying diabetic cardiovascular complications and identifies SESN2 as a potential therapeutic target for preventing endothelial dysfunction in diabetes. Our results indicate that SESN2 downregulation may contribute to the pathogenesis of diabetic vascular complications by promoting EndMT, increased oxidative stress, and inflammation. Full article

The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR–Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8–60.3% for RNAi and 88.1–89.3% for CRISPR–Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR–Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR–Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR–Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease. Full article

The host enzyme heparanase (HPSE) facilitates the release of herpes simplex virus type 2 (HSV-2) from target cells by cleaving the viral attachment receptor heparan sulfate (HS) from infected cell surfaces. HPSE 2, an isoform of HPSE, binds to but does not possess the enzymatic activity needed to cleave cell surface HS. Our study demonstrates that HSV-2 infection significantly elevates HPSE 2 protein levels, impacting two distinct stages of viral replication. We show that higher HPSE 2 negatively affects HSV-2 replication which may be through the regulation of cell surface HS. By acting as a competitive inhibitor of HPSE, HPSE 2 may be interfering with HPSE’s interactions with HS. We demonstrate that the enhanced expression of HPSE 2, either via viral infection or plasmid transfection, reduces HPSE’s ability to cleave HS, thereby hindering viral egress. Conversely, low HPSE 2 levels achieved through siRNA transfection allow HPSE to cleave more HS, reducing viral entry. Altogether, we propose a hypothetical model in which the modulation of HPSE 2 impedes HSV-2 replication by regulating HS availability on the cell surface. This dual role of HPSE 2 in viral replication and potential tumor suppression underscores its significance in cellular processes and viral pathogenesis. Full article

Purpose: Although chemotherapy is one of the standard treatments for gastric cancer, the disease’s resistance mechanisms continue to limit the survival rates. B7H6 (NCR3LG1), an immune checkpoint belonging to the B7 family, is significantly overexpressed in gastric cancer. This work investigated the possibility of using B7H6 suppression to improve the effectiveness of the widely used chemotherapy medication docetaxel. Materials and Methods: In this study, MKN-45 gastric cancer cells were transfected for 24 h with siRNA targeting B7H6, and then, docetaxel was added at optimal inhibitory doses (IC25 and IC50). To assess the impact of this combination therapy, cellular viability, proliferation, and migration were assessed using MTT assay, colony-forming unit assay, and wound-healing assay, respectively. Additionally, apoptosis and cell cycle status were evaluated by flow cytometry. Moreover, using qRT-PCR, the gene expression of B7H6 and indicators associated with apoptosis was also examined. Results: The sensitivity of MKN-45 cells to docetaxel was greatly increased by the siRNA-mediated knockdown of B7H6, resulting in a decrease in the drug’s IC50 value. When compared to each therapy alone, the combination of B7H6 siRNA plus docetaxel at IC50 levels exhibited a significant increase in apoptosis rate. The volume of cells arrested at the sub-G1 and G2-M phase was shown to rise when B7H6 siRNA transfection was combined with docetaxel. Furthermore, the combination treatment significantly decreased the ability of cells to migrate and form colonies. Conclusions: B7H6 suppression increases the susceptibility of MKN-45 gastric cancer cells to docetaxel treatment, resulting in decreased cellular proliferation and increased rates of apoptosis. The present work underscores the possibility of enhancing treatment results in gastric cancer by merging conventional chemotherapy with gene-silencing approaches. Full article

Oxysophocarpine (OSC), a quinolizidine alkaloid, shows neuroprotective potential, though its mechanisms are unclear. The aim of the present study was to investigate the neuroprotective effects of OSC through the nuclear factor erythroid 2−related factor 2 (Nrf2)/ heme oxygenase−1 (HO–1) signaling pathway using the HT–22 cell line. Assessments of cell viability were conducted utilizing the 3−(4,5−dimethylthiazol−2−yl)−2,5−diphenyltetrazolium bromide (MTT) assay. Assessments of oxidative stress (OS) were conducted through the quantification of reactive oxygen species (ROS). The integrity of the mitochondrial membrane potential (MMP) was scrutinized using fluorescent probe technology. Apoptosis levels were quantified using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The trafficking of Nrf2 within the cell nucleus was examined through immunofluorescence analysis. Furthermore, Western blotting (WB) was applied to evaluate the expression levels of proteins implicated in apoptosis and the Nrf2/HO–1 pathway. To further probe the influence of OSC on the overexpression of antioxidant enzymes, cells were subjected to transfection with HO–1 siRNA. The results showed that OSC inhibited glutamate-induced OS, as evidenced by reduced cell viability and ROS levels. Furthermore, the apoptotic condition induced by glutamate in HT–22 cells was significantly reduced following OSC treatment. More interestingly, the Nrf2/HO–1 signaling pathway was upregulated following OSC treatment. These results suggest that OSC can exert neuroprotective effects by regulating the Nrf2/HO–1 pathway to inhibit neuronal cell apoptosis, potentially aiding in the treatment of neurodegenerative diseases. Full article

