RNA-FISH Assays Research Articles

Rapid-response RNA-fluorescence in situ hybridization (FISH) assay platform for coronavirus antiviral high-throughput screening

Over the past 25 years, the global community has faced challenges posed by three distinct outbreaks of coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of a novel alphacoronavirus canine CoV (CCoV-HuPn2018) in human patients in Malaysia underscores the potential for crossover infections to humans. The threat of the ever-evolving nature of viral infections as well as the lingering health and socioeconomic effects of the recent SARS-CoV-2 pandemic emphasize the urgent need for advanced antiviral drug screening tools that can be quickly implemented to strengthen preparedness and preventive measures against future outbreaks. Here, we present the development and validation of a novel RNA-fluorescence in situ hybridization (FISH) imaging assay as a 384-well, high-throughput rapid response platform for antiviral drug discovery. RNA-FISH is a powerful tool to visualize specific mRNA in cultured cells using a high-content imaging platform. The flexibility of RNA-FISH probe sets allows for the rapid design of viral genome-specific probes, enabling in vitro assay development to test for inhibition of viral replication by either biologic or small molecule inhibitors. Screening of 170 antiviral compounds in concentration-response demonstrates a strong correlation between the RNA-FISH assay and an immunofluorescence assay (IFA) for both human coronaviruses HCoV-OC43 and HCoV-229E. Additionally, we successfully applied this methodology in the context of CCoV strain 1–71, proving rapid development and deployment, opening new avenues for the evaluation of antiviral drugs to potential future emerging threats.

FTX promotes esophageal cancer progression and desensitizes esophageal cancer cells to ionizing radiation by microRNA-99a/b-3p/WEE1/ERCC1 axis

Abstract Objectives This study was conducted to uncover the underlying cellular and molecular mechanisms of FTX dysregulation in EC. Methods The gene expression in tumor tissues was detected using western blot, immunohistochemistry, and quantitative real-time polymerase chain reaction (qPCR). The dual-luciferase reporter and RNA FISH assays confirmed the interaction between miRNA and target genes. Mouse models for Xenograft and lung metastasis were used to assess EC cell tumorigenesis and metastasis. Results This study finds that up-regulated FTX in patients with esophageal cancer correlates with poor clinical outcomes. Silencing FTX inhibits esophageal cancer cell growth and migration in vitro and tumor metastasis in vivo. miR-99a/b-3p sensitizes esophageal cancer cells to ionizing radiation by WEE1 (Wee1-like protein kinase) and ERCC1 (excision repair cross-complementation group 1) in vitro. Conclusions FTX promotes the malignant biological phenotype of esophageal cancer cells. Mechanistically, FTX acts as a ceRNA to regulate the transcription of WEE1 and ERCC1 by sponging hsa-miR-99a/b-3p.

CircPGM5 regulates Foxo3a phosphorylation via MiR-21-5p/MAPK10 axis to inhibit bladder cancer progression

Bladder cancer (BC) is one of the most prevalent malignant tumors worldwide, and the incidence is especially higher in males. Extensive evidence has demonstrated the pivotal role of circular RNAs (circRNAs) in BC progression. However, the exact regulatory mechanism of circRNAs in BC remains incompletely elucidated and warrants further exploration. This study screened a novel circRNA-circPGM5 from thousands of circRNAs by high-throughput sequencing. We found that circPGM5, originating from the PGM5 gene, was significantly lower expressed in BC tissues. Quantitative real-time PCR (qRT-PCR) verified that circPGM5 showed relatively low expression in 50 pairs of BC tissues and EJ and T24 cells. Notably, circPGM5 expression was correlated with stage, grade, and lymphatic metastasis of BC. Through RNA-FISH assay, we confirmed that circPGM5 predominantly localized in the cytoplasm. Functionally, overexpression of circPGM5 inhibited the proliferation, migration, and invasion of BC cells in vitro. Remarkably, circPGM5 demonstrated markedly significant tumor growth and metastasis suppression in vivo. Mechanistically, we discovered that circPGM5 upregulated the mitogen-activated protein kinase 10 (MAPK10) expression by influencing the oncogenic miR-21-5p activity through miR-21-5p absorption. This modulation of MAPK10 impacted the phosphorylation of the tumor suppressor Foxo3a in BC. In conclusion, our findings uncovered the tumor-suppressing role of circPGM5 in BC via the miR-21-5p/MAPK10/Foxo3a axis.

LINC01133 contributes to the malignant phenotypes of non-small cell lung cancer by targeting miR-30b-5p/FOXA1 pathway.

