The Effect of the cAMP Signaling Pathway on HTR8/SV-Neo Cell Line Proliferation, Invasion, and Migration After Treatment with Forskolin.

Pre-eclampsia (PE) is thought to be related to placental dysfunction, particularly poor extravillous trophoblast (EVT) invasion and migration abilities. However, the pathogenic mechanism is not fully understood. This article describes the impact of the cyclic adenosine monophosphate(cAMP) signaling pathway on EVT behavior, focusing on EVT proliferation, invasion, and migration. Here, we used the HTR8/SV-neo cell line to study human EVT function in vitro. HTR8/SV-neo cells were treated with different concentrations of forskolin (cAMP pathway-specific agonist) to alter intracellular cAMP levels, and dimethyl sulfoxide (DMSO) was used as the control. First, a cAMP assay was performed to measure the cAMP concentration in HTR8/SV-neo cells treated with different forskolin concentrations, and cell proliferation was assessed by constructing cell growth curves and assessing colony formation. Cell invasion and migration were observed by Transwell experiments, and intracellular epithelial-mesenchymal transition (EMT) marker expression was evaluated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting (WB). According to our research, the intracellular cAMP levels in HTR8/SV-neo cells were increased in a dose-dependent manner, and HTR8/SV-neo cell proliferation, invasion and migration were significantly enhanced. The expression of EMT and angiogenesis markers was upregulated. Additionally, with the increase in intracellular cAMP levels, the phosphorylation of intracellular mitogen-activated protein kinase (MAPK) signaling pathway components was significantly increased. These results suggested that the cAMP signaling pathway promoted the phosphorylation of MAPK signaling components, thus enhancing EVT functions, including proliferation, invasion, and migration, and to a certain extent, providing a novel direction for the treatment of PE patients.

Plasma TNF-α and phosphorylated α-syn are associated with fatigue in patients with Parkinson's disease.

BACKGROUD: Fatigue is one of the most common non-motor symptoms among patients with Parkinson's disease (PD).However, the pathogenesis keeps largely unknown. Moreover, it is lack of objective biomarker. OBJECTIVE: To investigate the relationship between plasma inflammatory cytokines and α-syn levels and fatigue in patients with PD. METHODS: A total of 63 PD patients were enrolled, including 35 patients with fatigue and 28 patients without fatigue. We compared the difference between plasma cytokines and alpha-synuclein (α-syn) in the two groups. Meanwhile, we analyzed the relationship between plasma cytokines and p-α-syn levels and fatigue. RESULTS: PD patients with fatigue had older age, longer disease duration, more severe motor scores. There were significant differences in the plasma levels of IL-1ß, IL-18, TNF-α, and phosphorylated α-syn (p-α-syn) between the two groups. The plasm inflammatory cytokine levels (IL-1ß, IL-18 and TNF-α) were positively associated with FSS scores. Moreover, the plasma p-α-syn level was significantly positively correlated with FSS scores. Furthermore, the higher PDQ-39 scores and higher plasma levels of TNF-α and p-α-syn were strongly associated with fatigue in PD. The ROC curve analysis showed the AUC of TNF-α for fatigue in PD was 0.663 with a sensitivity of 65.71% and specificity of 67.86%, while the AUC of p-α-syn was 0.786 with a sensitivity of 74.29% and specificity of 64.29%. The combination of TNF-α and p-α-syn improves the AUC to 0.803 with a sensitivity of 88.57% and specificity of 64.29%. CONCLUSION: The high plasma levels of TNF-α and p-α-syn were strongly associated with fatigue in PD.

Deciphering early human pancreas development at the single-cell level.

Understanding pancreas development can provide clues for better treatments of pancreatic diseases. However, the molecular heterogeneity and developmental trajectory of the early human pancreas are poorly explored. Here, we performed large-scale single-cell RNA sequencing and single-cell assay for transposase accessible chromatin sequencing of human embryonic pancreas tissue obtained from first-trimester embryos. We unraveled the molecular heterogeneity, developmental trajectories and regulatory networks of the major cell types. The results reveal that dorsal pancreatic multipotent cells in humans exhibit different gene expression patterns than ventral multipotent cells. Pancreato-biliary progenitors that generate ventral multipotent cells in humans were identified. Notch and MAPK signals from mesenchymal cells regulate the differentiation of multipotent cells into trunk and duct cells. Notably, we identified endocrine progenitor subclusters with different differentiation potentials. Although the developmental trajectories are largely conserved between humans and mice, some distinct gene expression patterns have also been identified. Overall, we provide a comprehensive landscape of early human pancreas development to understand its lineage transitions and molecular complexity.

