Non-specific siRNA Research Articles

Knockdown of DJ-1 Resulted in a Coordinated Activation of the Innate Immune Antiviral Response in HEK293 Cell Line.

PARK7, also known as DJ-1, plays a critical role in protecting cells by functioning as a sensitive oxidation sensor and modulator of antioxidants. DJ-1 acts to maintain mitochondrial function and regulate transcription in response to different stressors. In this study, we showed that cell lines vary based on their antioxidation potential under basal conditions. The transcriptome of HEK293 cells was tested following knockdown (KD) of DJ-1 using siRNAs, which reduced the DJ-1 transcripts to only 12% of the original level. We compared the expression levels of 14k protein-coding transcripts and 4.2k non-coding RNAs relative to cells treated with non-specific siRNAs. Among the coding genes, approximately 200 upregulated differentially expressed genes (DEGs) signified a coordinated antiviral innate immune response. Most genes were associated with the regulation of type 1 interferons (IFN) and the induction of inflammatory cytokines. About a quarter of these genes were also induced in cells treated with non-specific siRNAs that were used as a negative control. Beyond the antiviral-like response, 114 genes were specific to the KD of DJ-1 with enrichment in RNA metabolism and mitochondrial functions. A smaller set of downregulated genes (58 genes) was associated with dysregulation in membrane structure, cell viability, and mitophagy. We propose that the KD DJ-1 perturbation diminishes the protective potency against oxidative stress. Thus, it renders the cells labile and responsive to the dsRNA signal by activating a large number of genes, many of which drive apoptosis, cell death, and inflammatory signatures. The KD of DJ-1 highlights its potency in regulating genes associated with antiviral responses, RNA metabolism, and mitochondrial functions, apparently through alteration in STAT activity and downstream signaling. Given that DJ-1 also acts as an oncogene in metastatic cancers, targeting DJ-1 could be a promising therapeutic strategy where manipulation of the DJ-1 level may reduce cancer cell viability and enhance the efficacy of cancer treatments.

Plasmid DNA ionisable lipid nanoparticles as non-inert carriers and potent immune activators for cancer immunotherapy

Immunotherapy is currently a standard of care in the treatment of many malignancies. However, predictable side effects caused by systemic administration of highly immunostimulatory molecules have been a serious concern within this field. Intratumoural expression or silencing of immunogenic and immunoinhibitory molecules using nucleic acid-based approaches such as plasmid DNA (pDNA) and small interfering RNA (siRNA), respectively, could represent a next generation of cancer immunotherapy. Here, we employed lipid nanoparticles (LNPs) to deliver either non-specific pDNA and siRNA, or constructs targeting two prominent immunotherapeutic targets OX40L and indoleamine 2,3-dioxygenase-1 (IDO), to tumours in vivo.In the B16F10 mouse model, intratumoural delivery of LNP-formulated non-specific pDNA and siRNA led to strong local immune activation and tumour growth inhibition even at low doses due to the pDNA immunogenic nature. Replacement of these non-specific constructs by pOX40L and siIDO resulted in more prominent immune activation as evidenced by increased immune cell infiltration in tumours and tumour-draining lymph nodes. Consistently, pOX40L alone or in combination with siIDO could prolong overall survival, resulting in complete tumour regression and the formation of immunological memory in tumour rechallenge models. Our results suggest that intratumoural administration of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy.

Effect of antiviral siRNAs on the production of cytokines in vitro

Objectives. To evaluate the dynamics of the expression level of IL-1β and IL-28β (IFN-λ3) genes as a result of complex knockdown of some cellular genes, whose expression products play an important role in the reproduction of the influenza virus.Methods. Following the collection of virus-containing liquid and cell lysate within three days from the moment of transfection and infection, the intensity of viral reproduction was assessed using the cytopathic effect titration method. The concentration of viral ribonucleic acid (vRNA) and change in the expression of IL-1β and IL-28β (IFN-λ3) were determined by real-time reverse transcription quantitative polymerase chain reaction (real-time RT-qPCR). The nonparametric Mann–Whitney test was used to statistically calculate significant differences between groups.Results. The use of each small interfering ribonucleic acid (siRNA) complex led to a decrease in viral reproduction on the first day at the multiplicity of infection (MOI) of 0.001. The use of complex A (FLT4.2 + Nup98.1) and D (FLT4.2 + Nup98.1 + Nup205) led to a decrease in viral titer by 2.8 lgTCID50/mL and by 2.1 lgTCID50/mL relative to the use of nonspecific L2 siRNA and viral control (p ≤ 0.05). Transfection of complexes B (Nup98.1 + Nup205) and C (FLT4.2 + Nup205) also reduced the viral titer by 1.5 lgTCID50/mL and 1.8 lgTCID50/mL relative to nonspecific L2 siRNA and viral control (p ≤ 0.05). When conducting real-time RT-qPCR, a significant decrease in the concentration of viral RNA was also noted. When using complexes B, C, and D, the concentration of vRNA decreased on the first day by 14.5, 4.1, and 15 times, respectively. On the second day, a decrease in vRNA was observed in cells with B and D complexes by 17.1 and 18.3 times (p ≤ 0.05). Along with a decrease in the viral titer and vRNA, an increase in the expression of the IL-1β and IL-28β genes was observed on the first day when using all siRNA complexes relative to nonspecific and viral controls (p ≤ 0.05). On the second day, an increase was also observed in cells with A and D complexes, while on the third day, there was an increase in the expression of these genes in cells with complex D (p ≤ 0.05).Conclusions. The use of siRNA complexes is shown to have a pronounced antiviral effect while simultaneously suppressing the activity of cellular genes (FLT4, Nup98 and Nup205). In parallel, the transfection of complexes that block the formation of expression products necessary for viral reproduction is demonstrated to lead to an increase in the level of expression of the IL-1β and IL-28β genes. These results indicate not only that the use of siRNA has antiviral activity, but also immunomodulatory activity, which can contribute to a more effective immune response of the body.