This study aimed to explore the mechanism of action of LINC01133 in non-small cell lung cancer. LINC01133 expression in NSCLC patient tissues and cells was detected by qRT-PCR. After transfecting siRNA-LINC01133 in NSCLC cells, the proliferation and invasive migration ability of the cells were assessed via CCK-8 and Transwell assay, respectively. The sublocalization of LINC01133 in NSCLC cells was analyzed by bioinformatics prediction and nucleoplasm separation assay and RNA-FISH assay. Analysis of the binding relationship between LINC01133, FOXA1 and miR-30b-5p was all through bioinformatics website analysis, dual-luciferase reporter and RNA Pulldown assay. Functional rescue experiments confirmed the character of miR-30b-5p and FOXA1 in LINC01133 regulating the NSCLC cells biological behavior. LINC01133 high expressions were found in NSCLC tissues and cells. siRNA-LINC01133 treatment inhibited NSCLC cells malignant behavior. Mechanistically: LINC01133 promoted FOXA1 expression through adsorption binding of miR-30b-5p. Knocking down miR-30b-5p expression or up-regulating FOXA1 expression was able to reverse siRNA-LINC01133 inhibitory effect of tumor cell malignant behavior. LINC01133 promoted FOX1 expression by competitively binding miR-30b-5p, which attenuated the targeting inhibitory effect of miR-30b-5p on FOXA1 and ultimately promoted proliferation and invasive migration of NSCLC cells.

Abstract 465: Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors

Abstract Most humans infected with Epstein-Barr virus (EBV) remain asymptomatic throughout the whole life. However, EBV is linked to several human cancers, including Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC), and gastric carcinoma. Among these EBV-associated tumors, EBV genome is present in 100% of NPC tumor cells. In NPC cells, EBV establishes a latent infection, expressing limited number of viral proteins but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, also known as BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs includes BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). Although various functions of BART miRNAs have been identified, the role of EBV lnc-BARTs remains largely unknown. In this study, we provide evidence demonstrating that lnc-BARTs functions as a regulatory long non-coding RNA, referred to as lnc-BARTs. These lnc-BARTs play keys role in modulating the core networks that maintains EBV latency and promotes NPC oncogenesis through epigenetic mechanisms. Our study found that the apoptosis rate of NPC cells is significantly increased following the knockdown of EBV lnc-BARTs, indicating their anti-apoptotic role in EBV-associated tumors. Mass spectrometry analysis identified several host factors, including transcriptional regulators BRD4 and SC35’ interact with EBV lnc-BARTs in NPC cells. RNA-FISH assays showed lnc-BARTs co-localized with BRD4 and SC35 complexes in nuclear s peckles and such interaction were disrupted by JQ1, a BRD4 competitive inhibitor. Binding of lnc-BARTs to BRD4 RNA was confirmed using immunoprecipitation and pulldown assays. ATAC sequencing and transcriptome analyses showed that BART knockdown in EBV-harboring NPC cell line C666-1 led to reduced chromatin accessibility, downregulation of oncogenes, and decreased EBV episome replication and maintenance. Quantitative qPCR and DNA FISH analyses of cell samples treated with shRNA targeting lnc-BARTs or BRD4 reduced EBV copy numbers and EBNA1 RNA expression levels in EBV-infected NPC cells. Finally, ChIP sequencing analysis revealed that lnc-BARTs is critical for BRD4 binding to the oriP region of the EBV genome. These findings support a mechanism by which EBV lnc-BARTs epigenetically regulate host gene expression through functional interactions with elongation regulatory machinery and contribute to the maintenance of EBV latent genome by interaction with BRD4 complex. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Honglin Chen. Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 465.

CircRNA254 functions as the miR-375 sponge to inhibit coelomocyte apoptosis via targeting BAG2 in V. splendidus-challenged Apostichopus japonicus

Circular RNAs (circRNAs) function as immune regulators in many biological processes in mammals, while their function and underlying mechanisms in invertebrates are largely unexplored. In this study, the competing endogenous RNA (ceRNA) mechanism of circRNA that sponges miR-375 and thus regulates AjBAG2-mediated coelomocyte apoptosis was evaluated in Apostichopus japonicus. The results showed that circRNA254 (circ254) was significantly down-regulated in the intestines and coelomocytes after Vibrio splendidus challenge or Lipopolysaccharide exposure, which matched the RNA-seq results in A. japonicus within skin ulceration syndrome. Dual-luciferase and RNA FISH assays indicated that circ254 could directly combine with miR-375, in which circ254 possesses three binding sites of miR-375. Moreover, circ254 knockdown significantly promoted the coelomocyte apoptosis levels upon pathogen infection in vivo and in vitro. Furthermore, circ254 silencing could also down-regulate AjBAG2 expression and thereby promoting the levels of coelomocyte apoptosis levels and the expression of caspase 3, which the phenomenon could be reversed by treatment with miR-375 inhibitors. Taken together, our results confirmed that circ254 functions as a ceRNA of AjBAG2 by sponging miR-375, resulting in the inhibition of coelomocyte apoptosis in A. japonicus.

