Yunfei Xia

p53/p21 Pathway involved in mediating cellular senescence of bone marrow-derived mesenchymal stem cells from systemic lupus erythematosus patients.

Our and other groups have found that bone marrow-derived mesenchymal stem cells (BM-MSCs) from systemic lupus erythematosus (SLE) patients exhibited senescent behavior and are involved in the pathogenesis of SLE. Numerous studies have shown that activation of the p53/p21 pathway inhibits the proliferation of BM-MSCs. The aim of this study was to determine whether p53/p21 pathway is involved in regulating the aging of BM-MSCs from SLE patients and the underlying mechanisms. We further confirmed that BM-MSCs from SLE patients showed characteristics of senescence. The expressions of p53 and p21 were significantly increased, whereas levels of Cyclin E, cyclin-dependent kinase-2, and phosphorylation of retinoblastoma protein were decreased in the BM-MSCs from SLE patients and knockdown of p21 expression reversed the senescent features of BM-MSCs from SLE patients. Our results demonstrated that p53/p21 pathway played an important role in the senescence process of BM-MSCs from SLE.

Clinical and Biological Significance of Never in Mitosis Gene A-Related Kinase 6 (NEK6) Expression in Hepatic Cell Cancer

Nek6 is a cell cycle regulatory gene, which can control cell proliferation and survival. Recent studies suggested that desregulation of Nek6 expression plays a key role in oncogenesis. This study was aimed to investigate the potential roles of Nek6 in hepatocellular carcinoma (HCC) development. Immunohistochemistry and Western blot analysis was performed for Nek6 in 80 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were performed to determine the prognostic significance of Nek6 in HCC. In addition, Nek6 expression vector was used to detect its role in cell cycle control. Nek6 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Nek6 was associated with histological grade and the level of alpha fetal protein, and Nek6 was positively correlated with proliferation marker Ki-67. Univariate analysis showed that Nek6 expression was associated with poor prognosis. Multivariate analysis indicated that Nek6 and Ki-67 protein expression was an independent prognostic marker for HCC. While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Nek6 was upregulated. Overexpression Nek6 in Huh7 cell could promote the cell cycle. In conclusion, Nek6 is involved in the pathogenesis of hepatocellular carcinoma. It may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.

Suppression of the PI3K—Akt pathway is involved in the decreased adhesion and migration of bone marrow-derived mesenchymal stem cells from non-obese diabetic mice

T1DM (type 1 diabetes mellitus) is an autoimmune disease characterized by T‐cell‐mediated damage of islet β‐cells. The pathology of NOD (non‐obese diabetic) mouse involves the insulitis induced by infiltration of T‐cells, a similar pathogenic mechanism in T1DM patient. BM‐MSCs (bone marrow mesenchymal stem cells) are multipotent progenitor cells that can be isolated from a number of sources. Recent studies have shown that transplantation of MSCs to the NOD mice could prevent the process and have the therapeutic effects on T1DM. In our studies, we have found that migration and adhesion of BM‐MSCs from NOD mice were suppressed compared with the BM‐MSCs from ICR (imprinting control region) mice, accompanying with the abnormal distribution of FAK (focal adhesion kinase) and F‐actin (filamentous actin). Further, we have found that the activation of PI3K (phosphoinositide 3‐kinase)–Akt pathway was suppressed in BM‐MSCs from NOD mice. When the PI3K—Akt pathway was inhibited by LY294002, the adhesion and migration of BM‐MSCs from ICR mice were suppressed as well. These results indicated that the suppression of PI3K—Akt pathway is involved in the decreased adhesion and migration of BM‐MSCs from NOD mice.

Rapamycin reverses the senescent phenotype and improves immunoregulation of mesenchymal stem cells from MRL/lpr mice and systemic lupus erythematosus patients through inhibition of the mTOR signaling pathway.

We have shown that bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) from SLE patients exhibit senescent behavior and are involved in the pathogenesis of SLE. The aim of this study was to investigate the effects of rapamycin (RAPA) on the senescences and immunoregulatory ability of MSCs of MRL/lpr mice and SLE patients and the underlying mechanisms. Cell morphology, senescence associated β-galactosidase (SA-β-gal) staining, F-actin staining were used to detect the senescence of cells. BM-MSCs and purified CD4+ T cells were co-cultured indirectly. Flow cytometry was used to inspect the proportion of regulatory T (Treg) /T helper type 17 (Th17). We used small interfering RNA (siRNA) to interfere the expression of mTOR, and detect the effects by RT-PCR, WB and immunofluorescence. Finally, 1×106 of SLE BM-MSCs treated with RAPA were transplanted to cure the 8 MRL/lpr mice aged 16 weeks for 12 weeks. We demonstrated that RAPA alleviated the clinical symptoms of lupus nephritis and prolonged survival in MRL/lpr mice. RAPA reversed the senescent phenotype and improved immunoregulation of MSCs from MRL/lpr mice and SLE patients through inhibition of the mTOR signaling pathway. Marked therapeutic effects were observed in MRL/lpr mice following transplantation of BM-MSCs from SLE patients pretreated with RAPA.

