An effective and efficient method of transfecting primary human chondrocytes in suspension.
*Chondrocytes; *Osteoarthritis; *siRNA; *Transfection; Aggrecans/genetics; Articular/metabolism/pathology; Cartilage; Cells; Chondrocytes/cytology/*metabolism; Collagen Type II/genetics; Cultured; Humans; Messenger/*genetics; mRNA Cleavage and Polyadenylation Factors/antagonists & inhibitors/genetics; Nucleotidyltransferases/antagonists & inhibitors/genetics; Plasmids/*genetics; RNA; Small Interfering/*genetics; Transfection/*methods
Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80-90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes.
Makki Mohammad Shahidul; Akhtar Nahid; Haqqi Tariq M
Analytical biochemistry
2017
2017-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.ab.2017.03.009" target="_blank" rel="noreferrer noopener">10.1016/j.ab.2017.03.009</a>
Histone Deacetylase Inhibitor Vorinostat (SAHA) Suppresses IL-1beta-Induced Matrix Metallopeptidase-13 Expression by Inhibiting IL-6 in Osteoarthritis Chondrocyte.
Aged; Articular/drug effects/metabolism; Blotting; Cartilage; Chondrocytes/drug effects; Collagen Type II/drug effects/metabolism; Down-Regulation/drug effects; Female; Gene Expression Regulation/*drug effects; Histone Deacetylase Inhibitors/*pharmacology; Humans; Hydroxamic Acids/*pharmacology; Interleukin-1beta/*antagonists & inhibitors/genetics/metabolism; Knee Joint/metabolism; Male; Matrix Metalloproteinase 13/*drug effects/metabolism; Middle Aged; Osteoarthritis/*drug therapy; Proteoglycans/metabolism; Tumor Necrosis Factor-alpha/drug effects/metabolism; Vorinostat; Western
Osteoarthritis (OA) is the most common whole-joint disease and is characterized by progressive loss of the cartilage matrix. Matrix metallopeptidase-13 (MMP-13) is a highly active and an abundantly expressed protease in OA cartilage and chondrocytes and degrades type II collagen and proteoglycans. We investigated the mechanism of MMP-13 suppression by histone deacetylase inhibitor vorinostat (SAHA). OA chondrocytes were obtained from knee cartilage after enzymatic digestion and treated with IL-1beta in the absence or presence of various histone deacetylase inhibitors. Gene expression was quantified using quantitative RT-PCR. Protein expression and chromatin modifications were determined by Western immunoblotting using specific antibodies. The effect of IL-6 on the expression of
Makki Mohammad Shahidul; Haqqi Tariq M
The American journal of pathology
2016
2016-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.ajpath.2016.06.010" target="_blank" rel="noreferrer noopener">10.1016/j.ajpath.2016.06.010</a>
miR-139 modulates MCPIP1/IL-6 expression and induces apoptosis in human OA chondrocytes.
*Apoptosis; 3' Untranslated Regions; Aged; Chondrocytes/*metabolism/pathology; Down-Regulation; Female; Gene Expression Regulation; Humans; Interleukin-6/*genetics; Male; Messenger/genetics; MicroRNAs/*genetics; Middle Aged; Osteoarthritis/*genetics/pathology; Ribonucleases/*genetics; RNA; Transcription Factors/*genetics; Up-Regulation
IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3' untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 'seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1beta in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the 'seed sequence' located in the 3' UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes
Makki Mohammad Shahidul; Haqqi Tariq M
Experimental & molecular medicine
2015
2015-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1038/emm.2015.66" target="_blank" rel="noreferrer noopener">10.1038/emm.2015.66</a>
Deep sequencing and analyses of miRNAs, isomiRs and miRNA induced silencing complex (miRISC)-associated miRNome in primary human chondrocytes.
MicroRNAs, a group of small, noncoding RNAs that post-transcriptionally regulate gene expression, play important roles in chondrocyte function and in the development of osteoarthritis. We characterized the dynamic repertoire of the chondrocyte miRNome and miRISC-associated miRNome by deep sequencing analysis of primary human chondrocytes. IL-1beta treatment showed a modest effect on the expression profile of miRNAs in normal and osteoarthritis (OA) chondrocytes. We found a number of miRNAs that showed a wide range of sequence modifications including nucleotide additions and deletions at 5' and 3' ends; and nucleotide substitutions. miR-27b-3p showed the highest expression and miR-140-3p showed the highest number of sequence variations. AGO2 RIP-Seq analysis revealed the differential recruitment of a subset of expressed miRNAs and isoforms of miRNAs (isomiRs) to the miRISC in response to IL-1beta, including miR-146a-5p, miR-155-5p and miR-27b-3p. Together, these results reveal a complex repertoire of miRNAs and isomiRs in primary human chondrocytes. Here, we also show the changes in miRNA composition of the miRISC in primary human chondrocytes in response to
Haseeb Abdul; Makki Mohammad Shahidul; Khan Nazir M; Ahmad Imran; Haqqi Tariq M
Scientific reports
2017
2017-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1038/s41598-017-15388-4" target="_blank" rel="noreferrer noopener">10.1038/s41598-017-15388-4</a>
Modulation of ten-eleven translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) expression, alpha-Ketoglutarate (alpha-KG), and DNA hydroxymethylation levels by interleukin-1beta in primary human chondrocytes.