Single-cell RNA sequencing reveals the developmental program underlying proximal-distal patterning of the human lung at the embryonic stage.

The lung is the primary respiratory organ in human, in which the proximal airway and the distal alveoli are responsible for air conduction and gas exchange, respectively. However, the regulation of proximal-distal patterning at the embryonic stage of human lung development is largely unknown. Here we investigated the early lung development of human embryos at weeks 4-8 post fertilization (Carnegie stages 12-21) using single-cell RNA sequencing, and obtained a transcriptomic atlas of 169,686 cells. We observed discernible gene expression patterns of proximal and distal epithelia at week 4, upon the initiation of lung organogenesis. Moreover, we identified novel transcriptional regulators of the patterning of proximal (e.g., THRB and EGR3) and distal (e.g., ETV1 and SOX6) epithelia. Further dissection revealed various stromal cell populations, including an early-embryonic BDNF+ population, providing a proximal-distal patterning niche with spatial specificity. In addition, we elucidated the cell fate bifurcation and maturation of airway and vascular smooth muscle progenitor cells at the early stage of lung development. Together, our study expands the scope of human lung developmental biology at early embryonic stages. The discovery of intrinsic transcriptional regulators and novel niche providers deepens the understanding of epithelial proximal-distal patterning in human lung development, opening up new avenues for regenerative medicine.

Genetic correction of concurrent α- and ß-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology.

BACKGROUND: Thalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above. METHODS: In the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous ß41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C > G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays. RESULTS: These mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential. CONCLUSIONS: We designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent α- and ß-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent α- and ß-thalassemia.

Treatment and clinical outcomes of cervical cancer during pregnancy.

BACKGROUND: This study aims to investigate clinicopathological factors associated with survival rate and treatment of patients with cervical cancer during pregnancy (CCP). METHODS: A total of 92 patients diagnosed CCP were retrospectively reviewed. One patient was from Nanfang Hospital of Southern Medical University, 5 patients were from Tongji Hospital, and 86 patients were from case reports in the PubMed database from 1961 to 2019. Patients and tumor characteristics were evaluated. Kaplan-Meier and Cox regression methods were used to analyze the 5-year disease-specific survival (DSS). RESULTS: Most patients (73 cases) were stage I according to the 2018 International Federation of Gynecology and Obstetrics (FIGO) standards. Twelve patients (13.04%) terminated pregnancy once diagnosed. These patients were diagnosed at the mean gestational age (GA) of 11±3 weeks, during early pregnancy. For the rest of the patients (80 cases) who continued pregnancy, the mean GA was 35±2 weeks at delivery. There was a significant difference in survival whether the treatment was performed once diagnosed or not. The 5-year DSS was 75% in adenocarcinoma (AC), 68.5% in squamous cell carcinoma (SCC), and 43.7% in the rare subtype. Among the 38 patients who underwent neoadjuvant chemotherapy (NACT), one patient suffered from spontaneous abortion, and one baby experienced acute myeloid leukemia (AML) ex-FAB (French-American-British)-M7 subtype and received bone marrow transplantation. Other delivered newborns showed no abnormality or malformation. Cox multi-factorial analysis demonstrated that tumor size (2 cm) was an independent overall survival predictor for CCP patients (P<0.05). CONCLUSIONS: Tumor size was an independent prognostic factor of survival in CCP patients. Pregnancy has adverse effects on the prognosis of cervical cancer. Personalized treatment is a priority for CCP patients.

Cancer-derived exosomal miR-221-3p promotes angiogenesis by targeting THBS2 in cervical squamous cell carcinoma.

