scholarly journals Downregulation of miR-135b-5p Suppresses Progression of Esophageal Cancer and Contributes to the Effect of Cisplatin

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuzhu Di ◽  
Yanan Jiang ◽  
Xiuyun Shen ◽  
Jing Liu ◽  
Yang Gao ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
High Morbidity ◽  
Complementary Gene ◽  
Enhanced Expression ◽  
Direct Binding ◽  
Esophageal Epithelial Cells ◽  
Insight Into

Esophageal cancer (EC) is one of the commonest human cancers, which accompany high morbidity. MicroRNAs (miRNAs) play a pivotal role in various cancers, including EC. Our research aimed to reveal the function and mechanism of miR-135b-5p. Our research identified that miR-135b-5p was elevated in EC samples from TCGA database. Correspondingly real-time PCR assay also showed the miR-135b-5p is also higher expressed in Eca109, EC9706, KYSE150 cells than normal esophageal epithelial cells (Het-1A). CCK8, Edu, wound healing, Transwell assay, and western blot demonstrated miR-135b-5p inhibition suppresses proliferation, invasion, migration and promoted the apoptosis in Eca109 and EC9706 cells. Moreover, the miR-135b-5p inhibition also inhibited xenograft lump growth. We then predicted the complementary gene of miR-135b-5p using miRTarBase, TargetScan, and DIANA-microT. TXNIP was estimated as a complementary gene for miR-135b-5p. Luciferase report assay verified the direct binding site for miR-135b-5p and TXNIP. Real-time PCR and western blot assays showed that the inhibition of miR-135b-5p remarkably enhanced the levels of TXNIP in Eca109 and EC9706 cells. Furthermore, cisplatin (cis-diamminedichloroplatinum II, DDP) decreased miR-135b-5p expression and increased TXNIP expression. Enhanced expression of miR-135b-5p attenuated the inhibitory ability of cisplatin (cis-diamminedichloroplatinum II, DDP) in Eca109 cells, accompanied by TXNIP downregulation. In conclusion, the downregulation of miR-135b-5p suppresses the progression of EC through targeting TXNIP. MiR-135b-5p/TXNIP pathway contributes to the anti-tumor effect of DDP. These findings may provide new insight into the treatment of EC.

scholarly journals EGFL6 Modulates WNT Signaling to Drive Esophageal Cancer Metastasis and Proliferation

2020 ◽  
Author(s):  
Yanhong Wang ◽  
Jing Kang ◽  
Jihua Tian ◽  
Hongyan Jia ◽  
Juanjuan Wang ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Level ◽  
Marker Genes ◽  
Invasion And Migration ◽  
Ec Cells

Abstract Background Esophageal cancer (EC) is the sixth deadliest cancer in the world. There has been no breakthrough in the research on EC in the past few decades. Epidermal growth factor-like protein 6 (EGFL6), as a member of the epidermal growth factor superfamily, plays an important role in the occurrence and development of some tumors. However, the role of EGFL6 in the EC has never explored. Methods Immunohistochemical staining was used to evaluate the expression level of EGEC6 protein in human EC and its adjacent non-tumor tissues, and analyzed the correlation between the expression level of EGFL6 protein and clinical pathological indexes and survival rate. In vitro, by constructing EGFL6 silence and overexpressed EC cells,used CCK-8, clone formation, wound healing assays, transwell experiment and flow cytometry to explore the effects of EGFL6 on the proliferation, invasion, migration and apoptosis of EC. By using real-time PCR or western blot to detect the related marker genes of epithelial-mesenchymal transformation (EMT), tumor stem cells (TSCs) and Wnt/β-catenin. In vivo, established a nude mouse EC transplantation tumor model. Results The results showed that the expression level of EGFL6 in EC is significantly higher than that in adjacent non-tumor tissues, and is related to poor prognosis of patients. In vitro, CCK-8, clone formation, wound healing assays, transwell experiment and flow cytometry results show that EGFL6 overexpression can promotes proliferation, invasion and migration of EC cells and inhibits apoptosis. EGFL6 silencing inhibits proliferation, invasion and migration of EC cells and promotes apoptosis. Real-time PCR and Western-blot detection of EMT-related markers found that EGFL6 can induce EC cells EMT. Real-time PCR detection of esophageal cancer stem cell-related genes showed that EGFL6 may maintain the expression of esophageal cancer stem cell-like cell population. Western-blot detection of Wnt/β-catenin signaling marker genes showed that EGFL6 participated in the expression of Wnt/β-catenin signaling pathway. In vivo experiments found that knockout of EGFL6 could inhibit the formation of subcutaneous tumors in nude mice. Conclusion Taken together, our study identified a novel role and mechanism of EGFL6 in EC and provided epigenetic therapeutic strategies for the treatment of EC.

