Ziji Zhang

Expression of microRNAs during chondrogenesis of human adipose-derived stem cells

OBJECTIVES MicroRNAs (miRNAs) play an important role in the regulation of chondrogenesis of mesenchymal stem cells, but their expression still remains unknown in human adipose-derived stem cells (hADSCs). In this study the miRNA expression profile during chondrogenic differentiation of hADSC and the potential mechanism whereby miRNAs may affect the process of chondrogenesis are considered. METHODS hADSCs were isolated and cultured. The expression of chondrogenic proteins was detected using enzyme-linked immunosorbent assay (ELISA). miRNA expression profiles before and after chondrogenic induction were obtained using miRNA microarray essay and differently expressed miRNAs were primarily verified using quantitative real-time polymerase chain reaction (qRT-PCR). Putative targets of the miRNAs were predicted using online software programs MiRanda, TargetScan and miRBase. RESULTS Twelve miRNAs were found to be differentially expressed pre- and post-chondrogenic induction by over a two-fold change, including eight up-regulated miRNAs (miR-193b, miR-199a-3p/hsa-miR-199b-3p, miR-455-3p, miR-210, miR-381, miR-92a, miR-320c, and miR-136), and four down-regulated miRNAs (miR-490-5p, miR-4287, miR-BART8*, and miR-US25-1*). qRT-PCR analysis further confirmed these results. Predicted target genes of the differentially expressed miRNAs were based on the overlap of at least two online prediction algorithms, with the known functions of regulating chondrogenic differentiation, self-renewal, signal transduction and cell cycle control. CONCLUSIONS In this study we have identified a group of miRNAs and their target genes, which may play important roles in regulating chondrogenic differentiation of hADSCs. Our results provide the basis for further investigation into the molecular mechanism of chondrogenesis in hADSCs and their differentiation for cartilage engineering.

miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells.

Human adipose‐derived stem cells (hADSC) are capable of differentiating into an osteogenic lineage. It is believed that microRNAs (miRNAs) play important roles in regulating this osteogenic differentiation of human adipose‐derived cells, although its molecular mechanism remains unclear. We investigated the miRNA expression profile during osteogenic differentiation of hADSCs, and assessed the roles of involved miRNAs during the osteogenic differentiation. We obtained and cultured human adipose‐derived stems cells from donors who underwent elective liposuction or other abdominal surgery at our institution. miRNA expression profiles pre‐ and post‐osteogenic induction were obtained using microarray essay, and differently expressed miRNAs were verified using quantitative real‐time polymerase chain reaction (qRT‐PCR). The expression of osteogenic proteins was detected using an enzyme‐linked immunosorbent assay. Putative targets of the miRNAs were predicted using online software MiRanda, TargetScan, and miRBase. Eight miRNAs were found differently expressed pre‐ and post‐osteogenic induction, among which four miRNAs (miR‐17, miR‐20a, miR‐20b, and miR‐106a) were up‐regulated and four miRNAs (miR‐31, miR‐125a‐5p, miR‐125b, and miR‐193a) were down‐regulated. qRT‐PCR analysis further confirmed the results. Predicted target genes of the differentially expressed miRNAs based on the overlap from three public prediction algorithms: MiRanda, TargetScan, and miRBase Target have the known functions of regulating stem cell osteogenic differentiation, self‐renewal, signal transduction, and cell cycle control. We identified a group of miRNAs that may play important roles in regulating hADSC cell differentiation toward an osteoblast lineage. Further study of these miRNAs may elucidate the mechanism of hADSC differentiation into adipose tissue, and thus provide basis for tissue engineering. J. Cell. Biochem. 113: 888–898, 2012.

