scholarly journals Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

2020 ◽  
Author(s):  
yuehua yang ◽  
zunwen Lin ◽  
Jiangwei Chen ◽  
Sheng Ding ◽  
Weiwei Mao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Osteogenic Differentiation ◽  
Western Blotting ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pearson Correlation ◽  
Posterior Longitudinal Ligament ◽  
Interfering Rna ◽  
Collagen Type 1 ◽  
Spinal Ligament

Abstract Background: The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remains to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy associated gene expression was correlated with the expression of osteogenic differentiation genes. Methods: Expression of autophagy associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative Polymerase Chain Reaction (RT-qPCR), western blotting and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. Results: In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r=0.8233, P<0.001), alkaline phosphatase, biomineralization associated (ALP) (r=0.7821, P<0.001) and collagen type 1 (COL 1) (r=0.6078, P=0.001). Consistently, the upregulation of autophagy associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP and COL 1 by 63.2% (P<0.01), 52% (P<0.01) and 53.2% (P<0.01) in ligament fibroblasts from patients with OPLL, respectively. Conclusions: Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts, and promoted the development of OPLL.

scholarly journals Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Yuehua Yang ◽  
Zunwen Lin ◽  
Jiangwei Chen ◽  
Sheng Ding ◽  
Weiwei Mao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Osteogenic Differentiation ◽  
Western Blotting ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pearson Correlation ◽  
Posterior Longitudinal Ligament ◽  
Interfering Rna ◽  
Collagen Type 1 ◽  
Spinal Ligament

Abstract Background The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remain to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy-associated gene expression was correlated with the expression of osteogenic differentiation genes. Methods Expression of autophagy-associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy-associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy-associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. Results In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001), and collagen type 1 (COL 1) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy-associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP, and COL 1 by 63.2% (P < 0.01), 52% (P < 0.01), and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively. Conclusions Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.

scholarly journals Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

2020 ◽  
Author(s):  
yuehua yang ◽  
zunwen Lin ◽  
Jiangwei Chen ◽  
Sheng Ding ◽  
Weiwei Mao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Osteogenic Differentiation ◽  
Western Blotting ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pearson Correlation ◽  
Posterior Longitudinal Ligament ◽  
Interfering Rna ◽  
Collagen Type 1 ◽  
Spinal Ligament

Abstract Background The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remains to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy associated gene expression was correlated with the expression of osteogenic differentiation genes. Methods Expression of autophagy associated genes was detected in 21 samples from patients with OPLL patients and 16 non-OPLL patients. The correlation of autophagy associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy associated genes of fibroblasts was assessed by reverse transcription-quantitative Polymerase Chain Reaction (RT-qPCR), western blotting and immunofluorescence. The rate of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. Results In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001) and collagen type 1 (COL I) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy associated genes in fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The rate of autophagy was also increased in fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP and COL I by 63.2% (P < 0.01), 52% (P < 0.01) and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively. Conclusions Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts, and promoted the development of OPLL.

scholarly journals Integrated Transcriptomic Analysis Revealed Hub Genes and Pathways Involved in Sorafenib Resistance in Hepatocellular Carcinoma

2021 ◽  
Vol 27 ◽  
Author(s):  
Xili Jiang ◽  
Wei Zhang ◽  
Lifeng Li ◽  
Shucai Xie
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Pearson Correlation ◽  
Acquired Resistance ◽  
Rank Aggregation ◽  
Health Concern ◽  
Hub Genes ◽  
Multikinase Inhibitor

