RNA epigenetic modifications in ovarian cancer: The changes, chances, and challenges.

Ovarian cancer (OC) is the most common female cancer worldwide. Patients with OC have high mortality because of its complex and poorly understood pathogenesis. RNA epigenetic modifications, such as m6 A, m1 A, and m5 C, are closely associated with the occurrence and development of OC. RNA modifications can affect the stability of mRNA transcripts, nuclear export of RNAs, translation efficiency, and decoding accuracy. However, there are few overviews that summarize the link between m6 A RNA modification and OC. Here, we discuss the molecular and cellular functions of different RNA modifications and how their regulation contributes to the pathogenesis of OC. By improving our understanding of the role of RNA modifications in the etiology of OC, we provide new perspectives for their use in OC diagnosis and treatment. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Disease.

TCONS_00483150 as a novel diagnostic biomarker of systemic lupus erythematosus.

Aim: We aimed to identify differentially expressed Long noncoding RNAs (lncRNAs) and explore their functional roles in systemic lupus erythematosus (SLE). Materials & methods: We identified dysregulated lncRNAs and investigated their prognostic values and potential functions using MiRTarget2, catRAPID omics and Bedtools/blast/Pearson analyses. Results: Among the 143 differentially expressed lncRNAs, TCONS_00483150 could be used to distinguish patients with SLE from healthy controls and those with rheumatoid arthritis and patients with active/stable SLE from healthy controls. TCONS_00483150 was significantly correlated with anti-Rib-P antibody positivity and low C3 levels; TCONS_00483150 dysregulation might contribute to the metabolism of RNA and proteins in SLE patients. Conclusion: Overall, our findings offer a transcriptome-wide overview of aberrantly expressed lncRNAs in patients with SLE and highlight TCONS_00483150 as a potential novel diagnostic biomarker.

Downregulated miR­29a promotes B cell overactivation by upregulating Crk­like protein in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder; however, the pathogenesis is not fully understood. Accumulating evidence suggested an important role of microRNAs (miRNA/miR) in autoimmunity. The present study aimed therefore to determine the miRNA expression patterns in the B cells from the peripheral blood of 66 patients with SLE and 10 healthy controls (HCs) by using an Affymetrix GeneChip® miRNA 2.0 array. In addition, next­generation sequencing was used to obtain the peripheral blood mononuclear cell (PBMC) miRNA profiles from three patients with SLE and three HCs. Candidate miRNAs that were considered to contribute to the pathogenesis of SLE were obtained based on the intersection of miRNA profiles. The analysis revealed a significant downregulation in miR­29a expression levels in B cells from patients with SLE, which was subsequently verified using reverse transcription­quantitative PCR. Based on these results, the expression pattern of miR­29a in SLE was further investigated and its role in the hyperactivity of B cells was determined. miR­29a inhibitors and mimics were transfected into PBMCs obtained from HCs and patients with SLE, and an ELISA was used to demonstrate that miR­29a inhibition increased the production of IgG. Bioinformatics analysis predicted Crk­like protein (CRKL) as a target gene of miR­29a in patients with SLE. Therefore, CRKL expression levels were compared between patients with SLE and HCs by using western blotting, and its direct transcriptional regulation by miR­29a was determined using a dual­luciferase reporter assay. Low expression levels of miR­29a were revealed to upregulate the expression levels of CRKL in B cells, and the protein expression levels of CRKL in patients with SLE were significantly upregulated compared with the HCs. In conclusion, the results from the present study suggested that miR­29a may affect IgG antibody secretion in B cells by regulating CRKL, thereby contributing to the development and progression of SLE, which offers a novel candidate target for treatment.

Hsa_circ_0000479 as a Novel Diagnostic Biomarker of Systemic Lupus Erythematosus.

