miR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion, and migration in miscarriage†.

A large proportion of miscarriages are classified as unexplained miscarriages since no cause is identified. No reliable biomarkers or treatments are available for these pregnancy losses. While our transcriptomic sequencing has revealed substantial upregulation of miR-146b-5p in unexplained miscarriage villous tissues, its role and associated molecular processes have yet to be fully characterized. Our work revealed that relative to samples from normal pregnancy, miR-146b-5p was significantly elevated in villous tissues from unexplained miscarriage patients and displayed promising diagnostic potential. Moreover, miR-146b-5p agomir contributed to higher rates of embryonic resorption in ICR mice. When overexpressed in HTR-8/SVneo cells, miR-146b-5p attenuated the proliferative, invasive, and migratory activity of these cells while suppressing the expression of MMP9 and immune inflammation-associated cytokines, including IL1B, IL11, CXCL1, CXCL8, and CXCL12. Conversely, inhibition of its expression enhanced proliferation, migration, and invasion abilities. Mechanistically, IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19 were identified as miR-146b-5p targets regulating trophoblast function, and silencing IL-1 receptor-associated kinase-1 had similar effects as miR-146b-5p overexpression, while IL-1 receptor-associated kinase-1 overexpression could partially reverse the inhibitory impact of this microRNA on trophoblasts. miR-146b-5p may inhibit trophoblast proliferation, migration, invasion, and implantation-associated inflammation by downregulating IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19, participating in the pathogenesis of miscarriage and providing a critical biomarker and a promising therapeutic target for unexplained miscarriage.

Analysis of circRNAs and circRNA-associated competing endogenous RNA networks in ß-thalassemia.

The involvement of circRNAs in ß-thalassemia and their actions on fetal hemoglobin (HbF) is unclear. Here, the circRNAs in ß-thalassemia carriers with high HbF levels were comprehensively analyzed and compared with those of healthy individuals. Differential expression of 2183 circRNAs was observed and their correlations with hematological parameters were investigated. Down-regulated hsa-circRNA-100466 had a strong negative correlation with HbF and HbA2. Bioinformatics was employed to construct a hsa-circRNA-100466­associated competing endogenous RNA (ceRNA) network to identify hub genes and associated miRNAs. The hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified using both present and previously published data. The ceRNA network was verified by qRT-PCR analysis of ß-thalassemia samples, RNA immunoprecipitation of K562 cell lysates, and dual-luciferase reporter analysis. qRT-PCR confirmed that hsa-circRNA-100466 and SOX6 were significantly down-regulated, while miR-19b-3p was up-regulated. Hsa-circRNA-100466, miR-19b-3p, and SOX6 were co-immunoprecipitated by anti-argonaute antibodies, indicating involvement with HbF induction. A further dual-luciferase reporter assay verified that miR-19b-3p interacted directly with hsa-circRNA-100466 and SOX6. Furthermore, spearman correlation coefficients revealed their significant correlations with HbF. In conclusion, a novel hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified, providing insight into HbF induction and suggesting targets ß-thalassemia treatment.

Placental transcriptome sequencing combined with bioinformatics predicts potential genes and circular RNAs associated with hemoglobin Bart's hydrops fetalis syndrome.

AIM: Hemoglobin Bart's hydrops fetalis syndrome (BHFS) is the most severe form of α-thalassemia. Histological alternations can be observed in placenta, but placental transcriptome profile and circular RNAs have not been studied in this disease. The aim of this study was to define the placental transcriptional changes and find relevant circular RNAs in BHFS. METHODS: We performed high-throughput RNA sequencing to detect placental samples from fetuses affected by BHFS (n = 5) and normal fetuses (NF, n = 5), quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Sanger sequencing to validate the differentially expressed circRNAs and their potentially related miRNAs (BHFS, n = 22; NF, n = 11). Bioinformatics methods were performed for further analysis. RESULTS: Our results showed 152 differentially expressed genes (DEGs), 112 circRNAs, and 45 microRNAs that were differentially expressed. DEGs were found to be involved in Gene Ontology terms related to gas transport, cell adhesion, oxidative stress, organ development, hemopoiesis, and others. RT-qPCR results showed that hsa_circ_0003961 and hsa_circ_0006687 were upregulated (p < 0.05). The competing endogenous RNA and co-expression networks showed that hsa_circ_0003961 and hsa_circ_0006687 were connected with 3 miRNAs and some DEGs, including cell adhesion genes (e.g., CLDN19), hemoglobin related genes (e.g., SOX6 and HBZ) and angiogenesis related genes (e.g., EPHB2). Downregulations of hsa-miR-1299 and hsa-miR-625-5p in ceRNA network were also validated by RT-qPCR. Gene set enrichment analysis results for the two circRNAs showed that some gene sets associated with cell adhesion, hematopoietic system and apoptosis were significantly enriched. CONCLUSIONS: Our study characterized the placental transcriptome of BHFS. The circRNAs hsa_circ_0003961 and hsa_circ_0006687 in placenta may be relevant to BHFS.