MiR‐193b regulates early chondrogenesis by inhibiting the TGF‐beta2 signaling pathway

Cartilage generation and degradation are regulated by miRNAs. Our previous study has shown altered expression of miR‐193b in chondrogenic human adipose‐derived mesenchymal stem cells (hADSCs). In the current study, we investigated the role of miR‐193b in chondrogenesis and cartilage degradation. Luciferase reporter assays showed that miR‐193b targeted seed sequences of the TGFB2 and TGFBR3 3′‐UTRs. MiR‐193b suppressed the expression of early chondrogenic markers in chondrogenic ATDC5 cells, and TNF‐alpha expression in IL‐1b‐induced PMCs. In conclusion, MiR‐193b may inhibit early chondrogenesis by targeting TGFB2 and TGFBR3, and may regulate inflammation by repressing TNF‐alpha expression in inflamed chondrocytes.

Expression profile of long noncoding RNAs in cartilage from knee osteoarthritis patients

OBJECTIVES Long noncoding RNAs (lncRNAs) have emerged as a novel class of regulatory molecules involved in various biological processes, but their role in osteoarthritis (OA) remains unknown. Therefore, we aimed to reveal lncRNAs expression profile in human osteoarthritic cartilage and explore the potential functions of lncRNAs in OA. METHODS The expression profiles of lncRNAs and mRNAs in OA cartilage were obtained using microarray and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bioinformatics analyses including lncRNA classification and subgroup analysis, gene ontology (GO) analysis, pathway analysis, network analysis and target prediction were performed. RESULTS There were 3007 upregulated lncRNAs and 1707 downregulated lncRNAs in OA cartilage compared with normal samples (Fold change ≥ 2.0). In addition, 2136 mRNAs were upregulated and 2,241 mRNAs were downregulated in OA cartilage (Fold change ≥ 2.0). The qRT-PCR results of six dysregulated lncRNAs were consistent with the microarray data. 106 lncRNAs and 291 mRNAs composed the coding-non-coding gene co-expression network (CNC network). In the 600 top differentially expressed lncRNAs, 48 lncRNAs were predicted to have more than five cis-regulated target genes and up to 530 lncRNAs might regulate their trans target genes through collaboration with transcriptional factor (TF) SP1. The positive correlation between lncRNA uc.343 and predicted target homeobox gene C8 (HOXC8) expression in SW1353 cells treating with interleukin-1 beta confirmed the target prediction to some extent. CONCLUSIONS This study revealed the expression pattern of lncRNAs in OA cartilage and predicted the potential function and targets, which indicated that lncRNAs may be new biomarkers for diagnosis or novel therapeutic targets of OA.

The Role of MicroRNA-381 in Chondrogenesis and Interleukin-1-β Induced Chondrocyte Responses.

Aim: The molecular pathways regulating cartilage degradation are unclear. miR-381 was identified as a putative regulator of chondrogenesis related genes. Here, we examined its role in chondrogenesis and osteoarthritic cartilage degeneration. Methods: miR-381 expression was assessed in vitro in response to IL-1β stimulation in primary human (PHC) and mouse (PMC) chondrocytes, and ATDC5 derived chondrocytes; and in vivo in mouse embryos and human osteoarthritic cartilage. The effects of miR-381 on chondrogenesis and NF-kB signaling were assessed using a synthetic RNA mimic or inhibitor and luciferase assay, respectively. Upstream regulators of miR381 were probed using siRNA or overexpression plasmids for Sox9 and Runx2. Results: miR-381 expression was elevated in chondrogenic and hypertrophic ATDC5 cells. miR-381 was induced in vitro by IL-1β in ATDC5 cells, PMCs, and PHCs, and was expressed in areas of cartilage degradation or absorption in vivo. Overexpression of Runx2 or Sox9 increased miR-381 expression in ATDC5 cells. miR-381 suppressed expression of collagen, type II, alpha 1, and enhanced expression of metalloproteinase-13 (MMP-13), but did not regulate NFKBIA and NKRF activity. Conclusion: miR-381 was highly expressed during chondrogenesis and in arthritic cartilage. It may contribute to absorption of the cartilage matrix by repressing type II collagen and inducing MMP-13.

MiR-455-3p regulates early chondrogenic differentiation via inhibiting Runx2.