Indirubin relieves inflammatory injury of chondrocytes in a mouse model of osteoarthritis

To investigate the effect of indirubin for relieving joint inflammation and injury in a rat model of osteoarthritis. Articular cartilage chondrocytes were isolated from adult rat knee joint and cultured in the presence of interleukin-1β (IL-1β) and 0.1, 0.5, 1.0, or 2.0 μmol/L indirubin. The cells were transfected with NPAS2 siRNA or a non-specific siRNA, and the cell proliferation and apoptosis were evaluated using tetramethylthiazole blue staining and flow cytometry. The protein expression levels of Bax, Bcl-2, ACAN, COL2A1, MMP-13 and NPAS2 were detected with Western blotting, and the levels of NO, PGE2 and TNF-α in the culture supernatant were determined with ELISA. The mRNA expression levels of NPAS2, ACAN, COL2A1 and MMP-13 were detected using fluorescence quantitative PCR. In a C57BL/6 mouse model of osteoarthritis, the effect of indirubin on BAX, Bcl-2, ACAN and MMP-13 protein expressions in the bone and joint tissues were evaluated with Western blotting. Treatment with 0.1 μmol/L indirubin produced no significant changes in chondrocyte proliferation, apoptosis, caspase-3 activity, or BAX and Bcl-2 protein expressions. At higher doses (0.5, 1.0 and 2.0 μmol/L), indirubin significantly promoted cell proliferation, increased Bcl-2 protein expression, and lowered cell apoptosis rate, caspase-3 activity and Bax protein expression (P < 0.05). Indirubin treatment at 0.5 μmol/L up-regulated the protein and mRNA expressions of NPAS2, ACAN and COL2A1, and down-regulated the expressions of MMP-13, NO, PGE2 and TNF-α (P < 0.05). Interference of NPAS2 expression significantly attenuated the protective effect of 0.5 μmol/L indirubin against IL-1β-induced chondrocyte injury. The mouse model of osteoarthritis showed obviously increased protein levels of BAX and MMP-13 (P < 0.01) and decreased levels of Bcl-2 (P < 0.05) and ACAN (P < 0.01) in the knee joint, and indirubin treatment of the mouse models significantly inhibited the increase of BAX and MMP-13 protein expressions (P < 0.01) and up-regulated the protein expressions of Bcl-2 and ACAN (P < 0.05). Indirubin has a protective effect on osteoarthritis tissue and alleviates inflammation and damage of osteoarthritis chondrocytes possibly through NPAS2.

Knockdown of FLT4, Nup98, and Nup205 Cellular Genes Effectively Suppresses the Reproduction of Influenza Virus Strain A/WSN/1933 (H1N1) In vitro.