AIMS: Recently, cancer-derived exosomes were shown to have pro-metastasis function in cancer, but the mechanism remains unclear. Angiogenesis is essential for tumor progression and is a great promising therapeutic target for advanced cervical cancer. Here, we investigated the role of cervical cancer cell-secreted exosomal miR-221-3p in tumor angiogenesis. METHODS AND RESULTS: miR-221-3p was found to be closely correlated with microvascular density in cervical squamous cell carcinoma (CSCC) by evaluating the microvascular density with immunohistochemistry and miR-221-3p expression with in situ hybridization in clinical specimens. Using the groups of CSCC cell lines (SiHa and C33A) with miR-221-3p overexpression and silencing, the CSCC exosomes were characterized by electron microscopy, western blotting, and fluorescence microscopy. The enrichment of miR-221-3p in CSCC exosomes and its transfer into human umbilical vein endothelial cells (HUVECs) were confirmed by qRT-PCR. CSCC exosomal miR-221-3p promoted angiogenesis in vitro in Matrigel tube formation assay, spheroid sprouting assay, migration assay, and wound healing assay. Then, exosome intratumoral injection indicated that CSCC exosomal miR-221-3p promoted tumor growth in vivo. Thrombospondin-2 (THBS2) was bioinformatically predicted to be a direct target of miR-221-3p, and this was verified by using the in vitro and in vivo experiments described above. Additionally, overexpression of THBS2 in HUVECs rescued the angiogenic function of miR-221-3p. CONCLUSIONS: Our results suggest that CSCC exosomes transport miR-221-3p from cancer cells to vessel endothelial cells and promote angiogenesis by downregulating THBS2. Therefore, CSCC-derived exosomal miR-221-3p could be a possible novel diagnostic biomarker and therapeutic target for CSCC progression.

Cervical squamous cell carcinoma-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis by targeting VASH1.

Cancer-secreted exosomal miRNAs are emerging mediators of cancer-stromal cross-talk in the tumor environment. Our previous miRNAs array of cervical squamous cell carcinoma (CSCC) clinical specimens identified upregulation of miR-221-3p. Here, we show that miR-221-3p is closely correlated with peritumoral lymphangiogenesis and lymph node (LN) metastasis in CSCC. More importantly, miR-221-3p is characteristically enriched in and transferred by CSCC-secreted exosomes into human lymphatic endothelial cells (HLECs) to promote HLECs migration and tube formation in vitro, and facilitate lymphangiogenesis and LN metastasis in vivo according to both gain-of-function and loss-of-function experiments. Furthermore, we identify vasohibin-1 (VASH1) as a novel direct target of miR-221-3p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of VASH1 could respectively rescue and simulate the effects induced by exosomal miR-221-3p. Importantly, the miR-221-3p-VASH1 axis activates the ERK/AKT pathway in HLECs independent of VEGF-C. Finally, circulating exosomal miR-221-3p levels also have biological function in promoting HLECs sprouting in vitro and are closely associated with tumor miR-221-3p expression, lymphatic VASH1 expression, lymphangiogenesis, and LN metastasis in CSCC patients. In conclusion, CSCC-secreted exosomal miR-221-3p transfers into HLECs to promote lymphangiogenesis and lymphatic metastasis via downregulation of VASH1 and may represent a novel diagnostic biomarker and therapeutic target for metastatic CSCC patients in early stages.

Clinicopathological Aspects of Small Cell Neuroendocrine Carcinoma of the Uterine Cervix: a Multicenter Retrospective Study and Meta-Analysis.

BACKGROUND/AIMS: To evaluate the clinicopathologic aspects of small cell neuroendocrine carcinoma of the uterine cervix (SCNEC). METHODS: A retrospective review of 40 patients with SCNEC in 3 hospitals from 2009 to 2015 was conducted to assess the survival rates and examine the associations between clinicopathological variables and overall survival (OS). A meta-analysis of 22 studies containing 1901 patients was also conducted to further confirm the results. RESULTS: In the clinical group of 40 patients, the 5-year OS rate was 20%. Advanced International Federation of Gynecology and Obstetrics (FIGO) stage and radiation therapy (RT) were associated with poor survival. However, radical surgery was associated with prolonged survival. In the meta-analysis of 1901 patients, the 2-year disease-free survival (DFS) rate, 5-year DFS rate, 2-year OS rate, 3-year OS rate and 5-year OS rate of SCNEC were 48%, 35%, 62%, 35%, and 35% respectively. Advanced FIGO stage, larger tumor size, lymph node metastasis (LNM) (+), lymphovascular space involvement (LVSI) (+), parametrial involvement (PI) (+), depth of stromal invasion (DSI) > 2/3, and RT were associated with poor survival. However, a chemotherapy regimen similar to that for small cell lung cancer was associated with prolonged survival. CONCLUSION: Advanced FIGO stage, larger tumor size, LNM (+), LVSI (+), DSI > 2/3, PI (+), and RT were independent predictors of poor prognosis of SCNEC. Radical surgery combined with a chemotherapy regimen similar to that of small cell lung cancer may be a potential therapeutic approach for SCNEC.

Preoperative SCC-Ag and thrombocytosis as predictive markers for pelvic lymphatic metastasis of squamous cervical cancer in early FIGO stage.