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

scholarly journals Astragaloside positively regulated osteogenic differentiation of pre-osteoblast MC3T3-E1 through PI3K/Akt signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Wei Bing Jing ◽  
Hongjuan Ji ◽  
Rui Jiang ◽  
Jinlong Wang
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Akt Signaling ◽  
Calcium Deposition ◽  
Control Group ◽  
Akt Signaling Pathway ◽  
Western Blot Assay

Abstract Background Osteoporosis is a widespread chronic disease characterized by low bone density. There is currently no gold standard treatment for osteoporosis. The aim of this study was to explore the role and mechanism of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. Methods MC3T3-E1 cells were divided into control and different dose of Astragaloside (10, 20, 40, 50, and 60 μg/ml). Then, ALP and ARS staining were performed to identify the effects of Astragaloside for early and late osteogenic capacity of MC3T3-E1 cells, respectively. Real-time PCR and western blot were performed to assess the ALP, OCN, and OSX expression. PI3K/Akt signaling pathway molecules were then assessed by Western blot. Finally, PI3K inhibitor, LY294002, was implemented to assess the mechanism of Astragaloside in promoting osteogenic differentiation of MC3T3-E1 cells. Results Astragaloside significantly increased the cell viability than the control group. Moreover, Astragaloside enhanced the ALP activity and calcium deposition than the control groups. Compared with the control group, Astragaloside increased the ALP, OCN, and OSX expression in a dose-response manner. Western blot assay further confirmed the real-time PCR results. Astragaloside could significantly increase the p-PI3K and p-Akt expression than the control group. LY294002 partially reversed the promotion effects of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. LY294002 partially reversed the promotion effects of Astragaloside on ALP, OCN, and OSX of MC3T3-E1 cells. Conclusion The present study suggested that Astragaloside promoted osteogenic differentiation of MC3T3-E1 cells through regulating PI3K/Akt signaling pathway.

scholarly journals The Expression of Ptch1 and Ptch2 in the Stenotic Tissue of Congenital Ureteropelvic Junction Obstruction in Children

2020 ◽  
Author(s):  
Yanfang Yang ◽  
wenwen han ◽  
Weiping Zhang ◽  
Ning Sun
Keyword(s):  
Smooth Muscle ◽  
Western Blot ◽  
Smooth Muscle Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Ureteropelvic Junction Obstruction ◽  
Transmembrane Protein ◽  
Muscle Cells ◽  
Ureteropelvic Junction ◽  
Ureteral Polyps