Influenza is one of the most common infectious diseases, which affects the lower respiratory tract, and can lead to serious complications, including death. It is known that currently available therapeutic agents and vaccines do not provide 100% protection against influenza viruses. The development of drugs based on the RNA interference mechanism in the context of this problem is a promising area. This paper aims to assess the effect of FLT4, Nup98, and Nup205 cellular gene knockdown on the reproduction of the influenza A virus in human lung cell culture. Influenza virus strain A/WSN/1933 (St. Jude's Children's Research Hospital, USA) was used in this work as well as A549 cell culture (human lung adenocarcinoma, ATCC® CCL- 185, USA) and MDCK cell culture (dog kidney cells, Institut Pasteur, France). Small interfering RNAs (siRNAs) (Syntol, Russia) were synthesized for targeting the FLT4, Nup98, and Nup205 genes. Lipofectamin 2000 (Invitrogen, USA) was used for transfection. After 4 hours, the transfected cells were infected with the influenza virus at MOI = 0.1. Virus-containing fluid was collected within three days from the moment of transfection, and the intensity of viral reproduction was assessed by CPE titration and hemagglutination reactions. Viral RNA concentration was determined by RT-PCR. Mann- Whitney U test was used for statistical analysis. In cells treated with siRNA for FLT4, Nup98, and Nup205 genes, there was a significant decrease in the expression of target genes and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) at MOI = 0.1, although the cell survival rate did not decrease significantly. On the first day, the viral titer in cells treated with declared siRNA was lower, on average, by 1 Lg, and on the second and third days, by 2.2-2.3 Lg, compared to cells treated with nonspecific siRNA. During RT-PCR, a significant decrease in the concentration of viral RNA with Nup98.1 and Nup205 siRNA was detected: up to 190 times and 30 times on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies has been decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained while determining the hemagglutinating activity of the virus. The hemagglutinating activity decreased mostly (by 16 times) in cells treated with Nup205 and FLT4.2 siRNAs on the third day. In cells treated with FLT4.1, Nup98.1, and Nup98.2 siRNAs, the hemagglutinating activity decreased by 8 times. We identified a number of genes, such as FLT4, Nup98, and Nup205, whose expression can efficiently suppress viral reproduction when their expression is decreased. The original siRNA sequences were also obtained. These results are important for the creation of therapeutic and prophylactic agents, whose action is based on the RNA interference mechanism.

Abstract 2369: The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression

Abstract Basal-like breast cancer (BLBC) is an aggressive malignancy with a poor prognosis. Persistent activation of NRF2 and STAT3 is considered to stimulate the aggressive behavior of BLBC. An online database, NRF2-ome predicted the possibility of interplay between NRF2 and STAT3 based on domain motif interactions. However, cooperation between NRF2 and STAT3 and its implications for breast cancer progression remain elusive. Immunoprecipitation and in situ proximity analyses revealed that NRF2 and STAT3 directly interact with each other, which predominantly occurred in the nucleus of human breast cancer cells. After confirming the physical interaction between STAT3 and NRF2, we examined whether this could influence the expression or stability of each transcription factor. While silencing of NRF2 failed to alter the STAT3 expression, STAT3 knockdown facilitated degradation of NRF2. Of note, silencing of STAT3 did not affect the mRNA expression of NRF2, suggesting that STAT3-induced accumulation of NRF2 is attributable to stabilization of the preexisting protein. As the NRF2-STAT3 complex was found to accumulate predominantly in the nucleus, we speculate that NRF2 could bind to a functionally active phosphorylated form of STAT3 (P-STAT3Y705). The substitution of Y705 of STAT3 with the non-phosphorylatable phenylalanine (STAT3Y705F) abolished interaction with NRF2. Notably, the dimeric form, but not a monomeric form, of STAT3 interacts directly with the Neh1 and Neh3 domains of NRF2. Next, we investigated the functional significance of STAT3- NRF2 interaction in BLBC growth in vivo. STAT3 knockdown significantly reduced expression of NRF2 in the xenograft tumor tissues derived from MDA-MB-231 human breast cancer cells. Tumors derived from cells transfected with the non-specific control siRNA showed a pronounced interaction between STAT3 and NRF2, whereas such interaction was abolished in the STAT3-silenced xenograft tumor. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and NRF2 mRNA transcripts were correlated with elevated expression of P-STAT3Y705 and NRF2 target proteins in breast cancer patients. Next generation RNA sequencing analysis identified the gene encoding interleukin-23A (IL-23A) upregulated by concurrent binding of STAT3 and NRF2 to its promoter. IL-23A is markedly overexpressed in BLBC, compared with other subtypes of breast cancer. Combined silencing of NRF2 and STAT3 inhibited BLBC growth to a greater extent than that achieved with single siRNA transfection. IL-23A depletion by siRNA also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, these findings suggest that the STAT3-NRF2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer. Citation Format: Su-Jung Kim, Soma Saeidi, Nam-Chul Cho, Seung Hyeon Kim, Han-Byoel Lee, Wonshik Han, Hye-Kyung Na, Young-Joon Surh. The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2369.

Agonism of GPR120 Prevented High Glucose-Induced Apoptosis of Retinal Endothelial Cells through Inhibiting NLRP3 Inflammasome

