P–370 RPL-protease A as a potential biomarker for predicting recurrent pregnancy loss

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Z Pei ◽  
H B Park ◽  
H S Choi ◽  
B Choi ◽  
H Y Park ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pregnancy Loss ◽  
Recurrent Pregnancy Loss ◽  
Related Factor ◽  
Cellular Functions ◽  
Related Factors ◽  
Potential Biomarker ◽  
Β Hcg

Abstract Study question Could the reduction of RPL-protease A be involved in the dysfunctional trophoblast for resulting in recurrent pregnancy loss (RPL). Summary answer Low expression of RPL-protease A may result in RPL and low serum RPL-protease A level may be a potential biomarker for predicting RPL. What is known already The RPL-protease A is expressed and secreted by placenta. The RPL-protease A is involved in the pathogenesis of pre-eclampsia, and the serum RPL-protease A level is higher in the patients with pre-eclampsia than that of normal groups. In our previous study, we identified that the RPL-protease A mRNA level was lower in the villi of patients with RPL than that of normal groups. Study design, size, duration Using the CRISPR/Cas9 system, the RPL-protease A gene knockout BeWo cell (BeWo KO) line was established, and the wild type (BeWo WT) and BeWo KO cells were applied to investigate the roles of RPL-protease A in trophoblasts. The human serum RPL-protease A levels were investigated by Western blot analysis and ELISA kit. Participants/materials, setting, methods The cell-cell fusion, cell counting analysis, invasion and scratch wound assays, cell cycle analysis, and immunocytochemical analysis were used to investigate cellular functions of RPL-protease A in trophoblast. The sera were obtained from 32 normal pregnant women and 60 women with RPL. The Western blot analysis and ELISA were used for detection of serum RPL-protease A levels. Main results and the role of chance The β-hCG was detected in fused BeWo WT cells, while the BeWo KO cells cannot fuse and did not express the β-hCG. The ability of invasion was decreased, but the capacity of migration and proliferation was higher in BeWo KO cells than BeWo WT cells. Cell fusion related factor (β-hCG), and cell invasion related factors (MMP–2 and MMP–9) were highly expressed in BeWo WT cells, and cell related factor (FAK), and cell proliferation related factors (ERK, p38, JNK, MKK3, MKK6, Raf, and Ras) were highly expressed in BeWo KO cells. The Western blot analysis and ELISA indicate that the serum RPL-protease A level was decreased in patients with RPL compared to that of normal groups. Limitations, reasons for caution The results of this study have the limitation of RPL-protease A functions in vitro. Wider implications of the findings: The cellular functions of RPL-protease A in trophoblasts were investigated to explain the pathogenesis of RPL, and low serum RPL-protease A level can be used for a potential biomarker predicting RPL. Trial registration number Not applicable

scholarly journals MicroRNA-145 attenuates IL-6-induced enhancements of sensitivity to UVB irradiation by suppressing MyD88 in HaCaT cells

2018 ◽  
Vol 32 ◽  
pp. 205873841879594 ◽  
Author(s):  
Hui Dong ◽  
Wei Jiang ◽  
Hongquan Chen ◽  
Shui Jiang ◽  
Yunshu Zang ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Lupus Erythematosus ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
Ultraviolet B ◽  
Hacat Cells ◽  
Related Factors ◽  
Uvb Irradiation

