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BACKGROUND: Syndrome coronavirus-2 (SARS-CoV-2) has developed various strategies to evade the antiviral impact of type I IFN. Non-structural proteins and auxiliary proteins have been extensively researched on their role in immune escape. Nevertheless, the detailed mechanisms of structural protein-induced immune evasion have not been well elucidated. METHODS: Human alveolar basal epithelial carcinoma cell line (A549) was stimulated with polyinosinic-polycytidylic acid (PIC) and independently transfected with four structural proteins expression plasmids, including nucleocapsid (N), spike (S), membrane (M) and envelope (E) proteins. By RT-qPCR and ELISA, the structural protein with the most pronounced inhibitory effects on IFN-ß induction was screened. RNA-sequencing (RNA-Seq) and two differential analysis strategies were used to obtain differentially expressed genes associated with N protein inhibition of IFN-ß induction. Based on DIANA-LncBase and StarBase databases, the interactive competitive endogenous RNA (ceRNA) network for N protein-associated genes was constructed. By combining single-cell sequencing data (GSE158055), lncRNA-miRNA-mRNA axis was further determined. Finally, RT-qPCR was utilized to illustrate the regulatory functions among components of the ceRNA axis. RESULTS: SARS-CoV-2 N protein inhibited IFN-ß induction in human alveolar epithelial cells most significantly compared with other structural proteins. RNA-Seq data analysis revealed genes related to N protein inhibiting IFNs induction. The obtained 858 differentially expressed genes formed the reliable ceRNA network. The function of LINC01002-miR-4324-FRMD8 axis in the IFN-dominated immune evasion was further demonstrated through integrating single-cell sequencing data. Moreover, we validated that N protein could reverse the effect of PIC on LINC01002, FRMD8 and miR-4324 expression, and subsequently on IFN-ß expression level. And LINC01002 could regulate the production of FRMD8 by inhibiting miR-4324. CONCLUSION: SARS-CoV-2 N protein suppressed the induction of IFN-ß by regulating LINC01002 which was as a ceRNA, sponging miR-4324 and participating in the regulation of FRMD8 mRNA. Our discovery provides new insights into early intervention therapy and drug development on SARS-CoV-2 infection.
Assuntos
COVID-19 , MicroRNAs , RNA Longo não Codificante , Humanos , Células A549 , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , COVID-19/virologia , COVID-19/imunologia , Evasão da Resposta Imune , Interferon beta/genética , Interferon beta/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fosfoproteínas , RNA Endógeno Competitivo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
OBJECTIVE: To investigate the regulatory effect of miR-4324 on ankyrin 2(Talin2) expression and biological behaviors of breast cancer cells and the clinical implications of changes in miR-4324 and Talin2 expressions in breast cancer. METHODS: In breast cancer and adjacent tissues, the expressions of Talin2 and miR-4324 were examined with immunohistochemistry and qRT-PCR, respectively and the association of Talin2 expression levels with the prognosis and clinicopathological features of breast cancer patients was analyzed.The human breast cancer cell line SKBR-3 was transfected with miR-4324 mimic, miR-4324 inhibitor, si-Talin2, or both miR-4324 inhibitor and si-Talin2, and the changes in biological behaviors of the cells were examined; the cellular expression of Talin2at the mRNA and protein levels were detected with qRT-PCR and Western blotting.Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-4324 and Talin2.The effect of miR-4324-mediated regulation of Talin2 on SKBR-3 cell migration was assessed using Transwell assays. RESULTS: Talin2 expression was significantly higher in breast cancer tissues than in the adjacent tissues, and its expression level was correlated with lymph node metastasis and high HER-2 expression in breast cancer (P < 0.05) but not with the patient's age, clinical stage, histological grade or expressions of estrogen and progesterone receptors (P >0.05).The expression of miR-4324 was significantly reduced in breast cancer tissues as compared with the adjacent tissues (P < 0.01).In SKBR-3 cells, transfection with miR-4324 mimics significantly inhibited proliferation, migration and invasion (P < 0.05) and promoted apoptosis (P < 0.01) of the cells.Dual luciferase reporter gene assay confirmed that cotransfection with miR-4324 mimics significantly reduced luciferase activity of Talin2-3'-UTR WT reporter plasmid (P < 0.05).Transfection of the cells with miR-4324 mimics significantly reduced mRNA and protein expressions of Talin2(P < 0.05).Transwell migration assay showed that the migration ability of SKBR-3 cells was significantly enhanced after transfection with miR-4324 inhibitor (P < 0.01), lowered after transfection with si-Talin2(P < 0.01), and maintained at the intermediate level after co-transfection with miR-4324 inhibitor+si-Talin2 group (P < 0.05). CONCLUSIONS: High expression of Talin2 is associated with lymph node metastasis and HER-2 overexpression in breast cancer patients.Down-regulation of miR-4324 inhibits the proliferation, invasion and migration and induces apoptosis of breast cancer cells, and the inhibitory effect of miR-4324 knockdown on breast cancer cell migration is mediated probably by targeted inhibition of Talin2 expression.
