Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

BACKGROUND: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. METHODS: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. RESULTS: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. CONCLUSIONS: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

Knockdown of AMIGO2 suppresses proliferation and migration through regulating PPAR-γ in bladder cancer.

PURPOSE: This study aims to reveal the relationship between AMIGO2 and proliferation, migration and tumorigenicity of bladder cancer, and explore the potential molecular mechanisms. METHODS: The expression level of AMIGO2 is measured by qRT-PCR and immunohistochemistry (IHC). Stable AMIGO2 knockdown cell lines T24 and 5637 were established by lentivirus transfection. Cell Counting Kit (CCK-8 assay) was produced to determine cell proliferation, flow cytometry analysis was utilized to detect cell cycle, and wound healing assay was proceeded to test migration ability of bladder cancer cells. Xenograft mouse model was established for investigating the effect of AMIGO2 on tumor formation in vivo. The RNA Sequencing technology was applied to explore the underlying mechanisms. The expression level of PPAR-γ was measured by Western Blot. RESULTS: AMIGO2 was upregulated in bladder cancer cells and tissues. Inhibited expression of AMIGO2 suppresses cell proliferation and migration. Low AMIGO2 expression inhibited tumorigenicity of 5637 in nude mice. According to RNA-Seq and bioinformatics analysis, 917 DEGs were identified. The DEGs were mainly enriched in cell-cell adhesion, peroxisome proliferators-activated receptors (PPARs) signaling pathway and some other pathways. PPAR-γ is highly expressed in bladder cancer cell lines T24 and 5637, but when AMIGO2 is knocked down in T24 and 5637, the expression level of PPAR-γ is also decreased, and overexpression of PPAR-γ could reverse the suppression effect of cell proliferation and migration caused by the inhibition of AMIGO2. CONCLUSION: AMIGO2 is overexpressed in bladder cancer cells and tissues. Knockdown of AMIGO2 suppresses bladder cancer cell proliferation and migration. These processes might be regulated by PPAR-γ signaling pathway.

Knockdown of GNL3 inhibits LUAD cell growth by regulating Wnt-ß-catenin pathway.

BACKGROUND: Lung adenocarcinoma (LUAD) is a leading cause of tumor-associated mortality, and it is needed to find new target to combat this disease. Guanine nucleotide-binding -protein-like 3 (GNL3) mediates cell proliferation and apoptosis in several cancers, but its role in LUAD remains unclear. OBJECTIVE: To explore the expression and function of Guanine nucleotide-binding protein-like 3 (GNL3) in lung adenocarcinoma (LUAD) and its potential mechanism in inhibiting the growth of LUAD cells. METHODS: We evaluated the expression of GNL3 in LUAD tissues and its association with patient prognosis using databases and immunohistochemistry. Cell proliferation was assessed by CCK-8 assay as well as colony formation, while apoptosis was evaluated by FCM. The effect of GNL3 knockdown on the Wnt/ß-catenin axis was investigated by Immunoblot analysis. RESULTS: GNL3 is overexpressed in LUAD tissues and is correlated with poor prognosis. Knockdown of GNL3 significantly inhibited the growth as well as induced apoptosis in A549 as well as H1299 cells. Furthermore, we found that the inhibitory effect of GNL3 knockdown on LUAD cell growth is associated with the downregulation of the Wnt/ß-catenin axis. CONCLUSION: GNL3 is key in the progression of LUAD by metiating Wnt/ß-catenin axis. Targeting GNL3 may represent a novel therapeutic method for LUAD treatment.

Downregulation of ATP8B2 to Assess Plasmalogen Distribution and Far1 Expression in Primary Trabecular Meshwork Cells.

The trabecular meshwork (TM) from primary open-angle glaucoma (POAG) cases has been found to contain decreased levels of intracellular plasmalogens. Plasmalogens are a subset of lipids involved in diverse cellular processes such as intracellular signaling, membrane asymmetry, and protein regulation. Proper plasmalogen biosynthesis is regulated by rate-limiting enzyme fatty acyl-CoA reductase (Far1). ATPase phospholipid transporting 8B2 (ATP8B2) is a type IV P-type ATPase responsible for the asymmetric distribution of plasmalogens between the intracellular and extracellular leaflets of the plasma membranes. Here we describe the methodology for extraction and culturing of TM cells from corneal tissue and subsequent downregulation of ATP8B2 using siRNA transfection. Further quantification and downstream effects of ATP8B2 gene knockdown will be analyzed utilizing immunoblotting techniques.

