scholarly journals Διερεύνηση της έκφρασης γονιδίων τα οποία ενέχονται στο μεταβολισμό των λιπιδίων στην οστεοαρθρίτιδα και μηχανισμοί ρύθμισης αυτών

2014 ◽  
Author(s):  
Φωτεινή Κωστοπούλου
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Tgf Β1

Σκοπός: Αντικείμενο της διατριβής αποτέλεσε η διερεύνηση του μεταβολικού φαινοτύπου της ΟΑ μέσω μελέτης της έκφρασης γονιδίων που ενέχονται στο μεταβολισμό των λιπιδίων, όπως το SREBΡ–2 και το HMGCR, των μοριακών σηματοδοτικών μονοπατιών που ελέγχουν τη δράση τους, καθώς και η ρύθμιση της έκφρασης γονιδίων που ενέχονται στη σύνθεση και στην έξοδο της χοληστερόλης με τη χρήση microRNAs. Επιπλέον, δημιουργήθηκε πειραματικό ζωικό μοντέλο ΟΑ για την ανάδειξη πιθανών θεραπευτικών μορίων της νόσου in vivo. Μεθοδολογία: Ο προσδιορισμός της έκφρασης γονιδίων που συμμετέχουν στο μεταβολισμό των λιπιδίων έγινε με τις μεθόδους της real-time PCR και του Western blot. Η διερεύνηση του σηματοδοτικού μονοπατιού που ευθύνεται για την ενεργοποίηση της έκφρασής τους έγινε με τη χορήγηση TGF-β1 και αναστολέα των ιντεγκρινών στα χονδροκύτταρα. Επιπλέον, έγινε χορήγηση του miR-33a καθώς και του αναστολέα του anti-miR-33a στα χονδροκύτταρα προκειμένου να διερευνηθεί ο ρόλος του στη ρύθμιση της σύνθεσης της χοληστερόλης αλλά και στην έκφραση των γονιδίων που ενέχονται στην έξοδο της χοληστερόλης από τα χονδροκύτταρα. Τέλος, δημιουργήθηκε πειραματικό μοντέλο ΟΑ επαγόμενο με διατομή του πρόσθιου χιαστού συνδέσμου σε κουνέλια.Αποτελέσματα: Παρατηρήθηκε αυξημένη έκφραση του SREBP–2, του HMGCR, της ιντεγκρίνης aV, του TGF–β1 και των phosphor-PI3k και phosphor-Akt στα ΟΑ χονδροκύτταρα συγκριτικά με τα φυσιολογικά. Η επαγωγή χονδροκυττάρων με TGF–β1 προκάλεσε φωσφορυλίωση των PI3k και Akt και αύξηση της έκφρασης των γονιδίων SREBP–2 και HMGCR. Η αναστολή του TGF–β1 επέφερε μείωση των επιπέδων έκφρασης του SREBP–2, ενώ αναστολή της έκφρασης των ιντεγκρινών επέφερε σημαντική μείωση της έκφρασης των SREBP–2 και HMGCR, καθώς και της ενεργής μορφής της κινάσης PI3K. Στη συνέχεια, διαπιστώσαμε αυξημένη έκφραση του miR–33a στα ΟΑ χονδροκύτταρα συγκριτικά με τα φυσιολογικά και επαγωγή της έκφρασής του από τον TGF–β1, ενώ η χρήση αναστολέα του miR–33a σε επαγόμενα με TGF–β1 χονδροκύτταρα οδήγησε σε μείωση της φωσφορυλίωσης της πρωτεΐνης Akt. Επιπλέον, η χορήγηση του miR–33a στα φυσιολογικά χονδροκύτταρα επέφερε μείωση της έκφρασης των γονιδίων ABCA1 και ApoA1 και ταυτόχρονη αύξηση των επιπέδων της MMP–13, γεγονός που αντιστράφηκε μετά τη χρήση αναστολέα του miR–33a στα ΟΑ χονδροκύτταρα. Στο in vivo πειραματικό τμήμα της διατριβής διαπιστώσαμε στατιστικά σημαντική αύξηση των επιπέδων έκφρασης του SREBP–2 στα ΟΑ χονδροκύτταρα συγκριτικά με τα φυσιολογικά, ενώ η ενδοαρθρική χορήγηση αναστολέα των υποδοχέων του TGF–β προκάλεσε στατιστικά σημαντική μείωση των επιπέδων έκφρασης του SREBP–2, του COLX και της MMP–13, με ταυτόχρονη αύξηση των μεταγραφικών επιπέδων των αναβολικών μορίων COL2A1 και ACAN.Συμπεράσματα: Αναδείχτηκε για πρώτη φορά η εμπλοκή του SREBP–2 και του HMGCR στα ΟΑ χονδροκύτταρα καθώς και ο σηματοδοτικός μηχανισμός ρύθμισής τους μέσω των κινασών PI3K και Akt, ως αποτέλεσμα της ενεργοποίησης του TGF–β σηματοδοτικού μονοπατιού και της αυξημένης έκφρασης της ιντεγκρίνης aV στα ΟΑ χονδροκύτταρα. Επιπλέον, η αναστολή του miR–33a, η οποία επάγει την αυξημένη έκφραση γονιδίων που ευθύνονται για την έξοδο της περίσσειας χοληστερόλης από τα ΟΑ χονδροκύτταρα, αναδεικνύεται ως πιθανός θεραπευτικός μοριακός στόχος για την αναστολή της εξέλιξης της ΟΑ. Επίσης, η αναστολή του TGF–β in vivo, αναδεικνύεται ως μία νέα θεραπευτική προσέγγιση για την αντιμετώπιση της νόσου.