GPR120 has been reported to ameliorate inflammation in diabetes and diabetic complications. In this study, GW9508, the GPR120 agonist, was utilized in human retinal microvascular endothelial cells (HRMECs) exposed to high glucose (HG) to investigate the involvement of GPR120 in cellular viability and apoptosis as well as the association with the NLRP3 inflammasome. The expression of GPR120 in HRMECs cultured under HG was firstly detected by Western blotting. HRMECs were then assigned to the normal control, GW9508, HG, and HG + GW9508 groups. The expression of the NLRP3 inflammasome consists of NLRP3, ASC, and caspase-1 and was detected by Western blotting and the downstream IL-1β and IL-18 by ELISA. The cellular viability and apoptosis of HRMECs were detected by CCK-8 and flow cytometry, respectively. The expressions of apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. Finally, nonspecific siRNA (NS) or GPR120 siRNA (siGPR120) was transfected to the cells, followed by stimulation with or without GW9508 or HG, and the expression of NLRP3, ASC, and caspase-1 were detected by Western blotting in these groups. GPR120 is expressed in HRMECs, and HG can reduce its expression in a time-dependent manner. GW9508 can attenuate inflammation by reducing the expression of NLRP3, ASC, caspase-1, IL-1β, and IL-18 under HG. GW9508 rescues the viability of HRMCs and reduces cell apoptosis by preventing an increase in Bax expression and the reduction in Bcl-2 expression. Additionally, knockdown of GPR120 by siRNA weakened the effects of GW9508 on NLRP3 inflammasome expression. Activation of GPR120 protects retinal vascular endothelial cells from HG through inhibiting NLRP3 inflammasome. Thus, GPR120 might be a potential therapeutic target to reduce retinal endothelial damage in diabetic retinopathy.

A Mechanistic Role for RECK in the Regulation of Hepatocellular Inflammation

RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane‐anchored glycoprotein, modifies the extracellular matrix involved in various cancers, including hepatocellular carcinoma (HCC). Its role in regulating inflammatory and fibrogenic processes has also been postulated. Both inflammation and fibrosis contribute to progression of nonalcoholic steatohepatitis (NASH) to HCC. Here, we tested the hypothesis that inducing or sustaining RECK expression will inhibit proinflammatory amphiregulin and epidermal growth factor receptor (EGFR) signaling, progression of NASH and the development of HCC using an in vitro cell culture model employing isolated adult mouse primary hepatocytes. Ectopic RECK overexpression (gain‐of‐function) was achieved by the adenoviral (Adv) transduction of murine RECK cDNA. Adv.GFP served as a control. RECK expression was silenced (loss‐of‐function) by transducing RECK‐specific siRNA using lipofectamine. Nonspecific siRNA served as a control. Hepatocytes were exposed to TNFα in the presence or absence of inhibitors against ADAM 10 (A Disintegrin and Metalloproteinase Domain‐Containing Protein 10) and/or ADAM17 – two well‐established pro‐inflammatory sheddases. Results show that RECK overexpression inhibited TNFα‐induced ADAM10/17 activity, and amphiregulin (an EGFR ligand) and EGFR expression. In the presence of ADAM10/17 inhibitors, no further reduction was observed in amphiregulin and EGFR activity, suggesting a maximal inhibitory effect of RECK on EGFR activity. In contrast, silencing RECK significantly increased amphiregulin secretion, and this effect was reversed by inhibition of ADAM10/17, indicating RECK signals via regulation of these sheddases. Thus, RECK not only regulates sheddase activity of ADAM10 and 17, but also downstream amphiregulin and EGFR signaling under pro‐inflammatory stimuli. Because increased and sustained EGFR activity contributes to progression of NASH to HCC in preclinical models, our results indicate that inducing RECK has the potential to inhibit hepatocellular inflammation in the setting of NASH and HCC.

Effects of silencing neuropilin-2 on proliferation, migration, and invasion of colorectal cancer HT-29

ABSTRACT To investigate the effects of silencing neuropilin-2(NRP-2) on the proliferation, migration, and invasion of colorectal cancer(CRC) HT-29. Lipofectamine 2000 was used to transfect specific siRNA for NRP-2 and nonspecific control siRNA into human colorectal cancer HT-29 as the transfection group and meaningless sequence group. HT-29 cultured in a medium was used as the blank control group. The expression levels of NRP-2 mRNA in the cells were detected by real-time fluorescence quantitative PCR. The expressions of proliferation-associated protein Ki-67 in the cells were detected by immunochemical staining. Migration ability was assessed by a monolayer cell scratch wound damage and repair experiment. The Transwell chamber invasion experiment was adopted to determine invasive ability by measuring the number of tumor cells crossing the chamber membrane. Compared with the meaningless sequence group and blank control group, real-time fluorescence quantitative PCR showed that the relative expression level of NRP-2 mRNA in the transfection group was significantly decreased(P < 0.05). Results of immunochemical staining revealed that the expression of Ki-67 protein in the transfected cells was significantly reduced, and the proliferation ability was decreased(P < 0.05). The results further showed that the scratch healing rate of the transfected cells decreased after 24 h of healing(P < 0.05). Results of Transwell invasion assay showed that the number of cells passing through the stromal membrane of the upper chamber to the back of the chamber was significantly reduced in the transfection group(p < 0.05). Silencing NRP-2 could inhibit the proliferation, migration, and invasion of colorectal cancer HT-29.