Adeno-associated-virus-mediated delivery of CRISPR-CasRx induces efficient RNA knockdown in the mouse testis.

Rationale: In male mammals, many developmental-stage-specific RNA transcripts (both coding and noncoding) are preferentially or exclusively expressed in the testis, where they play important roles in spermatogenesis and male fertility. However, a reliable platform for efficiently depleting various types of RNA transcripts to study their biological functions during spermatogenesis in vivo has not been developed. Methods: We used an adeno-associated virus serotype nine (AAV9)-mediated CRISPR-CasRx system to knock down the expression of exogenous and endogenous RNA transcripts in the testis. Virus particles were injected into the seminiferous tubules via the efferent duct. Using an autophagy inhibitor, 3-methyladenine (3-MA), we optimized the AAV9 transduction efficiency in germ cells in vivo. Results: AAV9-mediated delivery of CRISPR-CasRx effectively and specifically induces RNA transcripts (both coding and noncoding) knockdown in the testis in vivo. In addition, we showed that the co-microinjection of AAV9 and 3-MA into the seminiferous tubules enabled long-term transgene expression in the testis. Finally, we found that a promoter of Sycp1 gene induced CRISPR-CasRx-mediated RNA transcript knockdown in a germ-cell-type-specific manner. Conclusion: Our results demonstrate the efficacy and versatility of the AAV9-mediated CRISPR-CasRx system as a flexible knockdown platform for studying gene function during spermatogenesis in vivo. This approach may advance the development of RNA-targeting therapies for conditions affecting reproductive health.

Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.

MiR-21-5p knockdown inhibits epithelial to mesenchymal transition in A549 lung adenocarcinoma cells by upregulating RhoB.

BACKGROUND: MiR-21-5p is a highly expressed microRNA that plays an important role in various cancer-promoting processes, including anchorage-independent growth, invasion, migration metastasis, and drug resistance in lung cancer. Studies indicate that miR-21-5p may contribute to these processes by promoting epithelial-mesenchymal transition (EMT). Ras homolog gene family member B (RhoB), a gene downregulated by miR-21-5p, has also been linked to EMT in lung cancer. However, the role of the miR-21-5p/RhoB axis in EMT regulation in lung adenocarcinoma remains unclear. In this study, we aimed to investigate the regulatory role of the miR-21-5p/RhoB axis in EMT and related in vitro functional characteristics such as migration, invasion, cisplatin resistance, and the formation of tumor spheroids. METHODS AND RESULTS: A549 cells were transfected with the miR-21-5p inhibitor, RhoB siRNA, and their corresponding negative controls. Wound healing, transwell invasion, Methyl thiazole tetrazolium (MTT), and sphere formation assays were also performed to evaluate the migration, invasion, cisplatin resistance, and anchorage-independent growth of A549 cells. RT-qPCR was used to determine the mRNA expression levels of EMT markers. MiR-21-5p knockdown inhibited migration, invasion, cisplatin resistance, and sphere formation while upregulating E-cadherin and downregulating Slug. Furthermore, RhoB silencing restored EMT and related in vitro functional characteristics in A549 cells. CONCLUSIONS: Knockdown of miR-21-5p inhibits EMT and related in vitro functional characteristics by upregulating RhoB, suggesting that miR-21-5p may promote EMT through downregulation of RhoB.

CDCA5 accelerates progression of breast cancer by promoting the binding of E2F1 and FOXM1.