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.

Abstract 1958: MicroRNA Mir-27b Rescues Impaired Angiogenic Function of Endothelial Progenitor Cells and Accelerates Wound Healing in Type 2 Diabetes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Jie-Mei Wang ◽  
Jun Tao ◽  
Alex F Chen
Keyword(s):  
Type 2 Diabetes ◽  
Wound Healing ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Real Time Pcr ◽  
Western Blot Analysis ◽  
Type 2 Diabetic

Endothelial progenitor cells (EPCs) play a key role in angiogenesis, which is dysfunctional in diabetes. MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level. However, whether miRNAs regulate EPC-mediated angiogenesis in diabetes is unknown. We tested the hypothesis that mir-27b rescues impaired EPC angiogenesis in vitro and in vivo via suppressing anti-angiogenic molecule thrombospondin-1 (TSP-1) in type 2 diabetes. Bone marrow-derived EPCs from adult male (C57BLKS/J, 9 weeks) type 2 diabetic db/db and their normal littermates db/+ mice (glucose 371.8±37.8 vs. 167.5±21.3 mg/dL, n=38, p<0.05) were used. miRNA processing enzyme Dicer in EPCs was decreased by >40% in db/db vs. db/+ mice (Western blot analysis, n=4 p<0.01), paralleled with >66% reduction of mir-27b expression (real-time PCR, n=4, p<0.05). Both TSP-1 mRNA and protein in EPCs were significantly higher in db/db vs. db/+ mice (real-time PCR, 130.1%, n=4, p<0.05, Western blot analysis, 127.4%, n=4 p<0.05), which were suppressed upon mir-27b mimic transfection (by 75%, real-time PCR and 69%, Western blot analysis, n=4 – 6, p<0.01). EPC-induced angiogenesis was decreased by >70% in db/db vs. db/+ mice (Matrigel tube formation assay, n=4, p<0.05), which was rescued upon mir-27b mimic transfection or silencing TSP-1 expression by its siRNA (both n=4, p<0.05). Furthermore, inhibition of mir-27b in normal EPCs increased their TSP-1 protein by 117.5% (n=6, p<0.05) and impaired their angiogenesis by 81.5% (n=4, p<0.01), both were reversed by silencing TSP-1 expression by its siRNA. Finally, excisional wound closure was markedly delayed in db/db vs. db/+ mice (4-mm punch biopsy, n=4, p<0.05), accompanied by impaired wound angiogenesis (perfusion index by Laser Doppler, n=4, p<0.05). Cell therapy of diabetic EPCs (3×10 5 cells) transfected with mir-27b mimic onto diabetic wounds significantly accelerated their closure rates (n=4, p<0.05 vs. diabetic EPCs alone), with a concomitant augmentation of in vivo wound angiogenesis (n=4, p<0.05). Mir-27b rescues impaired EPC angiogenesis and accelerates wound healing in type 2 diabetic mice, at least in part, via suppressing TSP-1 expression. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

scholarly journals Long Noncoding RNA uc.412 Promotes Mesangial Cell Fibrosis via Binding ELAVL1 in Glomerulosclerosis