Background/Objectives: The ribosomal S6 kinase 2 (S6K2) acts downstream of the mechanistic target of rapamycin complex 1 and is a homolog of S6K1 but little is known about its downstream effectors. The objective of this study was to use an unbiased transcriptome profiling to uncover how S6K2 promotes breast cancer cell survival. Methods: RNA-Seq analysis was performed to identify novel S6K2 targets. Cells were transfected with siRNAs or plasmids containing genes of interest. Western blot analyses were performed to quantify total and phosphorylated proteins. Apoptosis was monitored by treating cells with different concentrations of doxorubicin. Results: Silencing of S6K2, but not S6K1, decreased p21 in MCF-7 and T47D breast cancer cells. Knockdown of Akt1 but not Akt2 decreased p21 in MCF-7 cells whereas both Akt1 and Akt2 knockdown attenuated p21 in T47D cells. While Akt1 overexpression enhanced p21 and partially reversed the effect of S6K2 deficiency on p21 downregulation in MCF-7 cells, it had little effect in T47D cells. S6K2 knockdown increased JUN mRNA and knockdown of cJun enhanced p21. Low concentrations of doxorubicin increased, and high concentrations decreased p21 levels in T47D cells. Silencing of S6K2 or p21 sensitized T47D cells to doxorubicin via c-Jun N-terminal kinase (JNK)-mediated downregulation of Mcl-1. Conclusions: S6K2 knockdown enhanced doxorubicin-induced apoptosis by downregulating the cell cycle inhibitor p21 and the anti-apoptotic protein Mcl-1 via Akt and/or JNK. Full article

HPV/pap tests are widely used for cervical cancer screening, playing a crucial role in early diagnosis and guiding future treatment options. However, approximately 50% of cervical cancer patients are diagnosed at an advanced stage, which is associated with higher recurrence rates and poorer survival outcomes than early-stage diagnoses. This underscores the need for effective treatments for advanced-stage cervical cancer. Among the various oncogenes implicated in cancer, PIK3CA expression is known to cause cervical cancer, suggesting that inhibiting PIK3CA may impede cervical cancer progression. In this study, we transfected PIK3CA-overexpressing tumor cells (SiHa, C33A, and HeLa) with miR-29a, a microRNA extensively studied as a therapeutic candidate for oncogene suppression in various tumor types. We conducted RT-qPCR and Western blot analyses to assess changes in PIK3CA expression at the RNA and protein levels. Wound healing and cell migration assays were used to evaluate the effects of miR-29a on cell division and migration in HeLa cells. We confirmed a reduction in PIK3CA expression at both RNA and protein levels following miR-29a transfection. After transfecting miR-29a into HeLa cells, we observed a reduction in cell division and migration, as demonstrated by wound healing and cell migration assays. Additionally, we found that miR-29a binds to the 3′-UTR region of PIK3CA, leading to a reduction in its gene expression. Furthermore, we correlated the concentration of miR-29a in clinical histologic biopsy samples from cervical cancer patients with disease progression. These findings indicate that miR-29a can slow the progression of cervical cancer by targeting PIK3CA and potentially aid in its treatment. miR-29a shows promise as a therapeutic agent for inhibiting oncogene expression and controlling cervical cancer progression, especially in advanced-stage cases. Full article

Existing evidence indicates that LINCMD1 regulates muscle differentiation-related gene expression in skeletal muscle by acting as a miRNA sponge, though its role in leiomyoma development is still unknown. This study investigated LINCMD1′s involvement in leiomyoma by analyzing paired myometrium and leiomyoma tissue samples (n = 34) from patients who had not received hormonal treatments for at least three months prior to surgery. Myometrium smooth muscle cells (MSMCs) were isolated, and gene expression of LINCMD1 and miR-135b was assessed via qRT-PCR, while luciferase assays determined the interaction between LINCMD1 and miR-135b. To examine the effects of LINCMD1 knockdown, siRNA transfection was applied to a 3D MSMC spheroid culture, followed by qRT-PCR and Western blot analyses of miR-135b, APC, β-Catenin and COL1A1 expression. The results showed that leiomyoma tissues had significantly reduced LINCMD1 mRNA levels, regardless of patient race or MED12 mutation status, while miR-135b levels were elevated compared to matched myometrium samples. Luciferase assays confirmed LINCMD1′s role as a sponge for miR-135b. LINCMD1 knockdown in MSMC spheroids increased miR-135b levels, reduced APC expression, and led to β-Catenin accumulation and higher COL1A1 expression. These findings highlight LINCMD1 as a potential therapeutic target to modulate aberrant Wnt/β-Catenin signaling in leiomyoma. Full article