Human m6A-mRNA and lncRNA epitranscriptomic microarray reveal function of RNA methylation in hemoglobin H-constant spring disease.

The thalassemia of Hemoglobin H-Constant Spring disease (HbH-CS) is the most common type of Thalassemia in non-transfusion thalassemia. Interestingly, the clinical manifestations of the same genotype of thalassemia can be vastly different, likely due to epigenetic regulation. Here, we used microarray technology to reveal the epigenetic regulation of m6A in modifiable diseases and demonstrated a role of BCL2A1 in disease regulation. In this study, we revealed that methylating enzyme writers including METTL16, WTAP, CBLL1, RBM15B, and ZC3H13 displayed low expression and the demethylating enzyme ALKBH5, along with reader proteins including IGF2BP2 and YTHDF3 exhibited high expression. In addition, BCL2A1 was hypo-methylated and showed low expression. We also revealed that the BCL2A1 methylation level and IGF2BP2 expression were negatively correlated. Additionally, the mRNAs expression between ALKBH5 and IGF2BP2 were positively correlated. In HbH-CS, most genes were hypo-methylated. This included BCL2A1, which may play an important role in the process of red blood cell differentiation and development of HbH-CS. Moreover, the mRNA-M6A methylation status may be regulated by the demethylating enzyme ALKBH5 via IGF2BP2.

Human lymphatic endothelial cells contribute to epithelial ovarian carcinoma metastasis by promoting lymphangiogenesis and tumour cell invasion.

The microenvironment of a tumour is an important factor in ovarian cancer metastasis. The present study aimed to simulate the in vivo microenvironment of an ovarian carcinoma using a co-culture system consisting of human lymphatic endothelial cells (HLECs) and human ovarian carcinoma cells with directional high lymphatic metastasis (SKOV3-PM4s) in order to investigate the role of both cell types in ovarian carcinoma metastasis. The SKOV3-PM4s cultured in the HLEC-conditioned medium exhibited increased numbers of pseudopodia and mitotic figures, proliferated at a faster rate and exhibited enhanced invasion and migratory abilities. Furthermore, the HLECs cultured in SKOV3-PM4-conditioned medium exhibited significant morphological alterations and vacuolisation of the cytoplasm, as well as increased invasion, migratory and tube forming abilities. In addition, spontaneous fusion of the SKOV3-PM4s and HLECs was observed in the co-culture system using laser confocal microscopy. The gelatin zymography assay demonstrated that matrix metalloproteinase-2, which was downregulated in the SKOV3-PM4s, was upregulated in the co-culture system. The results of the present study suggested that the invasion ability of the SKOV3-PM4s was increased in the in vitro co-culture system of SKOV3-PM4 and HLECs. Therefore, alterations in the cell microenvironment may represent a novel strategy for ovarian cancer therapy.

[Characteristics change of the human directional highly lymphatic metastasis ovarian carcinoma cell and venous endothelial cell after establishment of their condition cultrue and co-culture cell system].