P-425 Biosilk and ovarioids: recombinant silk as a new tool for establishing a 3D-culture system for human ovarian primary cells

Abstract Study question Can Biosilk be used as scaffold for establishing a 3D-culture system to support attachment and growth of primary cells derived from adult ovarian biopsies? Summary answer The use of recombinant spider silk-based scaffold allowed the formation of 3D-structured ovarioids from both cortex and medulla isolated from 5 different patients. What is known already Fertility in women is adversely affected by several factors, such as age, environmental pollutants and diseases requiring gonadotoxic treatments. Understanding ovarian cell composition and organization may pave the way to novel fertility preservation methods with the ultimate goal of being applied to clinics. So far, there are no clinically established methods for in vitro growth and maturation of human ovarian follicles leading to mature competent oocytes. Therefore, exploring new 3D-culture systems for in vitro reconstruction of ovarian somatic cell niche could lead to development of novel tools to support growth of patient-specific follicles. Study design, size, duration Ovarian tissue was collected from gender reassignment patients (GRP) after informed written consent at Karolinska University Hospital Huddinge from 2019 to 2022. After separation of cortex and medulla, the samples were mechanically and enzymatically dissociated into single-cell suspensions and used to evaluate 3D- and 2D-culture methods. Freshly fixed biopsies from cortex and medulla (3x3x1 mm3) were used as control for the transcriptomic analysis and RNA-FISH assay. Participants/materials, setting, methods Tissue was obtained from five patients aged 23-31 years. Dissociated primary somatic cells seeded on Biosilk-foam scaffolds were kept in culture for 2 weeks, followed by detachment, equal division of the foams and suspension culture for additional 4 weeks (BioSilk-Ovarioids, BSO). BSOs were harvested in PFA and RNAlater (n = 6/patient, respectively) for morphological and transcriptomic analysis. Protein ZO1, and cell type-specific marker genes (AMHR2, PDGFRa, CLDN5, GJA4) were evaluated via immunodetection and RNA-FISH assay, respectively. Main results and the role of chance The sizes of BSOs from both cortex and medulla ranged between 0.5-1 mm at the end of the culture, appearing highly compacted under optical microscope. HE-stained BSO sections revealed that cells were distributed throughout the foams, showing good attachment and distribution. Marker genes were selected for specific cell types [AMHR2-granulosa, PDGFRa-stroma, CLDN5-endothelial, GJA4-perivascular cells (Wagner et al. 2020)] and used to study the representation of different somatic cells in the BSOs. The RNA-FISH analysis confirmed the presence of all cell type-specific marker genes, with predominating presence of stromal cells (PDGFRa). Newly formed ZO1-specific gap junctions were detected in both cortex- and medulla-derived BSOs, appearing mainly in the outer part of the structures. Interestingly, cell layers surrounding the original Biosilk scaffold were observed, especially in medulla-BSOs. Transcriptomic profiling of the samples showed clear separation to three main clusters by principal component analysis: freshly fixed tissue, 2D-cultures, and BSO. Moreover, clustering analysis of differentially expressed genes (DEGs) showed the presence of gene clusters in fresh tissues affected by both 3D/2D-cultures. Further analyses will focus on identification of significantly affected gene ontologies and pathways, which will further guide the optimization of the BSO culture system. Limitations, reasons for caution The tissue was derived from GRPs who always receive androgen treatments prior to surgery. As hormonal treatment may affect the ovarian environment, further trials with untreated patient samples will be needed to generalize the model to fertility preservation patients. Wider implications of the findings The establishment and development of the BSO 3D-culture system may enable the construction of patient-specific clinically significant tools for in vitro folliculogenesis. This would open new avenues for fertility restoration in patients who cannot receive auto-transplants and treatment of infertility in premature ovarian insufficiency if residual follicles remain in tissue. Trial registration number Not applicable

P-429 Transcriptomic heterogeneity within cortical ovarian follicle pool in child and adult