Objectives: To explore the clinical significance of squamous cell carcinoma antigen (SCC-Ag) and thrombocytosis to predict pelvic lymphatic metastasis (PLM) of squamous cervical cancer (SCC) in International Federation of Gynecology and Obstetrics (FIGO) stages IA-IIA. Methods: A retrospective clinicopathologic review of 782 patients of a primary cohort in three Chinese hospitals from 2010 to 2015, and 407 patients of a validation cohort in another institution from 2015 to 2017. A receiver operating characteristic curve was used to determine the optimal SCC-Ag threshold to predict PLM in the groups. Univariate and multivariate logistic analyses for PLM were performed to assess differences in outcome. Results: In the primary and validation cohort, 15.6% (122/782) and 25.3% (103/407) patients were classified into the thrombocytosis group (platelet count >300 × 109/L), respectively. Optimal cutoff values of SCC-Ag for predicting PLM of the thrombocytosis group and the normal group were 3.26 ng/mL (AUC 0.754; sensitivity 73.08%; specificity 72.92%; P = 0.000) and 4.58 ng/mL (AUC 0.706; sensitivity 53.26%; specificity 83.98%; P = 0.000), respectively, in the primary cohort, and 1.55 ng/mL (AUC 0.705; sensitivity 79.31%; specificity 55.41%; P = 0.000) and 1.75 ng/mL (AUC 0.655; sensitivity 69.57%; specificity 64.26%; P = 0.000), respectively, in the validation cohort. In multivariate logistic analysis, preoperative SCC-Ag over 3.26 ng/mL and lymphovascular space involvement were the significant predictors of PLM for SCC in FIGO stages IA-IIA. Conclusions: Preoperative SCC-Ag alone or combined with thrombocytosis might be used as predictive markers for PLM before initial treatment in early stage SCC.

[Association between HLA-A and HLA-DRB1 allele polymorphisms and susceptibility to tuberculosis in southern Chinese population].

OBJECTIVE: To investigate the correlation between tumor-associated macrophages (TAMs) and the development of high risk human papilloma virus (hr-HPV)-related cervical cancer. METHODS: A total of 112 cases of cervical tissue were collected, including 16 normal cervical tissues, 55 cervical intraepithelial neoplasia (CIN) tissues and 41 squamous cervical cancer (SCC) tissues. The expression of CD163+ macrophages in the cervical tissues was detected by immunohistochemical method, and the results were analyzed in relation with the clinical data of the patients. RESULTS: Immunohistochemical analysis showed that the cell density of CD163+ macrophages increased progressively with the increase in the tissue malignancy, in the order of normal cervical tissue, CIN Ⅰ, CIN Ⅱ-Ⅲ, and SCC. Correlation analysis revealed a positive relationship between CD163+ macrophage density and tissue malignancy (P=0.000). The density of CD163+ macrophages was significantly upregulated in HR-HPV-positive SCC tissue (P < 0.05). CD163+ macrophages were positively correlated with cervical lymph node metastasis (P=0.005) and FIGO stage (P=0.004) of SCC. CONCLUSIONS: The expression of CD163+ macrophages is positively correlated with malignant transformation of cervical tissues, and hr-HPV infection is significantly correlated with CD163 expression level in the macrophages. CD163+ macrophages can be used as predictors of the occurrence and progression of cervical cancer caused by hr-HPV infection.

Clinical Significance of CD163+ and CD68+ Tumor-associated Macrophages in High-risk HPV-related Cervical Cancer.

Objective. To explore the influence of M2-polarized tumor-associated macrophages (TAMs) on high-risk human papillomavirus (hr-HPV)-related cervical carcinogenesis and metastasis. Methods. CD68+ and CD163+ macrophages were examined immunohistochemically in a series of 130 samples, including 26 cases of normal cervical tissues, 59 cases of cervical intraepithelial neoplasia (CIN), and 45 cases of squamous cell carcinoma (SCC), and the results were statistically analyzed. The macrophage count was corrected for the epithelial and stromal compartments respectively. Clinical data were also obtained. Results. High counts of CD68+ and CD163+ macrophages were associated with hr-HPV infection (both p < 0.05) and positively correlated with cervical carcinogenesis (Spearman's rho = 0.478, p = 0.000; Spearman's rho = 0.676, p =0.000, respectively). The immunostaining pattern of CD163 exhibited clearer background than that of CD68. CD163+ macrophages showed a more obviously increasing migration into the epithelium along with the progression of CIN to invasive cancer. Notably, a high index of CD163+ macrophages was significantly associated with higher FIGO stages (p = 0.009) and lymph node metastasis (p = 0.012), but a similar finding was not found for CD68+ macrophages (p = 0.067, p = 0.079, respectively). Conclusions. Our study supported a critical role of TAMs as a prospective predictor for hr-HPV-related cervical carcinogenesis. CD163, as a promising TAMs marker, is superior to CD68 for predicting the malignant transformation and metastatic potential of cervical cancer.