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4’s potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption. Full article

Exposure to 4,4′-methylene diphenyl diisocyanate (MDI) in the workplace may lead to the development of occupational asthma (OA). However, the specific mechanism(s) by which MDI induces OA are poorly understood. Previous reports have demonstrated that MDI and MDI-glutathione (GSH) conjugate exposure downregulates endogenous human/murine (hsa/mmu)-microRNA(miR)-206-3p, resulting in the activation of mmu/hsa-miR-206-3p-regulated signaling pathways in macrophages. Circular RNAs (circRNAs) regulate many important biological processes by targeting endogenous miRs; however, whether MDI/MDI-GSH exposure may influence circRNA expressions is unknown. Several circRNAs have been identified that regulate hsa-miR-206-3p. We hypothesize that MDI-GSH conjugate exposure induces endogenous circRNA(s) to regulate hsa-miR-206-3p in macrophages. The expression of candidate hsa-miR-206-3p-binding circRNAs was determined from MDI-GSH conjugate-treated differentiated THP-1 macrophages using RT-qPCR. MDI-GSH exposures induced hsa_circ_0008726 and its host gene transcript DNAJB6, whereas other circRNA(s) examined were either not detected or unchanged. RNA-induced silencing complex-immunoprecipitation (RISC-IP) experiments confirm that hsa-miR-206-3p can bind to hsa_circ_0008726. The expressions of endogenous hsa-miR-206-3p, hsa-miR-206-3p-regulated KLF4, and KLF4-activated M2 macrophage-associated markers and chemokines were up-/down-regulated by transfection of hsa_circ_0008726 siRNAs or hsa_circ_0008726 overexpression plasmid in macrophages, respectively. These results suggest MDI-GSH exposure downregulates hsa-miR-206-3p via induction of endogenous hsa_circ_0008726/DNAJB6, resulting in the upregulation of hsa-miR-206-3p-mediated regulations in macrophages. Full article

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53–Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it. Full article

Lateolabrax maculatus iridovirus (LMIV) is a variant strain of red sea bream iridovirus (RSIV), causing serious economic losses in aquaculture. Claudins (CLDNs) are major components of tight junctions (TJs) forming an important line of defense against pathogens. Our pilot miRNA-mRNA joint analysis indicated the degradation of CLDN3, as well as its interaction with miR-181a during LMIV infection. To elucidate the miR-181a/CLDN3/LMIV interactions, in vitro assays were carried out on LMB-L cells. We first confirmed that LMIV infection could decrease the expression of CLDN3, accompanied by the enhancement of permeability, suggesting the dysfunction of TJs. Contrary to the inhibition of CLDN3, the activation of miR-181a was proved, presenting a negative correlation between miR-181a and CLDN3 (Pearson r = −0.773 and p < 0.01). In addition, the influence of CLDN3 on LMIV replication was analyzed by knockdown and over-expression of CLDN3. When CLDN3 was silenced in LMB-L cells with siCLDN3-623 at 9 days post transfection (dpt), LMIV copies and titers were significantly up-regulated by 1.59-fold and 13.87-fold, respectively. By contrast, LMIV replication in LMB-L cells was reduced by 60% and 71%, post transfection with pcDNA3.1-CLDN3 over-expressed plasmid at 6 dpt and 9 dpt, respectively. Ultimately, the regulatory relationship between miR-181a and CLDN3 was further validated by dual luciferase reporter assays. Taking into account the above-described results, we proposed a “miR-181a/CLDN3/LMIV” regulatory relationship. This study provides a new insight for understanding the mechanism of LMIV replication. Full article