Abstract Study question What are the transcriptomic differences between child and adult ovarian follicles in the human cortex? Summary answer Our results suggest that human ovarian cortical follicles show marked differences before and after puberty, with the greatest differences being present in the secondary stage. What is known already The ovaries and their main functional structures, follicles, are important for determining the reproductive health. Follicle growth and maturation are controlled by multiple mechanisms, including the hypothalamus pituitary ovarian axis and local paracrine signalling. Although dormant primordial follicles are activated to grow even in infancy, all growing follicles before the first menarche are bound to undergo atresia. Follicle growth activation in infancy and adulthood remains poorly understood. Additionally, child and adult follicles behave differently during in vitro culture suggesting differing biology, raising many questions e.g., about their suitability for fertility preservation. Study design, size, duration Ovarian cortical tissues from children were collected through fertility preservation program at the Helsinki Children’s hospital (Finland). Families of underage patients signed an informed written consent. Adult ovarian tissue was collected from gender reassignment patients at the Helsinki University hospital and Karolinska university hospital after informed written consent. Follicles at different maturation stages were isolated from ovarian cortex for single follicle transcriptomic analysis. Freshly-fixed cortical biopsies (3x3x1 mm3) were used for validation of transcriptomic analysis. Participants/materials, setting, methods Single viable follicles (n = 120, 60/age group) were mechanically and enzymatically isolated from frozen-thawed ovarian tissue from four adults (age >18 yrs) and five children (age 1-11 yrs) and processed for RNA-sequencing. Individual follicles were lysed, prepared for RNA libraries following Smart-Seq2 protocol and sequenced using NextSeq 500 platform. After quality assessment, 109 follicles were analysed (Adult n = 54, Child n = 55). DDX4, AMH, FOXL2 and DAZL transcripts and proteins localization were evaluated via immunodetection and RNA-FISH assay. Main results and the role of chance Principal component analysis (PCA) of all sequenced follicles demonstrated an expected timely progression of their stages based on maturation, and an overall similarity between child and adult. Interestingly, PCA displayed two distinct clusters of follicles in both adult and child samples. The first group contained only growing follicles and, after differentially expressed gene (DEG) analysis, demonstrated lower expression of traditional oocyte markers (group 1). The second group consisted of primordial and growing follicles and had lower expression of traditional granulosa marker (group 2). Group-related differential expression of main oocyte (DDX4, DAZL) and granulosa (FOXL2, AMH) marker genes were validated in tissue sections with RNA-FISH. Downstream analyses focused on group 2 follicles. When child and adult follicles were compared to each other, the highest number of DEGs were found in the secondary stage (primordial n = 32, primary n = 14, secondary n = 387). Pathway analysis of DEG displays enrichment of oestrogen-related related pathways in adult secondary follicles. Furthermore, we analysed DEGs between the follicular stages per age group, finding high number of DEGs between primordial and primary follicles (Adult n = 1663, Child n = 1914). The majority of those genes are unique to the age group (common=512). Limitations, reasons for caution Adult ovarian tissue is obtained from gender reassignment patients, who receive androgen treatments prior to surgery. Child ovarian tissue was collected from fertility preservation programme where some patients were exposed to chemotherapy prior to tissue collection. Wider implications of the findings The differences between child and adult follicles encourage further investigation of the suitability of child ovarian tissue in current fertility preservation protocols. Understanding transcriptomic changes during early folliculogenesis and reasons for follicle degeneration can help tailor protocols for in vitro growth of follicles. Trial registration number not applicable

CircNFATC3 promotes the proliferation of gastric cancer through binding to IGF2BP3 and restricting its ubiquitination to enhance CCND1 mRNA stability

BackgroundInsulin like growth factor II mRNA binding protein 3 (IGF2BP3) is an RNA binding protein with multiple roles in regulation of gene expression at the post-transcriptional level and is implicated in tumorigenesis and progression of numerous cancers including gastric cancer (GC). Circular RNAs (circRNAs) are a diverse endogenous noncoding RNA population that have important regulatory roles in cancer. However, circRNAs that regulate the expression of IGF2BP3 in GC is largely unknown.MethodsCircRNAs that bound to IGF2BP3 were screened in GC cells using RNA immunoprecipitation and sequencing (RIP-seq). The identification and localization of circular nuclear factor of activated T cells 3 (circNFATC3) were identified using Sanger sequencing, RNase R assays, qRT-PCR, nuclear-cytoplasmic fractionation and RNA-FISH assays. CircNFATC3 expression in human GC tissues and adjacent normal tissues were measured by qRT-PCR and ISH. The biological role of circNFATC3 in GC was confirmed by in vivo and in vitro experiments. Furthermore, RIP, RNA-FISH/IF, IP and rescue experiments were performed to uncover interactions between circNFATC3, IGF2BP3 and cyclin D1 (CCND1).ResultsWe identified a GC-associated circRNA, circNFATC3, that interacted with IGF2BP3. CircNFATC3 was significantly overexpressed in GC tissues and was positively associated with tumor volume. Functionally, the proliferation of GC cells decreased significantly after circNFATC3 knockdown in vivo and in vitro. Mechanistically, circNFATC3 bound to IGF2BP3 in the cytoplasm, which enhanced the stability of IGF2BP3 by preventing ubiquitin E3 ligase TRIM25-mediated ubiquitination, thereby enhancing the regulatory axis of IGF2BP3-CCND1 and promoting CCND1 mRNA stability.ConclusionsOur findings demonstrate that circNFATC3 promotes GC proliferation by stabilizing IGF2BP3 protein to enhance CCND1 mRNA stability. Therefore, circNFATC3 is a potential novel target for the treatment of GC.