BACKGROUND: Breast cancer is one of the most common malignant tumors in women. Cell division cycle associated 5 (CDCA5), a master regulator of sister chromatid cohesion, was reported to be upregulated in several types of cancer. Here, the function and regulation mechanism of CDCA5 in breast cancer were explored. METHODS: CDCA5 expression was identified through immunohistochemistry staining in breast cancer specimens. The correlation between CDCA5 expression with clinicopathological features and prognosis of breast cancer patients was analyzed using a tissue microarray. CDCA5 function in breast cancer was explored in CDCA5-overexpressed/knockdown cells and mice models. Co-IP, ChIP and dual-luciferase reporter assay assays were performed to clarify underlying molecular mechanisms. RESULTS: We found that CDCA5 was expressed at a higher level in breast cancer tissues and cell lines, and overexpression of CDCA5 was significantly associated with poor prognosis of patients with breast cancer. Moreover, CDCA5 knockdown significantly suppressed the proliferation and migration, while promoted apoptosis in vitro. Mechanistically, we revealed that CDCA5 played an important role in promoting the binding of E2F transcription factor 1 (E2F1) to the forkhead box M1 (FOXM1) promoter. Furthermore, the data of in vitro and in vivo revealed that depletion of FOXM1 alleviated the effect of CDCA5 overexpression on breast cancer. Additionally, we revealed that the Wnt/ß-catenin signaling pathway was required for CDCA5 induced progression of breast cancer. CONCLUSIONS: We suggested that CDCA5 promoted progression of breast cancer via CDCA5/FOXM1/Wnt axis, CDCA5 might serve as a novel therapeutic target for breast cancer treatment.

PD-L2 Expression in Breast Cancer Promotes Tumor Development and Progression.

Background: This work focused on investigating the role of programmed death ligand 2 (PD-L2) in the progression of breast cancer by utilizing breast cancer specimens and cells. Materials and Methods: The serum levels of soluble PD-L2 (sPD-L2) in breast cancer patients and healthy individuals were analyzed by means of the enzyme-linked immunosorbent assay, and the PD-L2 levels within 416 resected breast cancer specimens were assessed through immunohistochemistry. Concurrently, in vitro cell experiments and in vivo animal experiments were carried out to analyze the relationship between PD-L2 and the invasion and migration of breast cancer. Results: The concentration of sPD-L2 in breast cancer patients significantly increased compared to that in the control groups. Additionally, breast cancer patients with high concentrations of sPD-L2 had higher Ki67 values (≥30%) and tumor grades. PD-L2 was expressed in 79.09% of the cancer samples, which exhibited a positive correlation with the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Furthermore, we discovered that knockdown of PD-L2 inhibited the migratory and invasive abilities of both MCF-7 and MDA-MB231 cells. Conclusion: Our findings demonstrated that knockdown of PD-L2 suppressed tumor growth, providing novel insights into important biological functions.

Effect of NLRP3 gene knockdown on pyroptosis and ferroptosis in diabetic cardiomyopathy injury.

Diabetic cardiomyopathy (DCM) is a chronic disease caused by diabetes mellitus, which is recognized as a worldwide challenging disease. This study aimed to investigate the role and the potential mechanism of knocking down the NACHT-, LRR- and PYD domains-containing protein 3 (NLRP3), an inflammasome associated with onset and progression of various diseases, on high glucose or diabetes -induced cardiac cells pyroptosis and ferroptosis, two regulated non-necrosis cell death modalities discovered recent years. In the present study, both in vivo and in vitro studies were conducted simultaneously. Diabetic rats were induced by 55 mg/kg intraperitoneal injection of streptozotocin (STZ). Following the intraperitoneal injection of MCC950 (10 mg/kg), On the other hand, the DCM model in H9C2 cardiac cells was simulated with 35 mmol/L glucose and a short hairpin RNA vector of NLRP3 were transfected to cells. The results showed that in vivo study, myocardial fibers were loosely arranged and showed inflammatory cell infiltration, mitochondrial cristae were broken and the GSDMD-NT expression was found notably increased in the DM group, while the protein expressions of xCT and GPX4 was significantly decreased, both of which were reversed by MCC950. High glucose reduced the cell viability and ATP level in vitro, accompanied by an increase in LDH release. All of the above indicators were reversed after NLRP3 knockdown compared with the HG treated alone. Moreover, the protein expressions of pyroptosis- and ferroptosis-related fators were significantly decreased or increased, consistent with the results shown by immunofluorescence. Furthermore, the protective effects of NLRP3 knockdown against HG were reversed following the mtROS agonist rotenone (ROT) treatment. In conclusion, inhibition of NLRP3 suppressed DM-induced myocardial injury. Promotion of mitochondrial ROS abolished the protective effect of knockdown NLRP3, and induced the happening of pyroptosis and ferroptosis. These findings may present a novel therapeutic underlying mechanism for clinical diabetes-induced myocardial injury treatment.