Hepatocellular carcinoma (HCC), a high mortality malignancy, has become a worldwide public health concern. Acquired resistance to the multikinase inhibitor sorafenib challenges its clinical efficacy and the survival benefits it provides to patients with advanced HCC. This study aimed to identify critical genes and pathways associated with sorafenib resistance in HCC using integrated bioinformatics analysis. Differentially expressed genes (DEGs) were identified using four HCC gene expression profiles (including 34 sorafenib-resistant and 29 sorafenib-sensitive samples) based on the robust rank aggregation method and R software. Gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool. A protein–protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING), and small molecules reversing sorafenib resistance were searched for using the connectivity map (CMAP) database. Pearson correlation and survival analyses of hub genes were performed using cBioPortal and Gene Expression Profiling and Interactive Analysis (GEPIA). Finally, the expression levels of hub genes in sorafenib-resistant HCC cells were verified using quantitative polymerase chain reaction (q-PCR). A total of 165 integrated DEGs (66 upregulated and 99 downregulated in sorafenib resistant samples compared sorafenib sensitive ones) primarily enriched in negative regulation of endopeptidase activity, extracellular exosome, and protease binding were identified. Some pathways were commonly shared between the integrated DEGs. Seven promising therapeutic agents and 13 hub genes were identified. These findings provide a strategy and theoretical basis for overcoming sorafenib resistance in HCC patients.

Evaluation of Appropriate Reference Genes For Investigating Gene Expression in Chlorops oryzae (Diptera: Chloropidae)

2019 ◽  
Vol 112 (5) ◽  
pp. 2207-2214 ◽  
Author(s):  
Ping Tian ◽  
Lin Qiu ◽  
Ailin Zhou ◽  
Guo Chen ◽  
Hualiang He ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Reference Genes ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Housekeeping Genes ◽  
Future Research ◽  
Economic Damage ◽  
Physiological Processes

Abstract Reverse transcription quantitative polymerase chain reaction (PCR) has become an invaluable technique for analyzing gene expression in many insects. However, this approach requires the use of stable reference genes to normalize the data. Chlorops oryzae causes significant economic damage to rice crops throughout Asia. The lack of suitable reference genes has hindered research on the molecular mechanisms underlying many physiological processes of this species. In this study, we used quantitative real-time PCR to evaluate the expression of eight C. oryzae housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (βACT), beta-tubulin (βTUB), Delta Elongation factor-1 (EF1δ), ribosomal protein S11 (RPS11), RPS15, C-terminal-Binding Protein (CtBP), and ribosomal protein 49 (RP49) in different developmental stages and tissues in both larvae and adults. We analyzed the data with four different software packages: geNorm, NormFinder, BestKeeper, and RefFinder and compared the results obtained with each method. The results indicate that PRS15 and RP49 can be used as stable reference genes for quantifying gene expression in different developmental stages and larval tissues. GAPDH and βACT, which have been considered stable reference genes by previous studies, were the least stable of the candidate genes with respect to larval tissues. GAPDH was, however, the most stable reference gene for adult tissues. We verified the candidate reference genes identified and found that the expression levels of Cadherins (Cads) changed when different reference genes were used to normalize gene expression. This study provides a valuable foundation for future research on gene function, and investigating the molecular basis of physiological processes, in C. oryzae.

Inhibition of PLOD3 Suppresses Proliferation, Invasion and Migration of Breast Cancer Cells

2019 ◽  
Vol 9 (11) ◽  
pp. 1528-1534
Author(s):  
Shiqiong Su ◽  
Qing Ni ◽  
Jing Hou
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Western Blotting ◽  
Molecular Mechanisms ◽  
Treatment Strategies ◽  
Cell Counting ◽  
Matrix Metalloproteinase 2 ◽  
Invasion And Migration ◽  
And Migration ◽  
Interfering Rna

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) has been reported to be involved in various human cancers. However, the function of PLOD3 in breast cancer (BC) has not been addressed. This research attempted to probe the effects and molecular mechanisms of PLOD3 in BC. The expression of PLOD3 was examined by Western blotting and RT-qPCR in several BC cell lines and nontumorigenic breast MCF-10A cells. Then, PLOD3 was silenced by transfecting with small interfering RNA (siRNA). Cell proliferation was measured by Cell Counting Kit-8 assay and cell cycle was evaluated by flow cytometry assay after transfection. Subsequently, wound healing assay and Transwell assay were exploited for detecting the abilities of cell invasion and migration, respectively. In addition, the expression of proliferation- and migration-related genes were examined by Western blotting. The results revealed that the expression of PLOD3 was upregulated in BC cell lines compared with MCF-10A cells. PLOD3 silencing suppressed the proliferative ability of BC cells, enhanced the ratio of cells in the G1 and G2 phases and reduced those in the S phase. Moreover, the expression of Ki67 and cyclinD1 were significantly downregulated, accompanied by an upregulation in p27 expression after transfection with PLOD3 siRNA. Furthermore, inhibition of PLOD3 restrained invasion and migration of BC cells coupled with a reduced expression of matrix metalloproteinase 2 (MMP2) and MMP9. The explorations unveiled that PLOD3 silencing restrained proliferation, invasion and migration of BC cells, which provides theoretical basis and treatment strategies for the treatment of BC.