Identification of a Novel lncRNA LNC_001186 and Its Effects on CPB2 Toxin-Induced Apoptosis of IPEC-J2 Cells.

The Clostridium perfringens (C. perfringen) beta2 (CPB2) toxin produced by C. perfringens type C (CpC) can cause necrotizing enteritis in piglets. Immune system activation in response to inflammation and pathogen infection is aided by long non-coding RNAs (lncRNAs). In our previous work, we revealed the differential expression of the novel lncRNA LNC_001186 in CpC-infected ileum versus healthy piglets. This implied that LNC_001186 may be a regulatory factor essential for CpC infection in piglets. Herein, we analyzed the coding ability, chromosomal location and subcellular localization of LNC_001186 and explored its regulatory role in CPB2 toxin-induced apoptosis of porcine small intestinal epithelial (IPEC-J2) cells. RT-qPCR results indicated that LNC_001186 expression was highly enriched in the intestines of healthy piglets and significantly increased in CpC-infected piglets' ileum tissue and CPB2 toxin-treated IPEC-J2 cells. The total sequence length of LNC_001186 was 1323 bp through RACE assay. CPC and CPAT, two online databases, both confirmed that LNC_001186 had a low coding ability. It was present on pig chromosome 3. Cytoplasmic and nuclear RNA isolation and RNA-FISH assays showed that LNC_001186 was present in the nucleus and cytoplasm of IPEC-J2 cells. Furthermore, six target genes of LNC_001186 were predicted using cis and trans approaches. Meanwhile, we constructed ceRNA regulatory networks with LNC_001186 as the center. Finally, LNC_001186 overexpression inhibited IPEC-J2 cells' apoptosis caused by CPB2 toxin and promoted cell viability. In summary, we determined the role of LNC_001186 in IPEC-J2 cells' apoptosis caused by CPB2 toxin, which assisted us in exploring the molecular mechanism of LNC_001186 in CpC-induced diarrhea in piglets.

Abstract 3749: Oncogenic role of Epstein-Barr virus lnc-BARTs in EBV associated tumors

Abstract While majority of humans infected with Epstein-Barr virus (EBV) is asymptomatic for whole life, EBV is found to associate with several human cancers, including Burkitt’s lymphoma, Hodgkin’s diseases, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC) and gastric carcinoma. Among these EBV associated tumors, EBV genome is found in 100% of NPC tumor cells. EBV establishes a latent infection in NPC cells, expressing few viral proteins, but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, designated BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs comprises BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). While versatile functions of BART miRNAs have been revealed, little is known about the role of EBV lnc-BARTs. Here, we provided evidence to show that BARTs mRNA which derived from multiple exons of BART function as regulatory long non-coding RNA, namely lnc-BARTs, in modulating the core network for maintaining EBV latency and promoting NPC oncogenesis through epigenetic mechanisms. Our results showed that the apoptosis rate of NPC cell lines with knockdown of EBV lnc-BARTs was significantly increased, suggesting that lnc-BARTs have an anti-apoptotic effect in EBV associated tumors. MS analysis was performed to identify host factors which interact with EBV lnc-BARTs in NPC cells. Lnc-BARTs were shown to directly interact with several transcriptional regulators, including BRD4 and SC35. Notably, RNA-FISH assays demonstrated that lnc-BARTs co-localize with BRD4 and SC35 complexes in nuclear speckles, with these complexes being disrupted by treatment with JQ1, a BRD4 competitive inhibitor. RNA immunoprecipitation and RNA pulldown assays confirmed that lnc-BARTs bind to BRD4. ATAC sequencing analysis and transcriptome analysis further demonstrated that knockdown of BARTs in the EBV-harboring NPC cell line, C666-1, resulted in globally reduced chromatin accessibility downregulation of oncogenes like MYC and BCL2. In addition, ChIP sequencing analysis revealed that lnc-BARTs interfere PolII phosphorylation on promoters of genes. Our findings suggest that EBV lnc-BARTs are involved in epigenetic modulation of host gene expression through functional interaction with elongation regulatory machinery to maintain EBV latency and drive tumorigenesis in NPC. Citation Format: LIU JIAYAN, Bobo Wing-Yee Mok, Songtao He, Honglin Chen. Oncogenic role of Epstein-Barr virus lnc-BARTs in EBV associated tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3749.