Premature termination codons in SMN1 leading to spinal muscular atrophy trigger nonsense-mediated mRNA decay

Background and aimsSpinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder caused by SMN1 gene mutations. About 40% of SMN1 subtle mutations produced premature termination codons (PTC). This study aims to determine the capacity of these PTCs to trigger nonsense-mediated mRNA decay (NMD) pathway. MethodsThree nonsense mutations in SMN1, including c.43C > T, c.683T > A and c.844C > T, were investigated by using a minigene system and in vivo splicing assays. Two strategies were supplied: administration of cycloheximide (NMD inhibitor) and knockdown of UPF1 (a key NMD factor) in the cells carrying different minigenes. ResultsThe wild-type minigene exclusively produced correctly spliced transcripts (FL-SMN1). Both the 683T > A and 844C > T expressed remarkably lower FL-SMN1 than the wild-type cells. After cycloheximide treatment, the FL-SMN1 levels in both the 683T > A and 844C > T were increased significantly compared with that of untreated cells. UPF1 knockdown in both the mutant 683T > A and 844C > T caused a dramatically augmentation of FL-SMN1 as compared to that in the cells treated with non-specific control siRNAs. ConclusionOur data provide evidence that c.683T > A and c.844C > T, but not c.43C > T, in SMN1 leading to SMA trigger NMD using a minigene system. Therefore, NMD should be taken into consideration when exploring the pathogenetic mechanisms for these mutations.

Knockdown of &lt;i&gt;FLT4&lt;/i&gt;, &lt;i&gt;Nup98&lt;/i&gt;, and &lt;i&gt;Nup205&lt;/i&gt; cellular genes as a suppressor for the viral activity of Influenza A/WSN/33 (H1N1) in A549 cell culture

Objectives. To evaluate the effect of cellular genes FLT4, Nup98, and Nup205 on the reproduction of the influenza A virus in A549 human lung cancer cell line.Methods. The work was carried out using the equipment of the center for collective use of the I.I. Mechnikov Research Institute of Vaccines and Sera (Russia). The virus-containing fluid was collected within three days from the moment of transfection and infection and the intensity of viral reproduction was assessed by viral titration and hemagglutination reaction. The viral RNA concentration was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). To calculate statistically significant differences between groups, the nonparametric Mann–Whitney test was used.Results. In cells treated with small interfering RNAs (siRNAs) targeted at FLT4, Nup98, and Nup205 genes, a significant decrease in their expression and indicators of viral reproduction (virus titer, hemagglutinating activity, viral RNA concentration) was observed at a multiplicity of infection (MOI) = 0.1. Additionally, it was found that a decrease in the expression of target genes using siRNA does not lead to a significant decrease in cell survival. The viral titer in cells treated with siRNA FLT4.2, Nup98.1, and Nup205 on the first day was lower by an average of 1.0 lg, and on the second and third days, by 2.2–2.3 lg, compared to cells treated with nonspecific siRNA. During real-time RT-PCR, a significant decrease in the concentration of viral RNA was observed with siRNA Nup98.1 (up to 190 times) and Nup205 (up to 30 times) on the first day, 26 and 29 times on the second day, and 6 and 30 times on the third day, respectively. For FLT4.2 siRNA, the number of viral RNA copies decreased by 23, 18, and 16 times on the first, second, and third days. Similar results were obtained when determining the hemagglutinating activity of the virus. The hemagglutinating activity on the third day most strongly decreased in cells treated with siRNA Nup205 and FLT4.2 (16 times). In cells treated with siRNA FLT4.1, Nup98.1, and Nup98.2, hemagglutinating activity decreased by 8 times.Conclusions. In the present study, three cellular genes (FLT4, Nup98, and Nup205) were identified—the decrease in the expression of which effectively suppresses viral reproduction— and the original siRNA sequences were obtained. The results obtained are important for creating therapeutic and prophylactic medication, whose action is based on the RNA interference mechanism.

ARID1A knockdown enhances carcinogenesis features and aggressiveness of Caco-2 colon cancer cells: An in vitro cellular mechanism study

Loss of ARID1A, a tumor suppressor gene, is associated with the higher grade of colorectal cancer (CRC). However, molecular and cellular mechanisms underlying the progression and aggressiveness of CRC induced by the loss of ARID1A remain poorly understood. Herein, we evaluated cellular mechanisms underlying the effects of ARID1A knockdown on the carcinogenesis features and aggressiveness of CRC cells. A human CRC cell line (Caco-2) was transfected with small interfering RNA (siRNA) specific to ARID1A (siARID1A) or scrambled (non-specific) siRNA (siControl). Cell death, proliferation, senescence, chemoresistance and invasion were then evaluated. In addition, formation of polyploid giant cancer cells (PGCCs), self-aggregation (multicellular spheroid) and secretion of an angiogenic factor, vascular endothelial growth factor (VEGF), were examined. The results showed that ARID1A knockdown led to significant decreases in cell death and senescence. On the other hand, ARID1A knockdown enhanced cell proliferation, chemoresistance and invasion. The siARID1A-transfected cells also had greater number of PGCCs and larger spheroid size and secreted greater level of VEGF compared with the siControl-transfected cells. These data, at least in part, explain the cellular mechanisms of ARID1A deficiency in carcinogenesis and aggressiveness features of CRC.