scholarly journals Longitudinal Analysis of Gene Expression Changes During Cervical Carcinogenesis Reveals Potential Therapeutic Targets

2020 ◽  
Vol 16 ◽  
pp. 117693432092057
Author(s):  
Lijun Yu ◽  
Meiyan Wei ◽  
Fengyan Li
Keyword(s):  
Gene Expression ◽  
Western Blotting ◽  
Protein Interactions ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Enrichment Analysis ◽  
Gene Interaction ◽  
Gene Expression Omnibus ◽  
The Cancer Genome Atlas ◽  
Gene Expressions

Despite advances in the treatment of cervical cancer (CC), the prognosis of patients with CC remains to be improved. This study aimed to explore candidate gene targets for CC. CC datasets were downloaded from the Gene Expression Omnibus database. Genes with similar expression trends in varying steps of CC development were clustered using Short Time-series Expression Miner (STEM) software. Gene functions were then analyzed using the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Protein interactions among genes of interest were predicted, followed by drug-target genes and prognosis-associated genes. The expressions of the predicted genes were determined using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Red and green profiles with upward and downward gene expressions, respectively, were screened using STEM software. Genes with increased expression were significantly enriched in DNA replication, cell-cycle-related biological processes, and the p53 signaling pathway. Based on the predicted results of the Drug-Gene Interaction database, 17 drug-gene interaction pairs, including 3 red profile genes (TOP2A, RRM2, and POLA1) and 16 drugs, were obtained. The Cancer Genome Atlas data analysis showed that high POLA1 expression was significantly correlated with prolonged survival, indicating that POLA1 is protective against CC. RT-qPCR and Western blotting showed that the expressions of TOP2A, RRM2, and POLA1 gradually increased in the multistep process of CC. TOP2A, RRM2, and POLA1 may be targets for the treatment of CC. However, many studies are needed to validate our findings.

scholarly journals Endothelial progenitor cells promote osteogenic differentiation in co-cultured with mesenchymal stem cells via the MAPK-dependent pathway

2020 ◽  
Author(s):  
chu xu ◽  
haijie liu ◽  
yuanjia he ◽  
yuanqing li ◽  
xiaoning he
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Western Blotting ◽  
Nodule Formation ◽  
Endothelial Progenitor ◽  
Alp Activity

Abstract Background: The role of bone tissue engineering is to regenerate tissue using biomaterials and stem cell based approaches. Combination of two or more cell types is one of the strategies to promote bone formation. Endothelial progenitor cells (EPCs) may enhance the osteogenic properties of mesenchymal stem cells (MSCs) and promote bone healing, this study aimed to investigate the possible mechanisms of EPCs on promoting osteogenic differentiation of MSCs.Methods: MSCs and EPCs were isolated and co-cultured in Transwell chambers, the effects of EPCs on the regulation of MSC biological properties was investigated. Real-time PCR array and western blotting were performed to explore possible signaling pathways involved in osteogenesis. The expression of osteogenesis markers and calcium nodule formation was quantified by qRT-PCR, western blotting and Alizarin Red staining. Results: Results showed that MSCs exhibited greater alkaline phosphatase (ALP) activity and increased calcium mineral deposition significantly when co-cultured with EPCs. The mitogen-activated protein kinase (MAPK) signaling pathway was involved in this process. p38 gene expression and p38 protein phosphorylation levels showed significant up-regulation in co-cultured MSCs. Silencing expression of p38 in co-cultured MSCs reduced osteogenic gene expression, protein synthesis, ALP activity and calcium nodule formation. Conclusions: These data suggest paracrine signaling from EPCs influence the biological function and promote MSCs osteogenic differentiation. Activation of the p38MAPK pathway may be the key to enhancing MSCs osteogenic differentiation via indirect interactions with EPCs.