Background: Accumulating evidence suggests that differentially expressed non-coding circular RNAs (circRNAs) play critical roles in the progress of autoimmune diseases. However, the role of circRNAs in systemic lupus erythematosus (SLE) remains unclear. Methods: We initially used next-generation sequencing (NGS) to comprehensively analyze circRNA expression profiles in peripheral blood mononuclear cells (PBMCs) from 10 SLE patients, stratified by their disease activity characteristics (stable or active SLE), and 10 healthy controls (HCs). Candidate circRNAs identified were first validated by quantitative reverse-transcription (qRT)-PCR in PBMC samples from a training-phase cohort of five SLE patients and five HCs. The significantly dysregulated circRNAs were then confirmed by qRT-PCR in a validation cohort of 23 SLE patients and 21 HCs, and in an external validation cohort with 64 SLE patients, 58 HCs, and 50 patients with rheumatoid arthritis (RA). In addition, we conducted bioinformatics analysis and western blotting investigating the relationships between the candidate circRNAs and SLE progression. Results: Multilayer integrative analysis of circRNA regulation showed that 84 circRNAs were upregulated and 30 were downregulated in patients with SLE compared with HCs. We then analyzed the intersection of these differentially expressed circRNAs in an SLE-stable cohort, an SLE-active cohort, and HCs. This enabled us to narrow down dysregulated circRNAs to 15 upregulated circRNAs. Only hsa_circ_0000479 was significantly upregulated in PBMCs of patients with SLE compared with HCs (P < 0.05). Furthermore, the diagnostic potential of hsa_circ_0000479 expression to distinguish SLE patients from HCs and RA patients was also significantly increased in the validation-phase and external-validation-phase cohorts (P < 0.05). When distinguishing SLE patients from HCs, the diagnostic specificities of hsa_circ_0000479 were 0.619 and 1.0 in two validation cohorts, respectively (AUCs = 0.731 and 0.730, respectively). It was also significantly increased in either stable SLE patients or active SLE patients compared with HCs in these two cohorts (P < 0.05). We also used bioinformatics analysis to show that hsa_circ_0000479 regulates SLE progression by modulating metabolic pathways and the Wnt signaling pathway. Western blotting revealed that the expression of Wnt-16 protein was significantly decreased in SLE. Conclusion: Our results suggest that hsa_circ_0000479 has potential as a novel biomarker for the diagnosis of SLE.

MiR-199-3p promotes ERK-mediated IL-10 production by targeting poly (ADP-ribose) Polymerase-1 in patients with systemic lupus erythematosus.

MicroRNAs (miRNAs) have been implicated in both biological and pathological processes in patients with systemic lupus erythematosus (SLE). Previous studies have demonstrated dysregulated expression of miR-199-3p, interleukin (IL)-10, and poly (ADP-ribose) polymerase-1 (PARP-1) in SLE. However, the underlying mechanisms of these aberrations have not been fully elucidated. In this study, we investigated the mechanism through which miR-199-3p dysregulation contributed to the pathogenesis of SLE. Altered gene expression was assessed by ChIP analysis. We then silenced the expression of candidate genes using siRNA for functional analysis; mRNA expression, protein levels, and protein expression were determined by qRT-PCR, ELISA, and western blotting, respectively. According to ChIP and qRT-PCR results, miR-199-3p was up-regulated in SLE patients. Moreover, IL-10 was found to be highly expressed in SLE patients by ELISA. Further, PARP1 was significantly down-regulated in SLE patients based on western blotting. Our results also indicated that miR-199-3p inhibits PARP1 expression by activating the ERK1/2 pathway, thereby increasing IL-10 expression. Significantly up-regulated miR-199-3p was inversely related to PARP-1 expression and positively correlated with IL-10 levels in SLE. As miR-199-3p was shown to target PARP-1 to activate the ERK1/2 pathway and promote IL-10 production, restoring physiological miR-199-3p levels could represent a potential therapeutic strategy for SLE treatment.

NovelmiRNA-25 inhibits AMPD2 in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and represents a promising novel biomarker.