Abstract P130: P-cadherin activates metabolic coupling in the tumor-mesothelial niche: From glycolysis through lactate to lipogenesis

Abstract Peritoneal metastasis affects &amp;gt;70% ovarian cancer patients, and its effective management is an unmet medical goal. Successful adhesion to the peritoneal mesothelium is a key rate-limiting step for metastasis formation, but it is unclear the selective advantage of why ovarian cancer cells colonize specifically to the unique mesothelial environment. Metabolic reprogramming is a hallmark of cancers. Still their role in peritoneal metastasis remains to be fully understood. To overcome the limitations of a lack of tightly controlled experimental model to pinpoint key molecules causing metastasis, we have established an isogenic model of spontaneous human ovarian cancer metastasis. Metabolism is characterized using RNA-seq, RT-PCR, real-time cell metabolic assay, targeted lipidomic analysis and neutral lipid staining. Using coculture, we showed that HM but not NM specifically activated peritoneal mesothelium in a P-cadherin-dependent manner compared to other cadherins, suggesting that there is a unique role for P-cadherin. RNA-seq revealed distinct regulation of lipogenic pathway in non-specific or P-cadherin siRNA treated HM. For these, we have confirmed that P-cadherin could promote the expression of lipogenic genes ACLY, FASN and ACAT2 in HM. Concomitantly, P-cadherin activated glycolysis (HK2, Gpi and PGK1) in the mesothelial cells. Targeted lipidomic analysis further showed that lactate acid that was enriched in the mesothelium was shuttled from the mesothelium to the HM to fuel de novo lipid synthesis in HM, revealing a feed-forward positive loop. Suppressing lactate uptake by using a monocarboxylate transporter 1 inhibitor could abolish the metabolic crosstalk and subsequent metastatic outgrowth. Together, these findings reveal the metabolic differences between metastatic versus non-metastatic tumor cells in understanding ovarian cancer metastasis to the mesothelium, and suggest targeting P-cadherin could be a promising therapeutic approach (RGC17105919). Citation Format: Kun Wang, Jing Ma, Carmen C. L. Wong, Chi Bun Chan, Alice S. T. Wong. P-cadherin activates metabolic coupling in the tumor-mesothelial niche: From glycolysis through lactate to lipogenesis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P130.

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1

Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.

CHRNA1 promotes the pathogenesis of primary focal hyperhidrosis

The aim of the study was to elucidate the involvement of cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) in the pathogenesis of primary focal hyperhidrosis (PFH). The hyperhidrosis mouse model was constructed using pilocarpine injection. The expression levels of CHRNA1 in sweat gland tissues of PFH patients and hyperhidrosis mice were compared using Western blots and quantitative real-time PCR (qRT-PCR) analyses. Sweat secretion in hyperhidrosis mice treated with small-interfering RNA (siRNA) targeting CHRNA1 (si-CHRNA1) or non-specific siRNA were compared. Sweat secretory granules in the sweat gland cells of hyperhidrosis mice were examined using transmission electron microscopy. The serum level of acetylcholine was measured using enzyme-linked immunosorbent assay, while markers associated with PFH, including Aquaporin 5 (AQP5) and Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), were assessed using immunohistochemical assay and Western blots. Brain-derived neurotrophic factor (BDNF) and Neuregulin 1 (NRG-1) in sympathetic ganglia axons of hyperhidrosis mice were quantified using Western blots. CHRNA1 up-regulation is a characteristic of the sweat glands of PFH patients and Hyperhidrosis mice. Silencing CHRNA1 decreased sweat secretion and the number of sweat secretory granules of hyperhidrosis mice. Serum acetylcholine, as well as AQP5 and CACNA1C expression in the sweat glands, was reduced by siCHRNA1. BDNF1 and NRG-1 levels in the sympathetic ganglia axons were also attenuated by siCHRNA1 treatment. CHRNA1 up-regulation is a potential biomarker of PFH and downregulating CHRNA1 could alleviate the symptoms of PFH through inactivating the sympathetic system.