LILRB4 knockdown inhibits aortic dissection development by regulating pyroptosis and the JAK2/STAT3 signaling pathway.

Aortic dissection (AD) is a life-threatening condition with a high mortality rate and without effective pharmacological therapies. Our previous study illustrated that leukocyte immunoglobulin-like receptor B4 (LILRB4) knockdown promoted the contractile phenotypic switch and apoptosis of AD cells. This study aimed to further investigate the role of LILRB4 in animal models of AD and elucidate its underlying molecular mechanisms. Animal models of AD were established using 0.1% beta-aminopropionitrile and angiotensin II and an in vitro model was developed using platelet-derived growth factor BB (PDGF-BB). The effects of LILRB4 knockdown on histopathological changes, pyroptosis, phenotype transition, extracellular matrix (ECM), and Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) pathways were assessed using a series of in vivo and in vitro assays. The effects of the JAK2 inhibitor AG490 on AD cell function, phenotypic transition, and ECM were explored. LILRB4 was highly expressed in AD and its knockdown increased survival rate, reduced AD incidence, and alleviated histopathological changes in the AD mouse model. Furthermore, LILRB4 knockdown promoted contractile phenotype switch, stabilized the ECM, and inhibited pyroptosis. Mechanistically, LILRB4 knockdown inhibited the JAK2/STAT3 signaling pathway. JAK2 inhibitor AG490 inhibited cell viability and migration, enhanced apoptosis, induced G0/G1 cell cycle arrest, and suppressed S-phase progression in PDGF-BB-stimulated human aortic smooth muscle cells. LILRB4 knockdown suppresses AD development by inhibiting pyroptosis and the JAK2/STAT3 signaling pathway.

[Knock-down of long noncoding RNA H19 (lncRNA H19) promotes human colorectal cell autophagy and inhibits cell apoptosis induced by lipopolysaccharide].

Objective To investigate the expression levels of lncRNA H19 in ulcerative colitis (UC) patients and its role in UC. Methods Colonic mucosa and serum samples were collected from 25 UC patients and 25 healthy individuals at the General Hospital of Xizang Military Region. The expression levels of lncRNA H19 were detected, and the receiver operating characteristic (ROC) curve analysis was performed using serum samples. An in vitro inflammatory model was established in HT29 colorectal cells under lipopolysaccharide (LPS) stimulation, and the expression levels of lncRNA H19 were observed in HT29 cells through fluorescence quantitative PCR. HT29 cells with downregulated lncRNA H19 was constructed using lentivirus-mediated shRNA. The effect of lncRNA H19 on cell survival was analyzed through MTT assay. Cell apoptosis was detected by flow cytometry, and the protein expression levels of apoptosis and autophagy markers were analyzed through Western blot. After treatment with rapamycin, the survival of HT29 cells was observed by MTT assay. Results lncRNA H19 was highly expressed in the colonic mucosa and serum samples of UC patients with the ROC area being 0.786. Following LPS stimulation, the expression levels of lncRNA H19 was significantly increased in a time-dependent manner. Downregulation of lncRNA H19 can promote cell survival, inhibit cell apoptosis and increase autophagy level in HT29 cells. Treatment with rapamycin significantly increased the cell survival rate. Conclusion Knock-down of lncRNA H19 increases autophagy levels, inhibits LPS-induced apoptosis and promotes the survival of colon cells.

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos.

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

PKMYT1 knockdown inhibits cholesterol biosynthesis and promotes the drug sensitivity of triple-negative breast cancer cells to atorvastatin.

Triple negative breast cancer (TNBC) as the most aggressive molecular subtype of breast cancer is characterized by high cancer cell proliferation and poor patient prognosis. Abnormal lipid metabolism contributes to the malignant process of cancers. Study observed significantly enhanced cholesterol biosynthesis in TNBC. However, the mechanisms underlying the abnormal increase of cholesterol biosynthesis in TNBC are still unclear. Hence, we identified a member of the serine/threonine protein kinase family PKMYT1 as a key driver of cholesterol synthesis in TNBC cells. Aberrantly high-expressed PKMYT1 in TNBC was indicative of unfavorable prognostic outcomes. In addition, PKMYT1 promoted sterol regulatory element-binding protein 2 (SREBP2)-mediated expression of enzymes related to cholesterol biosynthesis through activating the TNF/ TNF receptor-associated factor 1 (TRAF1)/AKT pathway. Notably, downregulation of PKMYT1 significantly inhibited the feedback upregulation of statin-mediated cholesterol biosynthesis, whereas knockdown of PKMYT1 promoted the drug sensitivity of atorvastatin in TNBC cells. Overall, our study revealed a novel function of PKMYT1 in TNBC cholesterol biosynthesis, providing a new target for targeting tumor metabolic reprogramming in the cancer.