OBJECTIVE: To establish the condition cultrue cell system and co- culture cell system with SKOV3/PM4, HUVEC and to study the changes of their biological characteristics. METHODS: The cells of SKOV3/PM4 and HUVEC were labeled with green and red fluorescent respectively. The cell supernatant of SKOV3/PM4 and HUVEC were collected respectively as the condition medium (e.g: the cell supernatant of HUVEC cells was used as SKOV3/PM4 condition medium)and to establish the condition cultrue cell system and the co- culture cell system of the two cell lines. In the condition cultrue cell system, The morphological changes of cells were observed by HE staining to calculate the mitotic index. The ultrastructural changes of the two cells were observed by transmission electron microscopy (TEM). The growth curve of the cells was determined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry was used to analyzed the cell cycles. In the co-culture cell system, the interaction of the two cells were detected by laser scanning confocal microscope (LSCM). The expression of matrix metalloproteinase- 2 (MMP- 2) and matrix metalloproteinase- 9 (MMP- 9) were detected by gelatin zymography. RESULTS: Compared with the single culture SKOV3/PM4, the cells which cultured in HUVEC condition medium showed the increase of pseudopodia and nuclear division, the mitotic index respectively were [(4.8 ± 0.8)%, (11.2 ± 0.3)%; P < 0.05]. The growth rate was significantly increased. In cell cycles, it showed the declined cell ratio of G0/G1 phase, respectively [(69.4 ± 3.6)%, (48.4 ± 4.6)%; P < 0.05] and the raised cell ratio of G2/M phase, respectively [(5.2 ± 1.6)%, (24.9 ± 2.2)%; P < 0.05]. Compared with the single culture HUVEC, the cells which cultured in SKOV3/PM4 condition medium showed the significant morphological change and vacuolization in the cytoplasm, Nuclear division was increased and the mitotic index respectively were [(2.7 ± 0.5)%, (5.7 ± 0.6)%; P < 0.05]. The growth rate was slightly declined. In cell cycles, it showed the raised cell ratio in G0/G1 phase, respectively [(51.4 ± 2.2)%, (79.0 ± 4.1)%; P < 0.05] and the declined cell ratio in G2/M phase, respectively [(19.1 ± 1.2)%, (3.3 ± 0.5)%; P < 0.05]. After co-culture for 48 hours, spontaneous fusion between SKOV3/PM4 and HUVEC cell was observed by the laser confocal microscope. Gelatin zymography assay showed that MMP-2 was not expressed in HUVEC cells, low-expressed in SKOV3/PM4 cells and high-expressed in the co-culture SKOV3/PM4+HUVEC cells. The expression of MMP-2 in co-culture SKOV3/PM4+HUVEC cells and SKOV3/PM4 cells respectively were 1 885 ± 84 and 1 209 ± 114 (P < 0.05). But there were no MMP-9 expression in HUVEC cells, SKOV3/PM4 cells, and the co- culture SKOV3/PM4+HUVEC. CONCLUSION: The characteristics of SKOV3/PM4 and HUVEC show significant changes after condition culture and co-culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway.

Effects of HLEC on the secreted proteins of epithelial ovarian cancer cells prone to metastasize to lymph nodes.

OBJECTIVE: To study explores the effect of HLEC on the secreted proteins of epithelial ovarian cancer (EOC) cells (SKOV3-PM4) with directional highly lymphatic metastasis. METHODS: Supernatants of four groups of cultured cells, namely, SKOV3 (A), SKOV3+HLEC (B), SKOV3-PM4 (C), SKOV3-PM4+HLEC (D), were collected, and their proteins were detected by antibody arrays and iTRAQ-2D-LC-MALDI-TOF/TOF/MS. Significantly differential proteins were further analyzed via bioinformatics and validated in human serums and cell media via ELISA. RESULTS: Results of antibody arrays and mass spectrometry demonstrated that GRN and VEGFA were upregulated in group C (compared with group A), whereas IGFBP7 and SPARC were downregulated in group D (compared with group C). Comprehensive bioinformatics analysis results showed that IGFBP7 and VEGFA were closely linked to each other. Further validation with serums showed statistical significance in VEGFA and IGFBP7 levels among groups of patients with ovarian cancers, benign tumors, and control groups. Two proteins were upegulated in the first group. VEGFA in the control group was downregulated. For IGFBP, upregulation in the control group and down-regulation in the first group were also observed. CONCLUSION: The HLEC microenvironment is closely associated with directional metastasis to lymph nodes and with differential proteins including cell stromal proteins and adhesion factors. The upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 are closely associated with directional metastasis to lymph nodes in EOC cells.

[Screening of differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines using SELDI-TOF-MS technology].