64 SIN3 transcription regulator family member A regulates porcine early embryonic development by modulating CCNB1 expression

SIN3 transcription regulator family member A (SIN3A) is the central scaffold protein of the SIN3/HDAC (histone deacetylase) transcriptional repressor complex. We previously found that SIN3A participates in the mouse pre-implantation development by finetuning HDAC1 expression. However, it remains unresolved whether this functional significance of SIN3A is conserved in other mammals. The objective of this work was thus to characterise the expression profiles and the functional role of SIN3A in pre-implantation development using non-rodent animal models. RNA sequencing results show that a large amount of SIN3A mRNA is present in oocytes and early embryos before embryonic genome activation and a low amount thereafter, suggesting a maternal origin of SIN3A in all species examined. Interestingly, immunofluorescence data show that SIN3A protein level peaks at the 4-cell stage in pigs compared with the morula stage in cattle, suggesting a differential role of SIN3A among species. To explore the function of SIN3A in early embryonic development, we used a short interfering (si)RNA-mediated knockdown approach in porcine parthenogenetic activated (PA) embryos. Immunocytochemical analysis showed that SIN3A levels were diminished ∼80% compared with nonspecific siRNA (NC) injected control (n=3). To monitor the developmental potential of embryo depleted of SIN3A, we injected SIN3A-siRNA into MII stage oocytes, followed by parthenogenetic activation, and percent cleavage and blastocyst formation were recorded. We found that SIN3A knockdown (KD) did not affect the cleavage rate (NC vs. KD, 83.63±3.63% vs. 80.08±4.66%, n=5), but significantly reduced blastocyst rate compared with the NC group (NC vs. KD, 36.64±4.28% vs. 6.33±3.12%, n=5). Specifically, SIN3A depletion in early embryos causes developmental arrest at 2-cell stage in pigs but does not affect early embryonic development in bovines. In contrast with mouse data, SIN3A depletion results in only a slight decrease and even no difference in HDAC1 expression in porcine and bovine early embryos, respectively. In addition, HDAC1 knockdown does not cause 2-cell block but leads to a reduced blastocyst rate, suggesting that the effect of SIN3A depletion on porcine early embryos is independent of HDAC1. RNA-Seq analysis was used to compare the global transcript content between NC and KD 2-cell embryos. A total of 23 genes (14 upregulated and 9 downregulated) had undergone significant changes. Interestingly, cyclin B1 (CCNB1) ranked second among downregulated genes. To test whether knockdown of CCNB1 would display a similar phenotype in porcine early embryos, we injected CCNB1-siRNA into pronuclear stage. CCNB1 KD resulted in a similar phenotype as SIN3A depletion. Injection of exogenous CCNB1 mRNA into SIN3A-depleted embryos could partly rescue embryonic development. In conclusion, our results indicate SIN3A plays an essential role in porcine early embryonic development, probably involving the regulation of CCNB1 expression. This work was funded by National Natural Science Foundation of China, the Anhui Provincial Natural Science Foundation and China Postdoctoral Science Foundation.

Microinjection induces changes in the transcriptome of bovine oocytes

Gene knockdown techniques are widely used to examine the function of specific genes or proteins. While a variety of techniques are available, a technique commonly used on mammalian oocytes is mRNA knockdown by microinjection of small interfering RNA (siRNA), with non-specific siRNA injection used as a technical control. Here, we investigate whether and how the microinjection procedure itself affects the transcriptome of bovine oocytes. Injection of non-specific siRNA resulted in differential expression of 119 transcripts, of which 76 were down-regulated. Gene ontology analysis revealed that the differentially regulated genes were enriched in the biological processes of ATP synthesis, molecular transport and regulation of protein polyubiquitination. This study establishes a background effect of the microinjection procedure that should be borne in mind by those using microinjection to manipulate gene expression in oocytes.

Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α 2-Adrenergic Receptor-Dependent Suppression of Oxidative Stress.

Patients with orthotopic liver transplantation (OLT) frequently develop acute gut injury (AGI), and dexmedetomidine (Dex) has been reported to exert a protective effect against AGI. We investigated whether Dex protects against AGI through antioxidative stress effects by the Nrf2/HO-1 antioxidative signaling pathway. Rats were randomly allocated into a sham group and six orthotopic autologous liver transplantation (OALT) groups receiving different doses of Dex together with/without α2-adrenergic receptor (AR) blockers. Intestinal tissues were collected to visualize the barrier damage and to measure the indexes of oxidative stress. For in vitro studies, rat intestinal recess epithelial cells (IEC-6) underwent hypoxia/reoxygenation (H/R), and the protective role of Dex was evaluated after α2A-AR siRNA silencing. OALT resulted in increased oxidative stress, significant intestinal injury, and barrier dysfunction. Dex attenuated OALT-induced oxidative stress and intestinal injury, which was abolished by the pretreatment with the nonspecific α2A-AR siRNA blocker atipamezole and the specific α2A-AR siRNA blocker BRL-44408, but not by the specific 2B/C-AR siRNA blocker ARC239. Silencing of α2A-AR siRNA also attenuated the protective role of Dex on alleviating oxidative stress in IEC-6 cells subjected to H/R. Dex exerted its protective effects by activating Nrf2/HO-1 antioxidative signaling. Collectively, Dex attenuates OALT-induced AGI via α2A-AR-dependent suppression of oxidative stress, which might be a novel potential therapeutic target for OALT-induced AGI.