Abstract Background: Ptch1 and Ptch2 are expressed in tubular epithelium and stromal cells adjacent to the UPJ. They mediate inhibition of Smoothened, a transmembrane protein expressed on the cell surface. If the pathway is disturbed, UPJOcan occur. This aim study aimed to determine the expression of Ptch1 (P1) and Ptch2 (P2) in stenotic segments in children with congenital ureteropelvic junction obstruction (UPJO) compared with normal control subjects. Methods: Stenotic segments of ureter tissues were obtained from 20 UPJO patients.UPJO caused by other pathogenies, such as vessel and ureteral polyps, were excluded. The control ureter specimens were obtained from 10 patients with Wilm’s tumor, and the tissues were confirmed histologically to be unaffected. Immunofluorescence, western blot and real-time PCR were used to investigate the expression of P1 and P2. Statistical methods were used to find the differences between the two groupsResults: P1 and P2 were identified in the cytoplasm of smooth muscle in two groups through immunohistochemistry. However, there were no statistical differences between the two groups in P1 and P2 with immunohistochemistry (P=0.31 and P=0.3, respectively). There were also no statistical differences with western blot (P=0.75 and P=0.9, respectively) and real-time PCR (P=0.52 and P=0.45, respectively). However, with the immunofluorescence it was found that red-stained P1 were diffused in the controls group, but were mainly located in the intracellular perinuclear compartment of smooth muscle cells in UPJO. Conclusions: The expression of P1 and P2 between the two groups had no statistical significant. P1 were mainly located in the intracellular perinuclear compartment of smooth muscle cells in UPJO. The P1 pathway might be disturbed by the abnormal distribution rather than the quantity, which might be one probable pathogenesis of UPJO.

Effect of alendronate sodium on chondrocytes in joint inflammation through down-regulation of microRNA-184 (miR-184)

2021 ◽  
Vol 11 (7) ◽  
pp. 1132-1138
Author(s):  
Lin Shi ◽  
Xiuyun Li ◽  
Junfeng Tang
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Articular Chondrocytes ◽  
Cell Model ◽  
Joint Inflammation ◽  
Treg Cells ◽  
Inflammatory Factors ◽  
Model Group ◽  
Knee Arthritis

Chondrocytes participate in the progression of osteoarthritis (OA). Alendronate (ALN) can significantly improve the pathological changes of knee arthritis. However, whether alendronate affects chondrocytes of knee arthritis remains unclear. In this paper, the articular chondrocytes were assigned into model group. The inflammation cell model group was prepared using 10 ng/mL IL-1β, the alendronate group was co-cultured with 10 ng/mL IL-1β and 10 ng/mL ALN, and the miR-184 group was transfected with miR-184 siRNA on the basis of an inflammation model followed by the analysis of miR-184, BMP-2 and BMP-4 expression by real-time PCR, IL-1β and IL-22 levels were assayed by means of ELISA, Treg cells were detected by flow cytometry, IL-35 and TGF-β levels were checked by means of real-time PCR and western blot, and Wnt3, Wnt4 and β-Catenin protein levels were investigated by means of western blot. After alendronate and miR-184 siRNA were applied to the arthritis rat model, Treg cells was significantly decreased, IL-35 and TGF-β mRNA and secretion were reduced, miR-184 was down-regulated, BMP-2 and BMP-4 were upregulated, along with decreased IL-1β and IL-22 levels and expressions of Wnt3, Wnt4 and β-Catenin (P < 0.05). Alendronate inhibits Wnt/β-Catenin pathway by down-regulating miR-184, Treg cell and cytokines secretions these cytokines upregulate BMP-2 and BMP-4 in articular chondrocytes, and inhibits inflammatory factors secretion, thus ameliorating the progression of chondrocyte inflammation.

scholarly journals The Differential Expression of miRNAs and a Preliminary Study on the Mechanism of miR-194-3p in Keloids

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Zhishan Xu ◽  
Bingyu Guo ◽  
Peng Chang ◽  
Qiang Hui ◽  
Wei Li ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Differential Expression ◽  
Real Time Pcr ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cultured Fibroblasts ◽  
Gene Assay ◽  
And Migration ◽  
Related Proteins