Hsa_circRNA_007630 knockdown delays colon cancer progression by modulation of ferroptosis via miR-506-3p/AURKA axis.

Colon cancer contributes to high mortality rates internationally that has seriously endangered human health. Aurora kinase A (AURKA) served as a key molecule in colon cancer. However, its role of AURKA on regulating ferroptosis in colon cancer and their possible interactions with miRNAs and circRNAs remain still elusive. Comprehensive bioinformatics analysis after RNA-sequencing was conducted to determine the differentially expressed genes (DEGs), ferroptosis-related DEGs and hub genes. The direct relationship between miR-506-3p and hsa_circRNA_007630 or AURKA was predicted, then verified by dual luciferase reporter and quantitative real-time polymerase chain reaction. The rescue experiments were conducted by cotransfection with si-hsa_circRNA_007630, miR-506-3p inhibitor or pcDNA-AURKA in HT29 cells. Erastin was used to induce ferroptosis in HT29 cells and validated by detecting levels of intracellular Fe2+, lipid reactive oxygen species, glutathione, malondialdehyde and ferroptosis markers expression. We screened a total of 331 DEGs, 26 ferroptosis-related genes, among which 3 hub genes were identified through PPI network analysis. Therein, AURKA expression was elevated in colon cancer cells. Moreover, AURKA was targeted by miR-506-3p, and hsa_circRNA_007630 operated as miR-506-3p sponge. The effect of hsa_circRNA_007630 depletion on the inhibiting malignant phenotypes of HT29 cells was rescued by inhibition of miR-506-3p or AURKA overexpression. Additionally, AURKA reduced erastin-induced ferroptosis in HT29 cells. Depletion of circRNA_007630 exerts as a suppressive role in colon cancer through a novel miR-506-3p/AURKA pathway related to ferroptosis, and might become a novel marker for colon cancer.

Knockdown of swine leukocyte antigen expression in porcine lung transplants enables graft survival without immunosuppression.

Immune rejection remains the major obstacle to long-term survival of allogeneic lung transplants. The expression of major histocompatibility complex molecules and minor histocompatibility antigens triggers allogeneic immune responses that can lead to allograft rejection. Transplant outcomes therefore depend on long-term immunosuppression, which is associated with severe side effects. To address this problem, we investigated the effect of genetically engineered transplants with permanently down-regulated swine leukocyte antigen (SLA) expression to prevent rejection in a porcine allogeneic lung transplantation (LTx) model. Minipig donor lungs with unmodified SLA expression (control group, n = 7) or with modified SLA expression (treatment group, n = 7) were used to evaluate the effects of SLA knockdown on allograft survival and on the nature and strength of immune responses after terminating an initial 4-week period of immunosuppression after LTx. Genetic engineering to down-regulate SLA expression was achieved during ex vivo lung perfusion by lentiviral transduction of short hairpin RNAs targeting mRNAs encoding ß2-microglobulin and class II transactivator. Whereas all grafts in the control group were rejected within 3 months, five of seven animals in the treatment group maintained graft survival without immunosuppression during the 2-year monitoring period. Compared with controls, SLA-silenced lung recipients had lower donor-specific antibodies and proinflammatory cytokine concentrations in the serum. Together, these data demonstrate a survival benefit of SLA-down-regulated lung transplants in the absence of immunosuppression.

circPTP4A2 knockdown suppresses NSCLC progression via regulating proliferation and activating anti-tumor immunity.