MicroRNAs (miRNAs/miRs) play vital roles in various immune diseases including systemic lupus erythematosus (SLE). The current study aimed to assess the role of miR-145 in interleukin-6 (IL-6)-treated HaCaT cells under ultraviolet B (UVB) irradiation and further explore the potential regulatory mechanism. HaCaT cells were pretreated with IL-6 and then exposed to UVB to assess the effect of IL-6 on sensitivity of HaCaT cells to UVB irradiation. The levels of miR-145 and MyD88 were altered by transfection and the transfected efficiency was verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR)/western blot analysis. Cell viability, percentage of apoptotic cells and expression levels of apoptosis-related factors were measured by trypan blue assay, flow cytometry assay, and western blot analysis, respectively. In addition, the levels of c-Jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) signaling pathway-related factors were assessed by western blot analysis. IL-6 treatments significantly aggravated the reduction of cell viability and promotion of cell apoptosis caused by UVB irradiation in HaCaT cells. Interestingly, miR-145 level was augmented by UVB exposure and miR-145 mimic alleviated IL-6-induced increase of sensitivity to UVB irradiation in HaCaT cells, as dramatically increased cell viability and reduced cell apoptosis. Opposite effects were observed in miR-145 inhibitor-transfected cells. Meanwhile, MyD88 was negatively regulated by miR-145 and MyD88 mediated the regulatory effect of miR-145 on IL-6- and UVB-treated cells. In addition, miR-145 mimic inhibited the JNK and NF-κB pathways by down-regulating MyD88. In conclusion, the present study demonstrated that miR-145 alleviated IL-6-induced increase of sensitivity to UVB irradiation by down-regulating MyD88 in HaCaT cells.

Long non-coding RNA LINC01194 promotes the inflammatory response and apoptosis of LPS-treated MLE 12 cells through the miR-203a-3p /MIP-2 axis

2021 ◽  
Author(s):  
Yuyao Shen ◽  
Senwei Zhao ◽  
Minglei Hua
Keyword(s):  
Inflammatory Response ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Luciferase Reporter ◽  
Mouse Lung ◽  
Elisa Assay ◽  
Non Coding Rna ◽  
Potential Biomarker ◽  
Long Non Coding Rna

Acute lung injury (ALI) induced by bacteria LPS is characterized by the upregulation of the apoptosis rate of tissue cells and aggravation of inflammatory response. Although many studies have focused on the pathogenesis of this disease, its mechanism remains unknown. This study examined the regulatory role of long non-coding RNA (lncRNA) LINC01194 in the progression of ALI through various bioinformatics analyses and experimental work, including ELISA assay, dual-luciferase reporter assay, biotinylated RNA pull-down assay, and western blot analysis. The result showed that the LINC01194 was overexpressed in the ALI-induced mice model. We observed a significant upregulation of LINC01194 in LPS-treated Mouse lung epithelial type II cells (MLE-12 cells) after 24 hrs of induction. Bioinformatics analysis, Elisa assay, qRT-PCR analysis, Biotinylated RNA pull-down assay, apoptosis test, and western blot analysis demonstrated that the LINC01194 could act as a miR-203a-3p sponge to activate the inflammatory response in LPS-induced ALI model through post-transcriptional upregulation of MIP-2. We showed that LINC01194 regulates the inflammatory response and apoptosis of LPS-induced mice and MLE-12 cells via the miR-203a-3p/MIP-2 axis. LINC01194 could be a potential biomarker for early diagnosis and the treatment of ALI.

scholarly journals RNF216 Promotes Occurrence and Progression of Cholangiocarcinoma via Regulation of the DIAPH3 Ubiquitination

2020 ◽  
Author(s):  
Jianfei Tu ◽  
Weiqian Chen ◽  
Miaomiao Meng ◽  
Siyu Zhao ◽  
Fazong Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Migration And Invasion ◽  
Metastatic Progression ◽  
Loss Of Function ◽  
Invasion And Migration ◽  
Potential Biomarker