BACKGROUND: Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease with various clinical manifestations. MicroRNAs (miRNAs) and immunometabolism are recognized as key elements in SLE pathogenesis; however, the relationship between miRNAs in peripheral blood mononuclear cells (PBMCs) and metabolism in SLE remains unclear. METHODS: We detected PBMC miRNA and mRNA profiles from 3 pooled SLE patients and 3 healthy controls (HCs) using next-generation sequencing, predicted miRNA targets in dysregulated mRNAs, predicted functions and interactions of differentially expressed genes using bioinformatics analysis, validated candidate miRNAs using qRT-PCR, and investigated the association between the expression of candidate miRNAs and SLE clinical characteristics. Moreover, we validated the direct and transcriptional regulatory effect of NovelmiRNA-25 on adenosine monophosphate deaminase 2 (AMPD2) using a dual-luciferase reporter assay and western blot and confirmed AMPD2 mRNA and protein expression in PBMCs using qRT-PCR and western blot, respectively. RESULTS: Multilayer integrative analysis of microRNA and mRNA regulation showed that 10 miRNAs were down-regulated and 19 miRNAs were up-regulated in SLE patient PBMCs compared with HCs. Bioinformatics analysis of regulatory networks between miRNAs and mRNAs showed that 19 miRNAs were related to metabolic processes. Two candidate miRNAs, NovelmiRNA-25 and miR-1273h-5p, which were significantly increased in the PBMCs of SLE patients (P < 0.05), represented diagnostic biomarkers with sensitivities of 94.74% and 89.47%, respectively (area under the curve = 0.574 and 0.788, respectively). NovelmiRNA-25 expression in PBMCs was associated with disease activity in SLE patients, in both active and stable groups (P < 0.05). NovelmiRNA-25 overexpression downregulated AMPD2 expression in HEK293T cells through direct targeting of the AMPD2 3'UTR (P < 0.01), while inhibition of NovelmiRNA-25 activity led to increased AMPD2 expression (P < 0.01). NovelmiRNA-25 overexpression also downregulated AMPD2 protein expression in HEK293T cells; AMPD2 protein expression in SLE patient PBMCs was decreased. Our results show that differentially expressed miRNAs play an important role in SLE. CONCLUSIONS: Our data demonstrate a novel mechanism in SLE development that involves the targeting of AMPD2 expression by NovelmiRNA-25. miRNAs may serve as novel biomarkers for the diagnosis and evaluation of disease activity of SLE and represent potential therapeutic targets for this disease.

Correlation between systemic lupus erythematosus and cytomegalovirus infection detected by different methods.

Human cytomegalovirus (HCMV), a ß-herpes virus subfamily member, leads to a lifelong, latent infection in most humans, but the correlation between HCMV infection and systemic lupus erythematosus (SLE) remains controversial. We analyzed the relevance of HCMV infection in SLE by analyzing the peripheral blood leukocytes (PBLs) and serum samples of 60 patients with SLE and 111 healthy individuals. HCMV genes UL55 and UL138 were detected in PBLs by polymerase chain reaction (PCR), and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. The relationship between cellular HCMV infection in PBLs and common clinical indicators of SLE was further explored. Data indicated that the frequency of positive IgG and IgM anti-CMV antibodies was not significantly different in SLE patients and controls. However, compared to the healthy controls, the titers of IgG and IgM anti-CMV antibodies in SLE patients were significantly higher. The detection of cellular HCMV infection showed that almost all subjects were positive for UL138 gene in PBLs, but the positivity for UL55 gene was lower in PBLs. HCMV UL138 detection in PBLs was highly consistent with the frequency of the HCMV-specific IgG test and did not show significant difference in SLE patients and healthy controls. However, compared with that in healthy people, the positivity rate for cellular HCMV UL55 detection was significantly higher in SLE patients (P < 0.001). In addition, cellular HCMV UL55 with positive detection in PBLs was associated with significantly different clinical characteristics of SLE than that with negative detection. In conclusion, our data confirmed that the HCMV infection was related to the development of SLE. Especially, some clinical strains or substrains of HCMV, such as containing the UL55 gene in HCMV's genome, might play a vital role in the development of SLE.

Multiple-integrations of HPV16 genome and altered transcription of viral oncogenes and cellular genes are associated with the development of cervical cancer.