RNA interference (RNAi) is a promising approach for gene therapy in cancers, but it requires carriers to protect and deliver therapeutic small interfering RNA (siRNA) molecules to cancerous cells. Starch-based carriers, such as quaternized starch (Q-Starch), have been shown to be biocompatible and are able to form nanocomplexes with siRNA, but significant electrostatic interactions between the carrier and siRNA prevent its release at the target site. In this study, we aim to characterize the effects of the degree of substitution (DS) and molecular weight (Mw) of Q-Starch on the gene silencing capabilities of the Q-Starch/siRNA transfection system. We show that reducing the DS reduces the electrostatic interactions between Q-Starch and siRNA, which now decomplex at more physiologically relevant conditions, but also affects additional parameters such as complex size while mostly maintaining cellular uptake capabilities. Notably, reducing the DS renders Q-Starch more susceptible to enzymatic degradation by α-amylase during the initial Q-Starch pretreatment. Enzymatic cleavage leads to a reduction in the Mw of Q-Starch, resulting in a 25% enhancement in its transfection capabilities. This study provides a better understanding of the effects of the DS and Mw on the polysaccharide-based siRNA delivery system and indicates that the polysaccharide Mw may be the key factor in determining the transfection efficacy of this system. Full article

Neuron-specific Enolase 2 (Eno2) is an isozyme primarily distributed in the central and peripheral nervous systems and neuroendocrine cells. It promotes neuronal survival, differentiation, and axonal regeneration. Recent studies have shown that Eno2 localized on the cell membrane of motor neurons acts as a receptor for extracellular phosphoglycerate kinase 1 (ePgk1), which is secreted by muscle cells and promotes the neurite outgrowth of motor neurons (NOMN). However, interaction between Eno1, another isozyme of Enolase, and ePgk1 failed to return the same result. To account for the difference, we constructed seven point-mutations of Eno2, corresponding to those of Eno1, and verified their effects on NOMN. Among the seven Eno2 mutants, eno2-siRNA-knockdown NSC34 cells transfected with plasmid encoding the 419th aspartic acid mutated into serine (Eno2-[D419S]) or Eno2-[E420K] showed a significant reduction in neurite length. Moreover, the Eno2-ePgk1-interacted synergic effect on NOMN driven by Eno2-[D419S] was more profoundly reduced than that driven by Eno2-[E420K], suggesting that D419 was the more essential residue involved in NOMN mediated by Eno2-ePgk1 interaction. Eno2-ePgk1-mediated NOMN appeared to increase the level of p-Cofilin, a growth cone collapse marker, in NSC34 cells transfected with Eno2-[D419S] and incubated with ePgk1, thereby inhibiting NOMN. Furthermore, we conducted in vivo experiments using zebrafish transgenic line Tg(mnx1:GFP), in which GFP is tagged in motor neurons. In the presence of ePgk1, the retarded growth of axons in embryos injected with eno2-specific antisense morpholino oligonucleotides (MO) could be rescued by wobble-eno2-mRNA. However, despite the addition of ePgk1, the decreased defective axons and the increased branched neurons were not significantly improved in the eno2-[D419S]-mRNA-injected embryos. Collectively, these results lead us to suggest that the 419th aspartic acid of mouse Eno2 is likely a crucial site affecting motor neuron development mediated by Eno2-ePgk1 interaction, and, hence, mutations result in a significant reduction in the degree of NOMN in vitro and axonal growth in vivo. Full article

Tumors originating from thyroid follicular cells are the most common endocrine tumors, with rising incidence. Despite a generally good prognosis, up to 20% of patients experience recurrence and persistence, highlighting the need to identify the underlying molecular mechanisms. Dicer1 has been found to be altered in papillary thyroid cancer (PTC). Studies suggest that Dicer1 functions as a haploinsufficient tumor suppressor gene: partial loss promotes tumorigenesis, while complete loss prevents it. To investigate the effects of partial or total Dicer1 loss in PTC in vitro, we generated stable Dicer1 (+/−) cell lines from TPC1 using CRISPR-Cas9, though no Dicer1 (−/−) lines could be produced. Therefore, siRNA against Dicer1 was transfected into Dicer1 (+/−) cell lines to further decrease its expression. Transcriptomic analysis revealed changes in proliferation and cell locomotion. BrdU staining indicated a slow-down of the cell cycle, with fewer cells in S phase and more in G0-G1-phase. Additionally, transwell assays showed decreased invasion and migration after Dicer1 knockdown by siRNA. Moreover, Dicer1 overexpression led to decreased proliferation, invasion, and increased apoptosis. Our findings deepen the understanding of Dicer1’s role in thyroid cancer, demonstrating that both complete elimination and overexpression of Dicer1 inhibit thyroid oncogenesis, highlighting Dicer1 as a promising target for novel therapeutic strategies. Full article