BACKGROUND & OBJECTIVE: The ovarian serous papillary adenocarcinoma cell line SKOV3 and its subclones SKOV3-pm2 and SKOV3-pm3 are cell models to investigate the molecular mechanism of invasion and metastasis of ovarian cancers. This study was to screen differentially expressed proteins between ovarian carcinoma cell lines with directional (SKOV3-pm2 and SKOV3-pm3) and non-directional (SKOV3) highly lymphatic metastasis potentials using time-of-flight mass spectrometry technology and protein chips. METHODS: The lymphatic metastasis rates of the three cell lines were detected in animal models. Proteins in endochylema and supernatants of the three cell lines were screened using surface-enhanced laser desorption and ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Each sample was examined using weak cation exchange (CM-10) protein chip assay and immobilized metal affinity capture (IMAC-3) SELDI ProteinChip array. Detected protein peaks were filtrated and analyzed using Ciphergen proteinchip software 3.2.0 and Biomarker Wizard software. Differentially expressed proteins were defined as those whose absolute ratio values were greater than 0.5. RESULTS: Lymphatic metastasis rates in SKOV3, SKOV3-pm2 and SKOV3-pm3 cell xenografts in nude mice were 20%, 90% and 100%, respectively (P<0.05). Proteins in endochylema whose mass-to-charge ratio (m/z) were 6971, 7475, 9089, 9453, 10103, 11655, and the protein in supernatants whose m/z was 4746 were differentially expressed in SKOV3, SKOV3-pm2 and SKOV3-pm3 cells. CONCLUSION: Combined with weak cation exchange protein chip assay and immobilized metal affinity capture SELDI ProteinChip array, SELDI-TOF-MS technology can be used to screen and identify differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines.

[Establishment of human ovarian carcinoma cell lines with directional highly lymphatic metastasis and study of their biological characteristics].

OBJECTIVE: To establish a human ovarian carcinoma cell line with directional highly lymphatic metastasis and to study their biological characteristics. METHODS: The clone cells of ovarian carcinoma, SKOV3, were inoculated into the hind foot pad of nude mice. The cancer cells of lymph node metastatic foci were transplanted into nude mice again when the metastatic nude of mice were observed. After repetition of this procedure for 3 cycles, the metastatic rate and the metastatic paths were observed in nude mice of every passage. We used limited dilution method to separate and select colonial cells with directional highly lymphatic metastatic potentials from the lymphatic metastasis of human ovarian carcinoma cell line SKOV3. The cells with biological characteristics were assayed by growth curve, HE staining, karyotype analysis, nude mice transplantation and immunohistochemistry, respectively. RESULTS: We established a series of cell lines from lymph node metastasis and designated them as SKOV3-PM1, SKOV3-PM2 and SKOV3-PM3 cell strain. When the cells of SKOV3-PM3 were injected into the hind foot pad of nude mice, they produced 100% (10/10) spontaneous lymphatic metastasis. The lymphatic metastatic rates (26/10) were stable and higher than the mother cell line (1/10, P < 0.01). The metastatic paths were single, mostly to lymphatic nodes. The proliferation ability was increased in cancer cells of every passage in vivo and in vitro after passage of cancer cells of lymphatic metastatic foci for 3 cycles in nude mice. Cytogenetics study showed the karyotypes of SKOV3-PM3 had modes from 83 to 89, and the SKOV3 from 91 to 105. In comparison of SKOV3 with SKOV3-PM3, the number of chromosomes was significantly different (P < 0.05). Immunocytochemical study demonstrated all cell lines were still polygonal epithelial cells. The expression of epithelial membranes antigen (EMA) was positive, exhibiting features of human carcinoma. The cell of SKOV3-PM3 grew more quickly than SKOV3, their cell population doubling time being 22.7 hours and 49.6 hours, respectively (P < 0.05). Flow cytometry revealed the proportion of cells in DNA synthesis and mitosis was higher in SKOV3-PM1 (24.2%), SKOV3-PM2 (29.4%), and SKOV3-PM3 (36.7%) than in SKOV3 (21.5%; P < 0.05). CONCLUSIONS: The ovarian carcinoma sublines with directional highly lymphatic metastasis potential have been established successfully. The cells could provide a good experimental material for further investigation of the mechanism of metastasis and invasion of ovarian carcinoma.