MALAT-1 regulates the AML progression by promoting the m6A modification of ZEB1.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) is abnormally upregulated in various human cancers. However, the role of MALAT-1 in acute myeloid leukemia (AML) remains unclear. This study investigated the expression and function of MALAT-1 in AML. MTT assay was used to determine cell viability, qRT-PCR was applied to determine the RNA levels. Western blot was performed to detect the protein expression. Flow cytometry was conducted to measure cell apoptosis. RNA pull-down assay was carried out to detect the interaction between MALAT-1 and METTL14. RNA FISH assay was performed to determine the localization of MALAT-1 and METTL14 in AML cells. Our results have revealed the key role of MEEL14 and m6A modification in AML. Besides, MALAT-1 was significantly up-regulated in AML patients. MALAT-1 knockdown inhibited the proliferation, migration and invasion of AML cells, and induced cell apoptosis; additionally, MALAT-1 binding to METTL14 promoted the m6A modification of ZEB1. Besides, ZEB1 overexpression partially reversed the effect of MALAT-1 knockdown on the cellular functions of AML cells. Taken together, MALAT-1 promoted the aggressiveness of AML through regulating m6A modification of ZEB1.

Circular RNA circPGD contributes to gastric cancer progression via the sponging miR-16-5p/ABL2 axis and encodes a novel PGD-219aa protein

CircRNAs have critical effects on tumor development and progression. However, circPGD effect on gastric cancer (GC) is still elusive. Nuclear and cytoplasmic RNA fractionation, and RNA-FISH assay examined the localization of circPGD in MGC-803 cells. qRT-PCR was conducted to detect the expression and prognostic significance of circPGD, miR-16-5p, and ABL2 within GC tissues. Meanwhile, qRT-PCR, luciferase reporter assays, rescue, and western blotting assays confirmed the interactions between circPGD, miR-16-5p, and ABL2. Transwell, wound healing, and colony-formation assays, as well as CCK-8 and cell apoptosis assays, analyzed the functions of circPGD, miR-16-5p, ABL2, as well as PGD-219aa within GC cells. Western blotting and cell immunofluorescence experiments detected the differences in the expression of the related proteins. Finally, xenograft and metastatic mouse models were used to investigate circPGD function in vivo. Mass spectrometry was used to detect the existence of PGD-219aa in MGC-803 cells. CircPGD was localized in the cytoplasm and nucleus of MGC-803 cells. Compared with the control, circPGD and ABL2 expression increased within GC tissues and cells, and the miR-16-5p level was decreased. Functionally, circPGD promoted cell proliferation, migration and suppressed apoptosis in vitro. Mechanistically, circPGD sponged miR-16-5p for relieving miR-16-5p suppression on the corresponding target ABL2 via the SMAD2/3 and YAP signaling pathways. In addition, circPGD encodes a novel PGD-219aa protein that can enhance the growth and migration of GC cells, while inhibiting GC cells apoptosis via the SMAD2/3 and YAP signaling pathways. Furthermore, circPGD overexpression enhanced tumor aggressiveness, while circPGD knockdown inhibited tumor growth. Overall, circPGD has a novel oncogenic effect on GC cells, indicating the potential of circPGD as the tumorigenic factor and a promising diagnostic marker for GC.

CircHECTD1 attenuates apoptosis of alveolar epithelial cells in acute lung injury

AbstractCircular RNAs (circRNAs) play important roles in many lung diseases. This study aimed to investigate the role of circHECTD1 in acute lung injury (ALI). The mouse and cell models of ALI were induced by lipopolysaccharide (LPS). The apoptosis of alveolar epithelial cells (AECs) was detected by flow cytometry. The relationships between circHECTD1, miRNAs, and target genes were assessed by RNA pull-down, luciferase reporter gene, and RNA-FISH assays. circHECTD1 was downregulated in LPS-induced human and mouse AECs (HBE and MLE-12). The knockdown of circHECTD1 increased the apoptotic rates and the expressions of miR-136 and miR-320a, while its overexpression caused opposite effects in LPS-induced HBE and MLE-12 cells. Mechanistically, circHECTD1 bound to miR-320a and miR-136. miR-320a targeted PIK3CA and mediated the effect of circHECTD1 on PIK3CA expression. miR-136 targeted Sirt1 and mediated the effect of circHECTD1 on Sirt1 expression. Silencing PIK3CA and/or Sirt1 reversed the effect of circHECTD1 overexpression on the apoptosis of LPS-induced HBE and MLE-12 cells. In vivo, overexpression of circHECTD1 alleviated the LPS-induced ALI of mice. Our findings suggested that circHECTD1 inhibits the apoptosis of AECs through miR-320a/PIK3CA and miR-136/Sirt1 pathways in LPS-induced ALI.