Abstract BackgroundCholangiocarcinoma is a relatively uncommon malignant tumor with high mortality. However, the molecular underpinnings behind malignant progression of cholangiocarcinoma are incompletely understood. Here we demonstrate that RNF216 plays a suppressive role in cholangiocarcinoma occurrence and metastasis.MethodsIHC and Western blot analysis were performed to examine the expression pattern of RNF216 and DIAPH3 in the clinical CRC cholangiocarcinoma. The relationship between RNF216 and DIAPH3 was then validated using in western blot analysis. The mechanism of RNF216-mediated ubiquitination modification of DIAPH3 was analyzed via Co-IP analysis. Gain- or loss-of-function approaches were manipulated to evaluate the modulatory effects of RNF216 and DIAPH3 on cell growth and metastasis. The mediatory effects of RNF216 and DIAPH3 on cancerogenesis were validated in vivo.ResultsClinical data indicated that expression levels of RNF216 were associated with favorable clinical outcomes. RNF216 was downregulated in cholangiocarcinoma and inhibited cell proliferation and colony formation in vitro and xenograft tumorigenicity in vivo. Moreover, RNF216 suppressed Invasion and migration of cholangiocarcinoma. Mechanistic investigations further showed that RNF216 was involved in the ubiquitination of DIAPH3, a member of formin family related to assembly of actin cytoskeleton. RNF216 elicits tumor suppressor role by promoting degradation of DIAPH3. Importantly, expression of DIAPH3 rescued RNF216-mediated suppression of proliferation, cell migration, and invasion. ConclusionOur findings uncover a suppressive role for RNF216 in cholangiocarcinoma proliferation and metastatic progression and provide novel insight into that RNF216 is a potential biomarker or serves as a therapeutic target for cholangiocarcinoma.

scholarly journals Anti-laminin-1 Autoantibodies, Pregnancy Loss and Endometriosis

2004 ◽  
Vol 11 (3-4) ◽  
pp. 261-266 ◽  
Author(s):  
Junko Inagaki ◽  
Akane Kondo ◽  
Luis R. Lopez ◽  
Yehuda Shoenfeld ◽  
Eiji Matsuura
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pregnancy Loss ◽  
First Trimester ◽  
Subsequent Pregnancy ◽  
Basement Membranes ◽  
Non Invasive ◽  
Carboxyl Terminal ◽  
Laminin 1

Laminin-1 is a major component and multifunctional glycoprotein of basement membranes that consists of three different subunits, α1, β1 and γ1 chains. It is the earliest synthesized network-forming protein during embryogenesis and plays an important role in embryonic development, embryonic implantation and placentation. We have recently shown that IgG anti-laminin-1 antibodies were significantly associated with recurrent first-trimester miscarriages and with subsequent pregnancy outcome. Interestingly, these antibodies were also observed in patients with endometriosis-associated infertility but not in patients with other causes of infertility, including tubal factors, hormonal and uterine abnormalities. Laminin-α1, -β1 and -γ1 mRNAs have been detected in 90% of endometriotic lesions and all laminin-α1, -β1 and -γ1 chains were localized in the basement membranes of glandular epithelium in endometriotic peritoneal lesions. Western blot analysis showed that anti-laminin-1 antibodies from those patients reacted with all laminin-1's chains. ELISA also confirmed that one of the target epitopes for these antibodies was located in a particular region of the laminin-1 molecule, i.e. the carboxyl-terminal globular G domain of α1 chain. IgM monoclonal anti-laminin-1 autoantibody, that we recently established, also recognized the G domain. Anti-laminin-1 antibodies from mice immunized with –mouse— laminin-1, caused a higher fetal resorption rate with lower embryonic and placental weights. Thus, anti-laminin-1 antibodies may be important in development of autoimmune-mediated reproductive failures and the assessment of the antibodies may provide a novel non-invasive diagnosis of endometriosis.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

scholarly journals Protocol for determining protein cysteine thiol redox status using western blot analysis

2021 ◽  
Vol 2 (2) ◽  
pp. 100566
Author(s):  
Bikram Datt Pant ◽  
Sunhee Oh ◽  
Kirankumar S. Mysore
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Redox Status ◽  
Thiol Redox

Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

2021 ◽  
pp. 096032712110061
Author(s):  
D Cao ◽  
L Chu ◽  
Z Xu ◽  
J Gong ◽  
R Deng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

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