The constitutive expression of the high-risk HPV E6 and E7 viral oncogenes is the major cause of cervical cancer. To comprehensively explore the composition of HPV16 early transcripts and their genomic annotation, cervical squamous epithelial tissues from 40 HPV16-infected patients were collected for analysis of papillomavirus oncogene transcripts (APOT). We observed different transcription patterns of HPV16 oncogenes in progression of cervical lesions to cervical cancer and identified one novel transcript. Multiple-integration events in the tissues of cervical carcinoma (CxCa) are significantly more often than those of low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL). Moreover, most cellular genes within or near these integration sites are cancer-associated genes. Taken together, this study suggests that the multiple-integrations of HPV genome during persistent viral infection, which thereby alters the expression patterns of viral oncogenes and integration-related cellular genes, play a crucial role in progression of cervical lesions to cervix cancer.

Immunogenicity of a multiepitope plasmid DNA encoding T and B lymphocyte epitopes from latent membrane protein 2 (LMP2) of Epstein-Barr virus as a vaccine in mice.

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associating with several malignant diseases. Latent membrane protein 2 (LMP2) of EBV is considered to be an ideal candidate for immunotherapy or prophylactic EBV vaccine. We designed a LMP2 multiepitope containing T and B-cell epitope-rich peptides and constructed a recombinant plasmid containing mammalian codonoptimization EBV LMP2 multiepitope (pcDNA3.1+/EBV-LMP2 multiepitope). After pcDNA3.1+/EBV-LMP2 multiepitope was transfected into COS-7 cells, significant expression of the multiepitope in COS-7 cells was confirmed by RT-PCR and immunofluorescence assay. Western blot analysis indicated that serum from immunized mice could be discerned by the EBV-LMP2 protein and the EBV-LMP2 multiepitope specifically. The plasmid DNA of EBV-LMP2 multiepitope induced high levels anti-EBV membrane protein and anti-EBV LMP2 multiepitope IgG in mice. T lymphocytes from spleen of immunized mice showed a strong CTL activity. The present study suggested that plasmid DNA encoding EBV LMP2 multiepitope capable of stimulating enough cellular and humoral immunity could have potential for preventing or controlling EBV infection and EBV associated disease in mice.

[Correlation of expression of nuclear factor kappaB, inhibitor protein kappaB, and Bcl-2 in cervical cancer to human papillomavirus infection].

BACKGROUND & OBJECTIVE: It was proved that nuclear factor kappaB (NF-kappaB) and its inhibitor protein kappaB (I-kappaB) played critical roles in regulating the expression of proapoptotic genes, and NF-kappaB is overexpressed in some tumors and related with tumorigenesis. However, the expression of NF-kappaB and I-kappaB in cervical cancer and its correlation to human papillomavirus (HPV) infection have not been reported yet. This study was to explore the correlation of the expression of NF-kappaB, I-kappaB, and Bcl-2 in cervical cancer to human papillomavirus (HPV) infection. METHODS: The expression of NF-kappaB, I-kappaB, and Bcl-2 were detected by immunohistochemistry in 46 specimens of cervical cancer and 26 specimens of normal cervical tissue. HPV DNA was detected by polymerase chain reaction (PCR). RESULTS: The positive rates of NF-kappaB and Bcl-2 were significantly higher in cervical cancer than in normal cervical tissue (60.9% vs. 23.1%, 52.2% vs. 0.0%, P < 0.01). The positive rate of I-kappaB was significantly lower in cervical cancer than in normal cervical tissue (30.4% vs. 57.7%, P < 0.05). The expression of NF-kappaB was closely related to Bcl-2 (P < 0.05). The positive rate of NF-kappaB was significantly higher in HPV DNA-positive cervical cancer than in HPV DNA-negative cervical cancer (81.3% vs. 35.7%, P < 0.05), but the positive rates of I-kappaB and Bcl-2 between these 2 groups had no significant difference (P >0.05). CONCLUSION: The high expression of NF-kappaB and Bcl-2 and the low expression of I-kappaB may be related to cervical carcinogenesis, and NF-kappaB expression may be related to HPV infection.