CircARID1A binds to IGF2BP3 in gastric cancer and promotes cancer proliferation by forming a circARID1A-IGF2BP3-SLC7A5 RNA–protein ternary complex

BackgroundGastric cancer (GC) is one of the most common malignant tumors in China. Circular RNAs (circRNAs) are novel non-coding RNAs with important regulatory roles in cancer progression. IGF2BP3 has been found to play oncogenic roles in various cancers including GC, while the exact mechanism of IGF2BP3 is largely unknown.MethodsThe expression of IGF2BP3 in GC was evaluated by Western Blot and bioinformatics analysis. CircRNA expression profiles were screened via IGF2BP3 RIP-seq in GC. Sanger sequencing, RNase R digestion, nucleo-plasmic separation and RNA-FISH assays were used to detect the existence and expression of circARID1A. RNA ISH assay was employed to test the expression of circARID1A in paraffin-embedded GC tissues. Moreover, the function of circARID1A on cellular proliferation was assessed by CCK-8, plate colony formation, EdU assays and GC xenograft mouse model in vivo. Furthermore, the location or binding of circARID1A, IGF2BP3 protein and SLC7A5 in GC was evaluated by RNA-FISH/IF or RNA pull-down assays.ResultsWe identified a novel circRNA, circARID1A, that can bind to IGF2BP3 protein. CircARID1A was significantly upregulated in GC tissues compared with noncancerous tissues and positively correlated with tumor length, tumor volume, and TNM stage. CircARID1A knockdown inhibited the proliferation of GC cells in vitro and in vivo and circARID1A played an important role in the oncogenic function of IGF2BP3. Mechanistically, circARID1A served as a scaffold to facilitate the interaction between IGF2BP3 and SLC7A5 mRNA, finally increasing SLC7A5 mRNA stability. Additionally, circARID1A was able to directly bind SLC7A5 mRNA through complementary base-pairing and then formed the circARID1A-IGF2BP3-SLC7A5 RNA–protein ternary complex and promoted the proliferation of GC via regulating AKT/mTOR pathway.ConclusionsAltogether, our data suggest that circARID1A is involved in the function of IGF2BP3 and GC proliferation, and the circARID1A-IGF2BP3-SLC7A5 axis has the potential to serve as a novel therapeutic target for GC.

Circular RNA circ-TNPO3 inhibits clear cell renal cell carcinoma metastasis by binding to IGF2BP2 and destabilizing SERPINH1 mRNA.

BackgroundClear cell renal cell carcinoma (ccRCC) is a common malignant tumour of the urinary tract. The major causes of poor prognosis are the lack of early diagnosis and metastasis. Accumulating research reveals that circular RNAs (circRNAs) can play key roles in the development and the progression of cancer. However, the role of circRNAs in ccRCC is still uncertain.MethodsThe circRNAs microarray (n = 4) was performed to investigate the circRNAs with differential expression in ccRCC tissues. The candidate circRNA was selected based on the cut‐off criteria, such as circRNA expression abundance, circRNA size and the design of divergent primers. The circ‐transportin‐3 (TNPO3) levels in ccRCC tissues were tested by quantitative real‐time (qRT)‐PCR (n = 110). The characteristics and subcellular localization of circ‐TNPO3 were identified via RNase R assay, qRT‐PCR and fluorescence in situ hybridization (FISH). Then, we explored the biological roles of circ‐TNPO3 in ccRCC via the function experiments in vitro and in vivo. RNA pull‐down, RNA immunoprecipitation, bioinformatic analysis, RNA‐FISH assays and rescue assays were applied to validate the interactions between circ‐TNPO3, insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) and serpin family H member 1 (SERPINH1) to uncover the underlying molecular mechanisms of circ‐TNPO3.ResultsWe detected the obvious downregulation of circ‐TNPO3 in ccRCC compared to matched adjacent normal tissues (n = 110). The lower circ‐TNPO3 expression was found in ccRCC patients with distant metastasis, higher World Health Organization/International Society of Urologic Pathologists (WHO/ISUP) grade and more advanced tumour T stage. In vitro and in vivo, circ‐TNPO3 significantly suppressed the proliferation and migration of ccRCC cells. Mechanistically, we elucidated that circ‐TNPO3 directly bound to IGF2BP2 protein and then destabilized SERPINH1 mRNA. Moreover, IGF2BP2/SERPINH1 axis was responsible for circ‐TNPO3's function of inhibiting ccRCC metastasis. Epithelial splicing regulatory protein 1 (ESRP1) was probably involved in the biogenesis of circ‐TNPO3.ConclusionsCirc‐TNPO3 can suppress ccRCC progression and metastasis via directly binding to IGF2BP2 protein and destabilizing SERPINH1 mRNA. Circ‐TNPO3 may act as a potential target for ccRCC treatment.