Assuntos
Neoplasias da Mama , MicroRNAs , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Luciferases/genética , Metástase Linfática , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , RNA MensageiroRESUMO
The aim of the study was to evaluate the diagnostic utility of specific miRNAs in the preoperative assessment of thyroid nodules. One hundred and sixty thyroid fine needle aspiration biopsy (FNAB) samples with suspected thyroid carcinoma were collected. To detect the levels of miRNA expression in FNAB, next generation small RNA sequencing was performed in 60 samples. Based on the results obtained, three miRNAs (miR125A, miR200B, miR4324) were selected for further analysis. Based on the most frequently reported miRNAs in the literature associated with thyroid papillary carcinoma (PTC), two more miRNA (miR146B, miR221) were selected for further validation, using real-time reverse transcriptase polymerase chain reaction (RT-PCR) in 36 benign and 64 PTC samples. Expression of miR125A, miR146B, miR221, and miR4324 was significantly higher in patients with PTC compared with benign thyroid nodules (p Ë 0.05). miR125A and miR4324 were also significantly more highly expressed in patients with extrathyroidal tumor extension compared to those without extrathyroidal PTC extension (p < 0.001). We also found a significantly higher expression of miR221 (p = 0.043) in patients with multifocal carcinomas compared to patients with single foci carcinomas. This prospective study showed that the expression analysis of four miRNAs (miR125A, miR146B, miR221, and miR4324) improve accuracy of FNAB, which could allow a better pre-operative diagnostic and prognostic assessment of thyroid malignancies.
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BACKGROUND: Ovarian cancer is one of the most lethal malignancies, with a 1.9% mortality rate worldwide. The dysregulation of the FEN1 gene and miR-4324 has been associated with cancer progression. However, the relationship between miR-4324 and-FEN1 requires further investigation. METHODS: miR-4324 and FEN1 expressions in ovarian cancer tissues and cell lines were measured via RT-qPCR. The interaction between miR-4324 and FEN1 was assessed using luciferase and RNA pull-down assays. The effects of miR-4324 and FEN1 on cell proliferation, adhesion and apoptosis were determined by CCK-8, BrdU, colony formation, cell adhesion, Caspase-3 and western blot assays in ovarian cancer cell lines CaOV3 and OVCAR3, respectively. RESULTS: The results showed that miR-4324 expression was significantly decreased and FEN1 expression was enhanced in ovarian cancer tissues and cell lines. miR-4324 inhibitor promoted cell proliferation, adhesion and migration, and prevented apoptosis. Furthermore, the downregulation of FEN1 inhibited ovarian cancer cell growth and increased apoptosis. miR-4324 inhibited FEN1 expression and repressed ovarian cancer progression. CONCLUSION: Our study found that miR-4324 inhibited FEN1 expression, suppressed cell growth, and increased apoptosis in ovarian cancer cells. Therefore, we identified miR-4324 and FEN1 as potential therapeutic targets for ovarian cancer treatment.
Assuntos
MicroRNAs , Neoplasias Ovarianas , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
OBJECTIVE: MicroRNAs (miRNAs) are reported to have crucial roles in human cancers; however, their role in colorectal cancer (CRC) remains largely unknown. METHODS: In this study, we analyzed the expression of miR-4324 in CRC cell lines using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We also examined miR-4324 expression in CRC tumor tissues using a miRNA expression dataset obtained from the Gene Expression Omnibus. We validated the connection between miR-4324 and homeobox B2 (HOXB2) using a luciferase activity reporter assay and western blotting. The effects of miR-4324 and HOXB2 on CRC cell malignant behaviors in vitro were further investigated. RESULTS: miR-4324 expression was significantly decreased in both CRC tumor tissues and cell lines. Overexpression of miR-4324 suppressed CRC cell proliferation, migration, and invasion. In contrast, overexpression of HOXB2 promoted CRC malignant cell behaviors. Furthermore, we validated HOXB2 as a direct target of miR-4324. CONCLUSIONS: miR-4324 expression was decreased in CRC. miR-4324 regulates CRC cell proliferation, migration, and invasion by targeting HOXB2.
Assuntos
Neoplasias Colorretais , Proteínas de Homeodomínio , MicroRNAs , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Humanos , MicroRNAs/genética , Invasividade Neoplásica/genética , Fatores de TranscriçãoRESUMO
Considering the importance of microRNAs (miRNAs) in regulating cellular processes, we performed microarray analysis and revealed miR-4324 as one of the most differentially expressed miRNAs in bladder cancer (BCa). Then, we discovered that miR-4324 was a negative regulator of Rac GTPase activating protein 1 (RACGAP1) and that RACGAP1 functioned as an oncogenic protein in BCa. Our studies indicated that ectopic overexpression of miR-4324 in BCa cells significantly suppressed cell proliferation and metastasis and enhanced chemotherapy sensitivity to doxorubicin by repressing RACGAP1 expression. Further studies showed that estrogen receptor 1 (ESR1) increased the expression of miR-4324 by binding to its promoter, while the downregulation of ESR1 in BCa was caused by hypermethylation of its promoter. p-STAT3 induced the enrichment of DNMT3B by binding to the ESR1 promoter and then induced methylation of the ESR1 promoter. In turn, RACGAP1 induced STAT3 phosphorylation, increasing p-STAT3 expression and promoting its translocation to the nucleus. Therefore, the miR-4324-RACGAP1-STAT3-ESR1 feedback loop could be a critical regulator of BCa progression.