Orthotopic Xenograft Mouse Model of Cervical Cancer for Studying the Role of MicroRNA-21 in Promoting Lymph Node Metastasis.

Cervical cancer is the most frequent cause of gynecologic cancer-associated death worldwide. Animal models that demonstrate metastatic patterns consistent with the clinical course of cervical cancer are urgently needed to conduct studies focused on understanding the mechanisms of the disease and identifying optimal treatments. To address this, we established an orthotopic xenograft model of cervical cancer in female NOD-SCID mice using SiHa and ME180 cell lines stably expressing green fluorescent protein to evaluate the role of microRNA-21 (miR-21) in spontaneous lymph node metastasis in vivo. In this case, SiHa and ME180 cells were transduced by lentivirus to stably express green fluorescent protein and miR-21. Overexpression of miR-21 promoted proliferation, migration, and invasion of SiHa and ME180 cells in vitro. Finally, an orthotopic xenograft model of human cervical cancer was successfully established in NOD-SCID mice. Using this model, we confirmed that overexpression of miR-21 resulted in an increase in the size of primary tumors and in the frequency of spontaneous lymph node metastasis at the time of excision. Therefore, the use of the orthotopic xenograft model should allow for the investigation of novel factors that affect metastasis of cervical cancer and presents an opportunity to evaluate potential therapeutic agents that may inhibit the spread of the disease.

TGF-ß1-induced CK17 enhances cancer stem cell-like properties rather than EMT in promoting cervical cancer metastasis via the ERK1/2-MZF1 signaling pathway.

Tumor metastasis remains a major obstacle for improving overall cancer survival in cervical cancer (CC), which may be due to the existence of tumor microenvironment-related cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT). The mechanism underlying these processes needs to be further elucidated. Here, we report that TGF-ß1, one of the key microenvironmental stimuli, can enhance CSC characteristics, facilitate the EMT, and induce CK17. Silencing CK17 expression attenuated CSC-like properties without affecting the EMT markers induced by TGF-ß1, whereas forced overexpression of CK17 promoted lymphatic metastasis in vivo even without EMT inducement. Inhibitors of ERK1/2 signaling drastically decreased the induction of CK17 mediated by TGF-ß1. By combined computational and experimental approaches, we identified and validated that MZF1 was a key transcription factor binding to the promoter of CK17. Taken together, these results demonstrate that CK17 induced by the TGF-ß1-ERK1/2-MZF1 signaling pathway facilitates metastasis by promoting the acquisition of CSC properties rather than by inducing the EMT process in CC, suggesting that this CK17-related signaling pathway might be a suitable target for the development of therapy for CC metastasis.

Molecular characterization of HbCZF1, a Hevea brasiliensis CCCH-type zinc finger protein that regulates hmg1.

KEY MESSAGE: The HbCZF1 protein binds to the hmg1 promoter in yeast and this interaction was confirmed in vitro. The hmg1 promoter was activated in transgenic plants by HbCZF1. Biosynthesis of natural rubber is known to be based on the mevalonate pathway in Hevea brasiliensis. The final step in the mevalonate production is catalyzed by the branch point enzyme, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), which shunts HMG-CoA into the isoprenoid pathway, leading to the synthesis of natural rubber. However, molecular regulation of HMGR expression is not known. To study the transcriptional regulation of HMGR, the yeast one-hybrid experiment was performed to screen the latex cDNA library using the hmg1 (one of the three HMGR in H. brasiliensis) promoter as bait. One cDNA that encodes the CCCH-type zinc finger protein, designated as HbCZF1, was isolated from H. brasiliensis. HbCZF1 interacted with the hmg1 promoter in yeast one-hybrid system and in vitro. HbCZF1 contains a 1110 bp open reading frame that encodes 369 amino acids. The deduced HbCZF1 protein was predicted to possess a typical C-X7-C-X5-C3-H CCCH motif and RNA recognition motif. HbCZF1 was predominant in the latex, but little expression was detected in the leaves, barks, and roots. Furthermore, in transgenic tobacco plants, over-expression of HbCZF1 highly activated the hmg1 promoter. These results suggested that HbCZF1 may participate in the regulation of natural rubber biosynthesis in H. brasiliensis.