Exosomal circ-CACNG2 promotes cardiomyocyte apoptosis in multiple myeloma via modulating miR-197-3p/caspase3 axis

Multiple myeloma (MM) secreted exosomes are essential in MM-related complications such as osteolytic bone lesions and renal failure, but their role and underlying mechanism in cardiac complications has not yet been clarified. Here, we investigated the effects of U266 (a MM cell line) exosomes (U266-exo) on regulating the viability, cell cycle, oxidative stress and apoptosis of H9C2 cells and the role of circ-CACNG2 in these effects. We found that U266-exo coculture significantly inhibited viability and promoted apoptosis of H9C2 cells, and serum exosomes of MM patients harbored high level of circ-CACNG2. The clinical data analyses indicated that circ-CACNG2 was an independent prognostic and diagnostic indicator of MM-related cardiac complications. Also, in vitro experiments showed that circ-CACNG2 inhibited viability and promoted apoptosis of H9C2 cells. RIPA, pull-down assays, dual-luciferase reporter assays, and RNA FISH assays revealed that miR-197-3p could bind to circ-CACNG2 and caspase3 directly. Rescue experiments proved that circ-CACNG2 can increase the expression of caspase3 by binding to and decreasing the expression of miR-197-3p. In conclusion, MM-exosomes could inhibit cardiomyocyte viability and promote apoptosis partially through circ-CACNG2/miR-197-3p/caspase3 axis.

LncRNA LINC00152 promotes oral squamous cell carcinoma growth via enhancing Upstream Transcription Factor 1 mediated Mitochondrial Ribosomal Protein L52 transcription.

LINC00152 (long intergenic non-protein coding RNA 152) was identified as an oncogenic lncRNA in multiple cancers. In the current study, we aimed to explore the transcriptional profile of LINC00152 in oral squamous cell carcinoma (OSCC) and its regulations at the transcriptional level. Bioinformatic analysis was performed by extracting the OSCC subset from The Cancer Genome Atlas (TCGA)-Head and Neck Squamous Cell Carcinoma (HNSC). LINC00152subcellular localization and its interacting transcriptional factors (TFs) were explored. Dual-luciferase assay and ChIP-qPCR were applied to study transcriptional regulation. In vitro and in vivo tumor cell growth models were used for functional assays. NR_024206.2 was the dominant isoform that accounts for 80% of all transcripts of LINC00152. LINC00152 upregulation was associated with unfavorable survival of patients with OSCC. LINC00152knockdown significantly impaired OSCC cell growth in vitro and in vivo. RNA FISH assay confirmed nuclear and cytoplasmic distribution of LINC00152. It physically interacted with Upstream Transcription Factor 1 (USF1), a common transcription factor in mammalian cells. USF1 could bind to the promoter region of MRPL52 (Mitochondrial Ribosomal Protein L52) and activate its transcription. LINC00152 could enhance the binding, thereby indirectly elevating MRPL52 expression. USF1 or MRPL52knockdown slowed the proliferation of OSCC cells and partly canceled LINC00152-mediated growth-promoting effects. This study revealed a novel LINC00152-USF1/MRPL52 axis promoting OSCC tumor growth.

Long non-coding RNA HCG11 enhances osteosarcoma phenotypes by sponging miR-1245b-5p that directly inhibits plakophilin 2

ABSTRACT Long non-coding RNA (lncRNA) HCG11 can regulate various cancers through the ceRNA network. However, its role in osteosarcoma (OS) remains unknown. The HOS and Saos-2 cell lines were used for in vitro analyses. HCG11 and plakophilin 2 (PKP2) silencers, a miR-1245b-5p mimic, and a miR-1245b-5p inhibitor were utilized for the regulation analysis of lncRNA HCG11, miR-1245b-5p, and PKP2. Cell Counting Kit-8, wound healing, and transwell assays were used for cell proliferation, migration, and invasion analyses, and caspase-3 activity assay was used to measure cell apoptosis. The expression levels of lncRNA HCG11, miR-1245b-5p, and PKP2 were evaluated by quantitative real-time PCR and Western blotting. The distribution of lncRNA HCG11 was assessed using the RNA-FISH assay. The sponging and targeting roles of HCG11 and PKP2 on miR-1245b-5p were confirmed by dual-luciferase reporter analysis. An RNA immunoprecipitation assay was used to assess the binding between lncRNA HCG11 and miRNA-1245b-5p. We found that the lncRNA HCG11 was significantly upregulated in OS. LncRNA HCG11 silencing inhibits OS progression by repressing cell proliferation, migration, and invasion, and promoting cell apoptosis. RNA-FISH analysis indicated that lncRNA HCG11 was located in the cytoplasm. Mechanistic experiments showed that lncRNA HCG11 sponges miR-1245b-5p and negatively regulates miR-1245b-5p expression. Upregulated lncRNA HCG11 promotes proliferation, migration, and invasion, and inhibits apoptosis by inhibiting miR-1245b-5p in OS cells. PKP2 was verified as a target gene of miR-1245b-5p. Upregulated PKP2 promotes proliferation, migration, and invasion, and inhibits apoptosis by inhibiting miR-1245b-5p in OS. In conclusion, the HCG11/miR-1245b-5p/PKP2 axis promotes OS expression by promoting cell proliferation, migration, and invasion, and inhibiting apoptosis.

A CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.