Relationship between TIPE2 and caspase-11 during pyroptosis in macrophages of mice

Objective To evaluate the relationship between tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) and caspase-11 during pyroptosis in macrophages of mice. Methods J774A.1 macrophages of mice were divided into 4 groups (n=6 each) using a random number table method: non-specific siRNA (Scr-siRNA) group (S group), Scr-siRNA plus LPS/ATP group (S+ LPS/ATP group), TIPE2-siRNA group (T group) and TIPE2-siRNA plus LPS/ATP group (T+ LPS/ATP group). The corresponding siRNA and Lipofectamine2000 transfection reagents were added to each group, and transfection was performed for 24-48 h, and in addition LPS 1.0 μg/ml was then added, cells were incubated for 5 h, then ATP 5.0 mmol/L was added, and cells were incubated for 1 h in S+ LPS/ATP and T+ LPS/ATP groups.Cells were collected to detect the expression of TIPE2, caspase-11, NOD-like receptor familypyrin domain containing 3 (NLRP3) and caspase-1 (by Western blot). The supernatant was collected for determination of lactic dehydrogenase (LDH) and myeloperoxidase (MPO) activities and concentrations of interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results Compared with group S, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group S+ LPS/ATP (P<0.05). Compared with group T, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05). Compared with group S+ LPS/ATP, the expression of TIPE2 was significantly down-regulated, the expression of caspase-11, NLRP3 and caspase-1 was up-regulated, and and the activities of LDH and MPO and concentrations of IL-1β and IL-18 in supernatant were increased in group T+ LPS/ATP (P<0.05). Conclusion TIPE2 inhibits pyroptosis in macrophages through down-regulating the expression of caspase-11 in mice. Key words: Pyroptosis; Macrophages; Caspases; TIPE2

Effect of NLRP3 mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation injury

Objective: To investigate the effect of NACHT-LRR-PYD- containing proteins 3 (NLRP3) mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation (H/R) injury. Methods: In order to observe whether H/R-treatment could cause pyroptosis, H9c2 cells were divided into 2 groups randomly using the lottery method: control group(without H/R-treatment) and H/R group (in which the H9c2 cells were underwent H/R-treatment). In order to clarify the role of pyroptosis in H/R-injury, H9c2 cells were divided into 4 groups randomly using the lottery method: control group(in which the H9c2 cells were cultivated with normal medium); YVAD group(in which the H9c2 cells were pretreated with z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-YVAD-FMK) 20 μm for 4 hours, then replaced with normal medium); H/R group(H9c2 cells underwent H/R-treatment); YVAD+H/R group (in which the H9c2 cells were pretreated with 20 μm Z-YVAD-FMK for 4 hours before H/R-treatment). To determine whether H/R-induced cell pyroptosis is associated with NLRP3, H9c2 cells were divided into 4 groups randomly using the lottery method: control group (in which cells were transfected with a control nonspecific siRNA); si-NLRP3 group (in which cells were transfected with NLRP3-targeting siRNA); H/R group(in which cells were transfected with a control nonspecific siRNA before H/R-treatment); si-NLRP3+H/R group(in which the H9c2 cells were transfected with NLRP3-targeting siRNA before H/R-treatment). Pore formation on cell membrane was detected by propidium iodide (PI) staining. Cell viability was detected by CCK8 reagent. The protein expression of Caspase-1, interleukin-1β (IL-1β) and NLRP3 was detected by Western blot. Results: (1) The positive rate of PI staining ((26.46±5.15)% vs. (1.69±0.73)%,P<0.01), expression of NLRP3 (0.57±0.16 vs. 0.23±0.06,P<0.01), expression of Caspase-1 (1.07±0.13 vs. 0.37±0.08,P<0.01), and expression of IL-1β (0.38±0.08 vs. 0.16±0.05,P<0.01) were significantly higher in H/R group than in control group. (2)The cell vitality was significantly higher in YVAD+H/R group than in H/R group ((87.31±9.05)% vs. (73.30±7.19)%, P<0.05).The positive rate of PI staining was significantly decreased in YVAD+H/R group than in H/R group ((18.12±4.36)% vs. (26.45±4.60)%, P<0.05). The expression of Caspase-1 (0.72±0.12 vs. 1.07±0.15, P<0.05) and IL-1β(0.29±0.07 vs. 0.39±0.06, P<0.05) were significantly lower in YVAD+H/R group than in H/R group. (3) The cell vitality was significantly increased in si-NLRP3+H/R group than in H/R group ((85.46±7.71)% vs. (72.41±5.53)%, P<0.05). The positive rate of PI staining was significantly lower in si-NLRP3+H/R group than in H/R group ((18.22±4.20)% vs. (26.73±3.26)%, P<0.05). The expression of Caspase-1(0.87±0.07 vs. 1.15±0.15, P<0.05) and IL-1β(0.41±0.07 vs. 0.58±0.10, P<0.05) were significantly decreased in si-NLRP3+H/R group than in H/R group. Conclusion: NLRP3 mediated pyroptosis is involved in H/R injury of myocardial cells.