BACKGROUND: With a considerable variety of cancer subtypes, Non-small cell lung cancer (NSCLC) poses a substantial threat to public health, affecting a large number of individuals and resulting in a high mortality rate. Circular RNA (circRNA) has been applied in various diseases, including cancers. This study aims to investigate the clinial significance and functional role of circPTP4A2 in NSCLC. METHODS: The serum and tissue samples were collected for detecting circPTP4A2 expression in NSCLC using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Actinomycin D was used to treat NSCLC cells to detect circPTP4A2 stability. The CCK-8 and Transwell assays were utilized to assess the effects of circPTP4A2 in NSCLC cells. The ELISA assay and cytotoxicity analysis were used to detect the roles of circPTP4A2 in immune escape. RESULTS: The serum and tissue circPTP4A2 expression was upregulated in NSCLC. The high circPTP4A2 had a relatively high value in differentiating NSCLC patients from healthy individuals. The proliferation, invasion, and immune escape were repressed by circPTP4A2 knockdown. CONCLUSIONS: High circPTP4A2 has the potential to be a diagnostic biomarker in NSCLC. Silencing of circPTP4A2 receded the progression of NSCLC and enhanced antitumor immunity, which might provide potential targets and new ideas for improving the diagnosis and effect of immunotherapy in NSCLC patients.

Insights from different reproductive gene knockdowns via RNA interference in the lady beetle Eriopis connexa: Establishing a new model for molecular studies on natural enemies.

Insect pest control can be achieved by the application of RNA interference (RNAi), a key molecular tool in functional genomics. Whereas most RNAi research has focused on insect pests, few studies have been performed on natural enemies. Validating the efficacy of RNAi in natural enemies is crucial for assessing its safety and enabling molecular research on these organisms. Here, we assessed the efficacy of RNAi in the ladybird beetle Eriopis connexa Germar (Coleoptera: Coccinellidae), focusing on genes related to reproduction, such as vitellogenin (Vg) and its receptor (VgR). In the transcriptome of E. connexa, we found one VgR (EcVgR) and two Vg genes (EcVg1 and EcVg2). These genes have been validated by in silico analyses of functional domains and evolutionary relationships. Five-day-old females were injected with 500 ng/µL of a specific double-stranded RNA (dsRNA) (dsEcVg1, dsEcVg2, or dsEcVgR) for RNAi tests, while nonspecific dsRNA (dsGFP or dsAgCE8.1) was used as a control. Interestingly, dsEcVg2 was able to knockdown both Vg genes, while dsEcVg1 could silence only EcVg1. Additionally, the viability of the eggs was significantly reduced when both Vg genes were knocked down at the same time (after treatment with dsEcVg2 or "dsEcVg1+dsEcVg2"). Ultimately, malformed, nonviable eggs were produced when EcVgR was silenced. Interestingly, no dsRNA treatment had an impact on the quantity of eggs laid. Therefore, the feasibility of RNAi in E. connexa has been confirmed, suggesting that this coccinellid is an excellent Neotropical model for molecular research on natural enemies and for studying RNAi nontarget effects.

JARID2, a novel regulatory factor, promotes cell proliferation, migration, and invasion in oral squamous cell carcinoma.

BACKGROUND: Accurate regulation of gene expression is crucial for normal development and function of cells. The prognostic significance and potential carcinogenic mechanisms of the related gene JARID2 in OSCC are not yet clear, but existing research has indicated a significant association between the two. METHODS AND MATERIALS: The relationship between the expression of the JARID2 gene in tumor samples of OSCC patients and clinical pathological factors was analyzed using immunohistochemistry experiments and RT-qPCR analysis. Based on the clinical pathological data of patients, bioinformatics analysis was conducted using public databases to determine the function of JARID2 in OSCC. Knockdown OSCC cell lines were constructed, and the impact of JARID2 on the biological behavior of OSCC cell lines was assessed through CCK-8, wound healing assay, and transwell analysis. RESULTS: Immunohistochemistry experiments confirmed the correlation between JARID2 and the prognosis of OSCC patients, while RT-qPCR experiments demonstrated its expression levels in tissue and cells. CKK-8 experiments, wound healing assays, and Transwell experiments indicated that knocking down JARID2 had a negative impact on the proliferation, invasion, and migration of OSCC cells. Bioinformatics analysis results showed that the expression of JARID2 in OSCC is closely associated with patient gene co-expression, gene function enrichment, immune infiltration, and drug sensitivity. CONCLUSION: Our study indicates that JARID2 is a novel prognostic biomarker and potential therapeutic target for OSCC.

Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.