The aim of this study was to detect abnormally expressed microRNA (miRNA) in keloids and to study their functions. The differential expression of miRNAs in keloids and normal tissue was detected by gene microarray. MiRNA expression was verified by real-time PCR. A luciferase reporter gene assay, western blot, and real-time PCR were used to detect the effect of miR-194-3p on RUNX2. An MTT assay and a transwell assay were used to detect the effect of miR-194-3p in both primary cultured fibroblasts and HKF cells. Related proteins were analysed by western blot and real-time PCR. The expression of miR-194-3p was lower in keloids, and MiR-194-3p was shown to target RUNX2 directly. MiR-194-3p inhibited the proliferation and migration of fibroblasts through the inhibition of CDK4 and MMP2. MiR-194-3p and RUNX2 may become new targets for the prevention and treatment of keloids.

scholarly journals XPC Promotes Pluripotency of Human Dental Pulp Cells through Regulation of Oct-4/Sox2/c-Myc

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Lu Liu ◽  
Zhengjun Peng ◽  
Zhezhen Xu ◽  
Xi Wei
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Dental Pulp ◽  
Dental Pulp Cells ◽  
Multilineage Differentiation ◽  
Human Dental Pulp Cells ◽  
Human Dental Pulp ◽  
Pulp Cells

Introduction. Xeroderma pigmentosum group C (XPC), essential component of multisubunit stem cell coactivator complex (SCC), functions as the critical factor modulating pluripotency and genome integrity through interaction with Oct-4/Sox2. However, its specific role in regulating pluripotency and multilineage differentiation of human dental pulp cells (DPCs) remains unknown. Methods. To elucidate the functional role XPC played in pluripotency and multilineage differentiation of DPCs, expressions of XPC in DPCs with long-term culture were examined by real-time PCR and western blot. DPCs were transfected with lentiviral-mediated human XPC gene; then transfection rate was investigated by real-time PCR and western blot. Cell cycle, apoptosis, proliferation, senescence, multilineage differentiation, and expression of Oct-4/Sox2/c-Myc in transfected DPCs were examined. Results. XPC, Oct-4, Sox2, and c-Myc were downregulated at P7 compared with P3 in DPCs with long-term culture. XPC genes were upregulated in DPCs at P2 after transfection and maintained high expression level at P3 and P7. Cell proliferation, PI value, and telomerase activity were enhanced, whereas apoptosis was suppressed in transfected DPCs. Oct-4/Sox2/c-Myc were significantly upregulated, and multilineage differentiation in DPCs with XPC overexpression was enhanced after transfection. Conclusions. XPC plays an essential role in the modulation of pluripotency and multilineage differentiation of DPCs through regulation of Oct-4/Sox2/c-Myc.

HOXB7 Overexpression in Mesenchymal Cells Stimulates the Production of Pro-Angiogenic Molecules: Potential Role in Multiple Myeloma Associated Angiogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2743-2743
Author(s):  
Simona Colla ◽  
Nicola Giuliani ◽  
Paola Storti ◽  
Mirca Lazzaretti ◽  
Katia Todoerti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Line ◽  
Real Time ◽  
Real Time Pcr ◽  
Potential Role ◽  
Angiogenic Switch ◽  
Bone Biopsies