Induction of Apoptosis Coupled to Endoplasmic Reticulum Stress through Regulation of CHOP and JNK in Bone Marrow Mesenchymal Stem Cells from Patients with Systemic Lupus Erythematosus.

Previous studies indicated that bone marrow mesenchymal stem cells (BM-MSCs) from patients with systemic lupus erythematosus (SLE) exhibited the phenomenon of apoptosis. In this study, we aimed to investigate whether apoptosis of BM-MSCs from SLE patients were dysregulated. In this paper, endoplasmic reticulum stress (ERS) was evidenced by increased expression of phosphorylated protein kinase RNA-like ER kinase (PERK) and inositol-requiring protein-1 (IRE-1). We also found the activation of downstream target eukaryotic translation initiator factor 2α (eIF 2α) and CCAAT/enhancer-binding protein- (C/EBP-) homologous protein (CHOP) in BM-MSCs from SLE patients. Interestingly, we discovered that 4-phenylbutyric acid (4-PBA), a selective inhibitor of ERS, blocked the apoptosis of BM-MSCs from SLE patients and alleviated the level of Jun N-terminal kinase1/2 (JNK1/2) and CHOP. Furthermore, blockage of PERK signaling expression by siRNA not only significantly reduced the expression of CHOP, but also activated the anti-apoptotic regulator B-cell lymphoma-2 (Bcl-2). Blockage of IRE-1 or JNK1/2 by siRNA resulted in the decreased expression of JNK1/2 and proapoptosis protein Bcl-2 associated protein X (BAX). These results implicated that ERS-mediated apoptosis was a critical determinant of BM-MSCs from SLE patients.

p21 is Associated with the Proliferation and Apoptosis of Bone Marrow-derived Mesenchymal Stem Cells from Non-obese Diabetic Mice

Recent studies have shown that autologous and allogeneic transplantation of the BM-MSCs had therapeutic effects on T1DM, whereas the BM-MSCs from the NOD mice itself did not have this therapeutic effect. We previously demonstrated that Bone Marrow (BM) -MSCs from the non-obese diabetic (NOD) mice had the abnormal migration and adhesion. So we hypothesized that the proliferation and apoptosis of the BM-MSCs from the NOD mice were dysregulated. Our team compared the proliferation and apoptosis between NOD mice and imprinting control region (ICR) mice. Then we assessed whether the NF-κB-p53/p21 pathway was involved in the process. The cell proliferation ability of the BM-MSCs from the NOD mice were significantly decreased, while the percent of apoptotic cells was increased compared to those from the ICR mice. The p21 expression was significantly increased in the NOD-MSCs. The p65 level was enhanced in the BM-MSCs from the NOD mice when compared to the ICR mice, coincided with the expression of p21. Expressions of p65 and p21 were significantly decreased in the -BM-MSCs treated with p65 inhibitor. The knockdown p21 expression reversed the abnormal proliferation, colony formation and apoptosis of the BM-MSCs from the NOD mice. These data provide important preclinical references supporting the basis for further development of autologous MSC-based therapies for type1 diabetes mellitus (T1DM).

Treatment adherence to disease-modifying antirheumatic drugs in Chinese patients with rheumatoid arthritis.

Objective Nonadherence in rheumatoid arthritis (RA) patients using disease-modifying antirheumatic drugs (DMARDs) may lead to joint damage and function loss. The aim of this cross-sectional study was to explore Chinese RA patients’ adherence rates and investigate potential risk factors for nonadherence. Methods A total of 122 RA patients were recruited from the Affiliated Hospital of Nantong University from January 2014 to April 2015. Patients were asked to complete a set of standardized self-report questionnaires (Compliance Questionnaire on Rheumatology, Health Assessment Questionnaire, Short Form-36 questionnaire, 28-joint Disease Activity Score, Hospital Anxiety and Depression Scale, and Visual Analog Scale). Independent samples t-tests, chi-square analyses, and logistic regression modeling were used to analyze these data. Results Based on Compliance Questionnaire on Rheumatology, 38% of the patients adhered to DMARDs. Adherence was associated with education, income, depression, and the total number of DMARDs. Other demographic and clinical characteristics were not associated with adherence. Logistic regression models identified income, depression, and the total number of DMARDs as predictors of medication nonadherence. Conclusion In this study, 62% of patients with RA were not adherent to their DMARD prescription. Education, income, depression, and the total number of DMARDs were associated with medication adherence, and income, depression, and the total number of DMARDs were independent predictors of medication adherence in patients with RA. These findings could help medical personnel develop helpful interventions to improve adherence in RA patients by paying more attention to the patients with these accompanying risk factors and, finally, improve RA patients’ quality of life.