2020 ◽  
Author(s):  
Jialing Ji ◽  
Ju Rong ◽  
Ming Sun ◽  
Chan Huang ◽  
Jin Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Noncoding Rna ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Critical Role ◽  
Convincing Evidence ◽  
Tgf Β1

Abstract Background: Glomerulosclerosis is a characteristic pathologic feature in chronic kidney disease (CKD). Convincing evidence indicates that the mesangial cells (MCs) play critical role in this process. However, the exact mechanism remains unclear. Using RNA-seq analysis, we previously found that lncRNA uc.412 was involved in the MC proliferation. Here, the effect of uc.412 on glomerular fibrosis and the potential mechanism were explored. Methods: In vivo, CKD mice models were established by 5/6 nephrectomy. The expression of lncRNA uc.412 in CKD was detected by Real-Time PCR. In vitro, MCs were intervened with TGF-β1 (10ng/mL). The uc.412 expression in MCs was detected by in site hybridization. MCs were transfected with uc.412 siRNA or a lentivirus targeting uc.412 and then examined using western blot, Real-Time PCR, RNA pull down assay and immunofluorescence staining. Results: We found that the expression of uc.412 was significantly increased in CKD mice and is induced by TGF-β1 via Smad3- dependent signal pathway. Overexpressing uc.412 caused MCs fibrosis and knockdown of uc.412 alleviated TGF-β1-induced MCs fibrosis. Using RNA pull down analysis, we found that the ELAVL1 was the specific binding protein for uc.412. Moreover, ELAVL1 expression was increased in TGF-β1-treated MCs and silencing ELAVL1 expression attenuated MCs fibrosis. Conclusions: Thus, here, we demonstrated that uc.412, which is regulated in a Smad3-dependent mechanism, is significantly increased during progression of CKD via regulating ELAVL1 expression. Our findings provided the therapeutic strategy for treatment of CKD.

scholarly journals JMJD2C promotes colorectal cancer metastasis via regulating histone methylation of MALAT1 promoter and enhancing β-catenin signaling pathway

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Xinnan Wu ◽  
Ruixiao Li ◽  
Qing Song ◽  
Chengcheng Zhang ◽  
Ru Jia ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Signaling Pathway ◽  
Histone Methylation ◽  
Real Time Pcr ◽  
Methylation Level ◽  
Promoter Activity ◽  
Histone Demethylase

Abstract Background Our previous work demonstrated that lncRNA-MALAT1 was overexpressed in recurrent colorectal cancer (CRC) and metastatic sites in post-surgical patients. However, the upstream regulatory mechanism of MALAT1 is not well-defined. Histone demethylase JMJD2C holds great potential of epigenetic regulating mechanism in tumor diseases, especially the moderating effect on the promoter activity of targeted genes associated closely with tumor development. Therefore, we herein investigated whether JMJD2C could epigeneticly regulate the promoter activity of MALAT1 and the downstream β-catenin signaling pathway, thereby affecting the metastatic abilities of CRC cells. Methods JMJD2C expressions in human CRC samples were detected by real-time PCR and immunohistochemistry staining. Gene silencing and overexpressing efficiencies of JMJD2C were confirmed by real-time PCR and western blot. The migration of CRC cells in vitro were tested by transwell and wound healing assays. The protein expression and cellular localization of JMJD2C and β-catenin were characterized by immunofluorescence staining and western blot. The histone methylation level of MALAT1 promoter region (H3K9me3 and H3K36me3) was tested by ChIP-PCR assays. The promoter activity of MALAT1 was detected by luciferase reporter assay. The expressions of MALAT1 and the downstream β-catenin signaling pathway related genes in CRC cells were detected by real-time PCR and western blot, respectively. The nude mice tail vein metastasis model was established to observe the effect of JMJD2C on the lung metastasis of CRC cells in vivo. Results Our present results indicated that histone demethylase JMJD2C was overexpressed in matched CRC tumor tissues of primary and metastatic foci, and CRC patients with lower JMJD2C expression in primary tumors had better prognosis with longer OS (Overall Survival). The following biological function observation suggested that JMJD2C promoted CRC metastasis in vitro and in vivo. Further molecular mechanism investigation demonstrated that JMJD2C protein translocated into the nuclear, lowered the histone methylation level of MALAT1 promoter in the sites of H3K9me3 and H3K36me3, up-regulated the expression of MALAT1, and enhanced the β-catenin signaling pathway in CRC cells. Conclusion Our data demonstrated that JMJD2C could enhance the metastatic abilities of CRC cells in vitro and in vivo by regulating the histone methylation level of MALAT1 promoter, thereby up-regulating the expression of MALAT1 and enhancing the activity of β-catenin signaling pathway, providing that JMJD2C might be a novel therapeutic target for CRC metastasis.