The expression of miR‐455‐3p has been shown to be up‐regulated in chondrogenesis of mesenchymal stem cell, but its role in different stages during chondrogenesis remains unknown. Here, we show that miR‐455‐3p is increased in ATDC5 cells from 0 d to 21 d, but rapidly decreases at 28 d, and a similar expression kinetic is detected in the development of mouse embryos. We show that miR‐455‐3p functions as an activator for early chondrogenic differentiation, most likely by inhibiting the expression of Runt‐related transcription factor 2 (Runx2) as indicated by luciferase reporter assays. In conclusion, miR‐455‐3p may activate early chondrogenesis by directly targeting Runx2.

Akt phosphorylates Prohibitin 1 to mediate its mitochondrial localization and promote proliferation of bladder cancer cells

Bladder cancer (BC) is very common and associated with significant morbidity and mortality, though the molecular underpinnings of its origination and progression remain poorly understood. In this study, we demonstrate that Prohibitin 1 (PHB) was overexpressed in human BC tissues and that PHB upregulation was associated with poor prognosis. We also found that PHB was necessary and sufficient for BC cell proliferation. Interestingly, the overexpressed PHB was primarily found within mitochondria, and we provide the first direct evidence that phosphorylation by Akt at Thr258 of PHB induces this mitochondrial localization. Inhibiton of Akt reverses these effects and inhibited the proliferation of BC cells. Finally, the phosphorylation of PHB was required for BC cell proliferation, further implicating the importance of the Akt in BC. Taken together, these findings identify the Akt/PHB signaling cascade as a novel mechanism of cancer cell proliferation and provide the scientific basis for the establishment of PHB as a new prognostic marker and treatment target for BC.

MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression.

Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3′-untranslated region (3′-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.

IRAK-M in macrophages around septically and aseptically loosened hip implants†

The most common long-term complication of joint arthroplasty is loosening, which is mediated by chronic inflammatory cytokines produced by macrophages stimulated by implant-derived debris and eventually bacterial components adherent to such debris. In this study, antiinflammatory interleukin-1 receptor-associated kinase-M (IRAK-M) was studied in macrophages in interface membranes in vivo using immunohistochemical staining and in titanium particle-stimulated macrophages in vitro using reverse transcriptase-polymerase chain reaction. Results show that the interface membranes of septically and aseptically loosened prosthesis express more IRAK-M protein than control membranes from osteoarthritic patient and that IRAK-M mRNA-levels increase upon particle stimulation. These findings suggest that, the upregulation of IRAK-M in macrophages is involved in the local immunosuppression around implants, and may contribute to septic and aseptic implant loosening.

Identification and Characterization of Long Non-Coding RNAs in Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Background/Aims: Long noncoding RNAs (lncRNAs) play important roles in stem cell differentiation. However, their role in osteogenesis of human adipose-derived stem cells (ASCs), a promising cell source for bone regeneration, remains unknown. Here, we investigated the expression profile and potential roles of lncRNAs in osteogenic differentiation of human ASCs. Methods: Human ASCs were induced to differentiate into osteoblasts in vitro, and the expression profiles of lncRNAs and mRNAs in undifferentiated and osteogenic differentiated ASCs were obtained by microarray. Bioinformatics analyses including subgroup analysis, gene ontology analysis, pathway analysis and co-expression network analysis were performed. The function of lncRNA H19 was determined by in vitro knockdown and overexpression. Quantitative reverse transcription polymerase chain reaction was utilized to examine the expression of selected genes. Results: We identified 1,460 upregulated and 1,112 downregulated lncRNAs in osteogenic differentiated human ASCs as compared with those of undifferentiated cells (Fold change ≥ 2.0, P < 0.05). Among these, 94 antisense lncRNAs, 85 enhancer-like lncRNAs and 160 lincRNAs were further recognized. We used 12 lncRNAs and 157 mRNAs to comprise a coding-non-coding gene expression network. Additionally, silencing of H19 caused a significantly increase in expression of osteogenesis-related genes, including ALPL and RUNX2, while a decrease was observed after H19 overexpression. Conclusion: This study revealed for the first time the global expression profile of lncRNAs involved in osteogenic differentiation of human ASCs and provided a foundation for future investigations of lncRNA regulation of human ASC osteogenesis.