Abstract Bone marrow (BM) neo-angiogenesis has a critical role in multiple myeloma (MM) progression. It is well established that the angiogenic process in MM is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment cells. However the molecular mechanisms at the basis of the angiogenic process in MM are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is critical in tumor-associated angiogenic switch in solid tumors as breast cancer. Actually the potential role of HOXB7 in MM-induced angiogenesis is not known. In this study we have investigated the expression of HOXB7 by MM and BM microenvironment cells and its potential role in the regulation of the angiogenic process. First, by microarray analysis in a large database of MM patients (n°= 132) we found that HOXB7 was overexpressed by MM cells in about 10% of patients as compared to healthy donors and MGUS subjects. On the other hand HOXB7 mRNA was expressed in 18 out of 23 human myeloma cell lines tested. Moreover, we found that isolated BM mesenchymal (MSC) and osteoblastic (OB) cells, obtained from bone biopsies in a subgroup of MM patients (n°=24) expressed HOXB7 gene by microarray analysis and real time PCR. HOXB7 expression was also investigated at protein level by immunohistochemistry on bone biopsies of MM patients finding that MSC and OB as well as endothelial cells expressed HOXB7 protein mainly at nuclear level. In order to investigate the potential role of HOXB7 in the angiogenic process we enforced HOXB7 expression by lentivirus vectors in MSC using both primary BM MSC and the human MSC cell line HS-5 to obtain a stable transduced cell line. The overexpression of HOXB7 in HOXB7 transduced MSC as compared to the empty vector-transduced MSC cells was confirmed by real time PCR, western blot and immunohistochemistry. By Gene chips U133 plus 2.0 (Affymetrix) we evaluated the gene expression profiling of HOXB7 over-expressing MSC finding that proangiogenic cytokines, metalloproteinases and chemokines were significantly modulated in HOXB7-transduced MSC cells as compared to control cells. Data were validated either by real time PCR or by western blot and by an angiogenesis antibody array showing that bFGF and VEGF production was induced in MSC by HOXB7 overexpression. Consistently, we found that conditioned media of HOXB7-transduced MSC cells significantly stimulated vessel formation as compared to controls using an in vitro angiogenic model. Finally we observed that the angiogenic in vitro differentiation of HOXB7-transduced MSC was significantly increased as compared to controls. In conclusion our data suggest the HOXB7 overexpression in MSC regulates the angiogenic switch and could be a potential therapeutic target in MM-induced angiogenesis.

miR-184 Inhibits Inflammation Through Targeting Toll-Like Receptor 4 (TLR4)/NLR Family Pyrin Domain Containing 3 (NLRP3) Signaling Pathway in Neonatal Purulent Meningitis

2020 ◽  
Vol 10 (4) ◽  
pp. 569-575
Author(s):  
Jiang Wu ◽  
Shixiong Yang ◽  
Jin Shi ◽  
Yibing Shi
Keyword(s):  
Cell Proliferation ◽  
Cerebrospinal Fluid ◽  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Caspase 3 ◽  
Caspase 1 ◽  
Purulent Meningitis ◽  
Inflammation Group

Neonatal purulent meningitis (NPM) leads to higher mortality and neurological sequelae rates. miR184 involves in inflammation and tumor, but the role of miR-184 in NPM remains unclear. NPM patients and non-intracranial infected neonates were collected and miR-184 expression in cerebrospinal fluid was assessed by real-time PCR. The Neuro-2a cell line was cultured and divided into control group, inflammation group (treated with LPS), and miR-184 inhibitor group, which was transfected with miR-184 inhibitor on the basis of inflammation followed by analysis of miR-184 and TLR4 expression by Real time PCR, Caspase 3 activity, cell proliferation by MTT assay, secretion of IL-1β and IL-6 by ELISA, NLRP3 expression by real time PCR and western blot, and Caspase-1 p20 and NF- B level by western blot. miR-184 expression level was significantly increased in cerebrospinal fluid of NPM group (P < 0 05) and also elevated in inflammation group along with significantly inhibited cell proliferation was inhibited, increased Caspase 3 activity, IL-1β and IL-6 secretion, and decreased TLR4, NLRP3, Caspase-1 p20 and NFκ- B expression (P < 0 05). miR-184 inhibitor significantly down-regulated miR-184 expression in the inflammation group, promoted cell proliferation, decreased Caspase 3 activity, IL-1β and IL-6 secretion, and increased TLR4, NLRP3, Caspase1 p20 and NF- κB expression (P < 0 05). miR-184 expression is increased in neonatal purulent meningitis and it can inhibit inflammation by targeting TLR4/NLRP3 signaling pathway, leading to amelioration of the progression of neonatal purulent meningitis.

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