*DNA Methylation; *Epigenesis; 5-hmC; 5-Methylcytosine/analogs & derivatives; Chondrocytes; Chondrocytes/chemistry/*metabolism; Cytokine; Cytosine/*analogs & derivatives/analysis/metabolism; Dioxygenase; DNA Methylation; DNA-Binding Proteins/*biosynthesis/genetics; Epigenetics; Genetic; Humans; IL-1; Interleukin-1beta/pharmacology/*physiology; Isocitrate Dehydrogenase/*biosynthesis/genetics; Ketoglutaric Acids/*metabolism; Messenger/biosynthesis/genetics; Mixed Function Oxygenases; Primary Cell Culture; Proto-Oncogene Proteins/*biosynthesis/genetics; RNA; TNF-alpha; Tumor Necrosis Factor-alpha/pharmacology/physiology
5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1-3 (TET1-3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1beta and TNF-alpha are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1beta and TNF-alpha on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be approximately 0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1beta alone or in combination with TNF-alpha. We observed a dramatic (10-20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2-3-fold) in TET3 expression in chondrocytes stimulated with IL-1beta and TNF-alpha. IL-1beta and TNF-alpha significantly suppressed the activity and expression of IDHs, which correlated with the reduced alpha-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1beta and TNF-alpha, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.
Haseeb Abdul; Makki Mohammad Shahidul; Haqqi Tariq M
The Journal of biological chemistry
2014
2014-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M113.512269" target="_blank" rel="noreferrer noopener">10.1074/jbc.M113.512269</a>
Overexpression of beta-catenin and cyclinD1 predicts a poor prognosis in ovarian serous carcinomas.
Adult; Female; Humans; Middle Aged; Aged; Young Adult; Immunohistochemistry; Neoplasm Staging; Prognosis; Kaplan-Meier Estimate; Up-Regulation; Biomarkers; beta-catenin; beta Catenin/analysis/*biosynthesis; Cyclin D1/analysis/*biosynthesis; cyclinD1; Ovarian Neoplasms/*metabolism/mortality/pathology; ovarian serous carcinoma; prognosis; Cystadenocarcinoma; Serous/*metabolism/mortality/pathology; Tumor/*analysis
UNLABELLED: Ovarian serous cancer is the most common subtype of epithelial ovarian cancer, and is the leading cause of death from gynecologic cancer. There is an important need for exploration of diagnostic and prognostic markers for this disease. beta-catenin and cyclinD1 play central roles in the tumorigenesis for certain cancers. The role of beta-catenin and cyclinD1 in diagnosis and prognosis of ovarian serous carcinoma is uncertain. In the present study, the expression of beta-catenin and cyclinD1 was examined in 60 ovarian serous carcinomas patients with immunohistochemical staining. The relationship between expression of beta-catenin and cyclinD1 and FIGO stage, pathological grade was analyzed. Kaplan-Meier survival function was used to analyze the prognosis. Overexpression of beta-catenin is more often detected in patients with FIGO stage III and IV than in those with stage I, and II (P=0.003). No significant relationship was found between expression of beta-catenin and pathological grade (P=0.817). Positive expression of beta-catenin related to lower survival rate (P=0.034). The expression of cyclinD1 had no relationship with FIGO stage (P=0.829). Overexpression of cyclinD1 was positively to pathological grade (P=0.017) and survival rate (P=0.009). There is a significantly positive relationship between expression of beta-catenin and cyclinD1 (P=0.014). No statistical significance was found between expression of beta-catenin and cyclinD1 and other pathological parameters. CONCLUSIONS: Expression of beta-catenin and cyclinD1 may be used as predict markers for poor prognosis.
Wang Hai; Wang Haiyan; Makki Mohammad Shahidul; Wen Juanjuan; Dai Yingqing; Shi Qunli; Liu Qi; Zhou Xiaojun; Wang Jiandong
International journal of clinical and experimental pathology
2014
1905-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).