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 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 Periplaneta Americana Extract may Attenuate 2,4,6-Trinitrobenzenesulfonic Acid-Induced Intestinal Fibrosis by Inhibiting TGF-β/Smad Signaling Pathway

2021 ◽  
Author(s):  
Jing Liu ◽  
Pin Lv ◽  
Xiang Rao ◽  
Jiajia Wang
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Periplaneta Americana ◽  
Collagen I ◽  
Smad Signaling ◽  
Intestinal Fibrosis ◽  
Smad Signaling Pathway

Abstract PurposeIntestinal fibrosis is an incurable digestive disease accompanied by stricture formation, and it has an increasing incidence in recent years. Periplaneta americana is one of the medicinal insects with a long history. There are few reports on the effect of intestinal fibrosis. This study aims to evaluate the inhibitory effect of PA treatment on intestinal fibrosis. MethodsTNBS was used to establish intestinal fibrosis model by enema in BALB/c mice. The mice were treated with PA (50, 100, 200 mg/kg body weight) and 5-aminosalicylic acid (5-ASA) (40mg/kg) by gavage once a day for 6 weeks. At the end of the last week, the mice were sacrificed. Colon samples were collected for H&E and Masson staining. The mRNA and protein expression of α-smooth muscle actin (α-SMA), collagen I and the transforming growth factor-β (TGF-β) / Smad signaling pathway were conducted by real-time PCR and western blot analysis. In vitro, TGF-β1 was used to induce intestinal fibrosis at human colon fibroblasts (CCD-18Co). And using real-time PCR and western blot methods to detect the expression of α-SMA and collagen I. ResultsPA inhibited the expression of α-SMA and collagen I in vivo and in vitro. But the difference was that PA inhibited the TGF-β/Smad signaling pathway in vivo, and the same results had not been obtained in vitro. Conclusion: PA may attenuate intestinal fibrosis by inhibiting TGF-β/Smad signaling pathway, but more experiments were needed to prove it in vitro. ConclusionsPA has potential pharmacological effects in inhibiting intestinal fibrosis, and the TGF-β/Smad signaling pathway seemed promising.

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 Μελέτη της επίδρασης του ενεργοποιού του πλασμινογόνου του τύπου της ουροκινάσης στην παθογένεια της κολίτιδας

2013 ◽  
Author(s):  
Ελισάβετ Καραμανάβη
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Ki 67 ◽  
Tnf Α ◽  
Tgf Β1