scholarly journals MMP14 Contributes to HDAC Inhibition-Induced Radiosensitization of Glioblastoma

2021 ◽  
Vol 22 (19) ◽  
pp. 10403
Author(s):  
Yuchuan Zhou ◽  
Hongxia Liu ◽  
Wang Zheng ◽  
Qianping Chen ◽  
Songling Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Treatment Method ◽  
Gene Expression Omnibus ◽  
Primary Brain Tumor ◽  
Study Gene Expression ◽  
Radiation Induced ◽  
Human Gbm

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Radiotherapy has long been an important treatment method of GBM. However, the intrinsic radioresistance of GBM cells is a key reason of poor therapeutic efficiency. Recently, many studies have shown that using the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) in radiotherapy may improve the prognosis of GBM patients, but the underlying molecular mechanisms remain unclear. In this study, Gene Expression Omnibus (GEO) datasets GSE153982 and GSE131956 were analyzed to evaluate radiation-induced changes of gene expression in GBM without or with SAHA treatment, respectively. Additionally, the survival-associated genes of GBM patients were screened using the Chinese Glioma Genome Atlas (CGGA) database. Taking the intersection of these three datasets, 11 survival-associated genes were discovered to be activated by irradiation and regulated by SAHA. The expressions of these genes were further verified in human GBM cell lines U251, T98G, and U251 homologous radioresistant cells (U251R) by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). It was found that MMP14 mRNA was considerably highly expressed in the radioresistant cell lines and was reduced by SAHA treatment. Transfection of MMP14 siRNA (siMMP14) suppressed cell survivals of these GBM cells after irradiation. Taken together, our results reveal for the first time that the MMP14 gene contributed to SAHA-induced radiosensitization of GBM.

scholarly journals Gene Expression Profiling of Blood in Ruptured Intracranial Aneurysms: in Search of Biomarkers

2013 ◽  
Vol 33 (7) ◽  
pp. 1025-1031 ◽  
Author(s):  
Joanna Pera ◽  
Michal Korostynski ◽  
Slawomir Golda ◽  
Marcin Piechota ◽  
Jaroslaw Dzbek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intracranial Aneurysms ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Relevant Information ◽  
Specific Pattern ◽  
Differentially Expressed ◽  
Specific Gene ◽  
Ruptured Intracranial Aneurysms

The molecular mechanisms underlying the systemic response to subarachnoid hemorrhage (SAH) from ruptured intracranial aneurysms (RAs) are not fully understood. We investigated whether the analysis of gene expression in peripheral blood could provide clinically relevant information regarding the biologic consequences of SAH. Transcriptomics were performed using Illumina HumanHT-12v4 microarrays for 43 RA patients and 18 controls (C). Differentially expressed transcripts were analyzed for overrepresented functional groups and blood cell type-specific gene expression. The set of differentially expressed transcripts was validated using quantitative polymerase chain reaction in an independent group of subjects (15 RA patients and 14 C). There were 135 differentially expressed genes (false discovery rate ≤1%, absolute fold change ≤1.7): the abundant levels of 78 mRNAs increased and 57 mRNAs decreased. Among RA patients, transcripts specific to T lymphocyte subpopulations were downregulated, whereas those related to monocytes and neutrophils were upregulated. Expression profiles of a set of 16 genes and lymphocyte-to-monocyte-and-neutrophil gene expression ratios distinguished RA patients from C. These results indicate that SAH from RAs strongly influences the transcription profiles of blood cells. A specific pattern of these changes suggests suppression in lymphocyte response and enhancements in monocyte and neutrophil activities. This is probably related to the immunodepression observed in SAH.

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