General Transcription Factor IIB Overexpression and a Potential Link to Proliferation in Human Hepatocellular Carcinoma

The general transcription factor IIB (TFIIB) plays a central role in preinitiation complex (PIC) assembly, providing a bridge between promoter-bound TFIID and RNA Polymerase II (RNA POLII). TFIIB functionally counteracts the transcriptional activation of hepatitis B virus X protein (HBx), which has been shown to play a role in the development of human hepatocellular carcinoma (HCC). However, the function of TFIIB in HCC remains unclear. In this article, we demonstrate that TFIIB plays an important role in HCC pathogenesis. TFIIB expression was immunohistochemically examined in a series of 100 HCC tissue specimens. The expression level of TFIIB showed significant correlation with the histological grade (P = 0.030), the level of AFP (P = 0.011) and the proliferation marker Ki-67 (P = 0.0002). High TFIIB expression level correlated with poor survival. Western blot analysis also confirmed that the TFIIB protein was overexpressed in HCC tissue compared to benign normal tissue. Additionally, Western blot and qRT-PCR analyses showed a high expression level of TFIIB protein in the HCC cell lines SMMC7721, HepG2, BEL7404, and Huh7 and the immortalized normal line BEL7702 but a lower expression in the normal Chang hepatocyte cell line. Following the release of Huh7 cells from serum starvation, the expression of TFIIB was upregulated. A cell growth assay suggested that TFIIB was involved in the proliferation and growth of HCC cells. In conclusion, our results demonstrate that TFIIB overexpression may play essential roles in the pathogenesis of hepatocellular carcinoma by affecting the proliferation of HCC cells.

Involvement of autophagy in the procedure of endoplasmic reticulum stress introduced apoptosis in bone marrow mesenchymal stem cells from nonobese diabetic mice.

Recent studies showed that bone marrow mesenchymal stem cells (BM‐MSCs) from nonobese diabetic (NOD) mice exhibited the phenomenon of apoptosis. However, the mechanisms of apoptosis remained largely unknown. In this study, endoplasmic reticulum (ER) stress and autophagy were evidenced in BM‐MSCs from NOD mice for the first time. We found the ER stress‐mediated apoptosis was supported by the up‐regulation of ER stress markers including augmented phosphorylation of phosphorylated protein kinase RNA‐like ER kinase and eukaryotic translation initiator factor 2α as well as cleavage of caspase‐3. Evidence of autophagy included the formation of the acidic vesicular organelles and increase of LC3 accumulation. Intriguingly, blockage of ER stress could reduce the apoptosis of BM‐MSCs from NOD mice and alleviated accumulation of LC3, which indicated that ER stress induced apoptosis and autophagy. Furthermore, our results showed that the mechanism of ER stress‐induced autophagy was associated with the decrease of p‐S6 (a marker of mTOR activity). Here, we demonstrated that ER stress‐induced cell death was mediated by autophagy that was partly attributed to the inactivation of the mammalian target of rapamycin.

JAK-STAT signaling mediates the senescence of bone marrow-mesenchymal stem cells from systemic lupus erythematosus patients

Previous studies have revealed that bone marrow-mesenchymal stem cells (BM-MSCs) from systemic lupus erythematosus (SLE) patients exhibited early signs of senescence, which may participate in the development of SLE. However, the molecular mechanisms about this phenomenon have not been fully elucidated. In the current study, we aimed to investigate whether Janus kinase (JAK)-signaling transducers and activators of transcription (STAT) signaling mediated the senescence of BM-MSCs from SLE patients. Twelve female SLE patients and healthy subjects were enrolled in the study. All BM-MSCs were isolated by density gradient centrifugation. Western blot analysis was used to test the expression of JAK-STAT signaling molecules. We observed the activity of β-gal of cells, the changes of cytoskeletal structure by F-actin staining, and the distribution of cell cycle by flow cytometry. BM-MSCs from SLE patients showed prominent features of senescence, and abnormal activation of JAK-STAT signaling transduction, high level of phosphorylated JAK2, and STAT3. After stimulation of IFN-γ in normal MSCs, JAK-STAT signaling was activated. The cell volume and the number of senescence-associated β-galactosidase (SA-β-gal) positive in SLE BM-MSCs were increased. The organization of cytoskeleton was nearly disordered. The rate of cell proliferation was decreased. AG490, the inhibitor of JAK2, and knockdown of STAT3 in BM-MSCs, could significantly reverse the senescence. In summary, our study indicated that JAK-STAT signaling pathway may play a critical role in the senescence of SLE BM-MSCs.