Η ύπαρξη συσχέτισης μεταξύ φλεγμονής και καρκίνου είναι πλέον ευρέως αποδεκτή. Τα αποτελέσματα πρόσφατων ερευνών δείχνουν ότι η ανάπτυξη και η επιβίωση των καρκινικών κυττάρων εξαρτώνται σε μεγάλο βαθμό από τους παράγοντες της φλεγμονής. Ορισμένοι ερευνητές θεωρούν τον καρκίνο σαν μια «πληγή» που ο οργανισμός αποτυγχάνει να επουλώσει. Ο ενεργοποιός του πλασμινογόνου του τύπου της ουροκινάσης (uPA) είναι μία πρωτεάση που συμμετέχει τόσο στη φλεγμονή όσο και στην επούλωση. Πολλές εργασίες καταδεικνύουν το ρόλο του συστήματος της ινωδόλυσης στην εξέλιξη, διείσδυση και μετάσταση του καρκίνου, ενώ ο uPA θεωρείται παράγοντας που προάγει την εξέλιξη του καρκίνου. Ωστόσο, δεν υπάρχουν μελέτες που να εξετάζουν τον πιθανό ρόλο του uPA στα πρώιμα στάδια της καρκινογένεσης. Εξαίρεση σε αυτό αποτελεί μία έρευνα, σύμφωνα με την οποία τα Apcmin/+ ποντίκια από τα οποία λείπει το γονίδιο που κωδικοποιεί τον uPA εμφανίζουν λιγότερα αδενώματα στο έντερο. Ο uPA ενεργοποιεί τον Tgf-β1, ο οποίος στα πρώιμα στάδια της καρκινογένεσης έχει ογκο-κατασταλτική δράση. Στόχος της συγκεκριμένης διατριβής ήταν η διερεύνηση της επίδρασης της απουσίας του uPA στην κολίτιδα που προκαλείται από το DSS. Φυσιολογικοί (WT) και uPA-/- BALB/cJ ποντικοί έλαβαν DSS με το πόσιμο νερό. Η νεκροτομή των πειραματόζωων πραγματοποιήθηκε μία εβδομάδα ή επτά μήνες μετά την παύση χορήγησης του DSS. Η φλεγμονή, η ακεραιότητα του επιθηλίου και οι δυσπλαστικές/προ-νεοπλασματικές αλλοιώσεις βαθμονομήθηκαν ξεχωριστά. Σε τομές παραφίνης σημάνθηκαν με ανοσοϊστοχημικά η β-κατενίνη, η Ε-καντχερίνη, ο Tgf-β1, η IL-6 και η IL-17. Για τη σήμανση των κυττάρων σε πολλαπλασιασμό και απόπτωση χρησιμοποιήθηκαν αντισώματα κατά του ki-67 και της κασπάσης-3, αντίστοιχα. Με ανοσοϊστοχημεία σημάνθηκαν επίσης τα Τ-λεμφοκύτταρα, τα ρυθμιστικά Τ-λεμφοκύτταρα, τα μακροφάγα, τα σιτευτικά κύτταρα και τα ουδετερόφιλα. Η ενεργός μορφή του Tgf-β1 και ο uPA προσδιορίστηκαν ποσοτικά στο βλεννογόνο του εντέρου με τη μέθοδο ELISA. Επίσης, χρησιμοποιώντας real-time PCR έγινε ποσοτικός προσδιορισμός των γονιδίων IL-6, IL-10, Tgf-β1, Tgf-βRII και SMAD-4. Τα φλεγμονικά κύτταρα και οι κυτταροκίνες αξιολογήθηκαν ποσοτικά με κατάλληλη μορφομετρική μέθοδο. Επτά μήνες μετά τη χορήγηση του DSS οι πέντε από τους έντεκα (5/11) uPA-/-+DSS ποντικοί εμφάνισαν πολυποειδή αδενώματα, ενώ κανένας από τους WT+DSS ποντικούς δεν εμφάνισε πολύποδα. Επιπλέον, οι uPA-/-+DSS ποντικοί είχαν περισσότερα φλεγμονικά κύτταρα και περισσότερες αλλοιώσεις δυσπλασίας από τους WT+DSS ποντικούς. Προκειμένου να διερευνηθεί γιατί οι uPA-/-+DSS ποντικοί εμφάνισαν πολύποδες, ενώ οι WT+DSS ποντικοί όχι, πραγματοποιήθηκε εξέταση του παχέος εντέρου σε πιο πρώιμη χρονική στιγμή. Μία εβδομάδα μετά τη διακοπή χορήγησης του DSS, και οι δύο ομάδες που έλαβαν DSS εμφάνισαν τις τυπικές αλλοιώσεις της κολίτιδας που προκαλεί το DSS. Ωστόσο, οι uPA-/-+DSS ποντικοί εμφάνισαν περισσότερες διαβρώσεις και πιο προχωρημένου βαθμού αλλοιώσεις δυσπλασίας. Στο βλεννογόνο του παχέος εντέρου των uPA-/-+DSS ποντικών παρατηρήθηκαν περισσότερα ουδετερόφιλα και μακροφάγα, λιγότερα ρυθμιστικά Τ-λεμφοκύτταρα, αύξηση των κυτταροκινών IL-6, IL-17, TNF-α και IL-10 και σημαντική μείωση των επιπέδων του ενεργού Tgf-β1 συγκριτικά με τους WT+DSS ποντικούς. Τα μη λειτουργικά ρυθμιστικά Τ-λεμφοκύτταρα, η πιο επίμονη φλεγμονική αντίδραση και η κληρονομούμενη ανικανότητα παραγωγής επαρκούς ποσότητας ενεργού Tgf-β1 εξωκυτταρικά, λόγω της έλλειψης του uPA, αποτελούν τις πιθανές εξηγήσεις για τη νεοπλασματογένεση που σχετίζεται με τη φλεγμονή στο παχύ έντερο των uPA-/-+DSS ποντικών.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

Sign in / Sign up

Export Citation Format

Share Document