Currently, the overall therapeutic efficiency of mesenchymal stem cells (MSCs) transplantation for the treatment of cardiovascular disease is not satisfactory. The low viability and angiogenic capacity of the implanted cells in the local infarct tissues restrict their further application. Evidence shows that long noncoding RNA H19 (lncRNA-H19) mediates cell survival and angiogenesis. Additionally, it is also involved in MSCs biological activities. This study aimed to explore the functional role of lncRNA-H19 in MSCs survival and angiogenic capacity as well as the underlying mechanism. MSCs were obtained from C57BL/6 mice and cultured in vitro. Cells at the third passage were divided into the following groups: MSCs+H19, MSCs+H19 NC, MSCs+si-H19, MSCs+si-H19 NC and MSCs. The MSCs+H19 and MSCs+H19 NC groups were transfected with lncRNA-H19 and lncRNA-H19 scramble RNA respectively. The MSCs+si-H19 and MSCs+si-H19 NC groups were transfected with lncRNA-H19 siRNA and lncRNA-H19 siRNA scramble respectively. MSCs were used as the blank control. All groups were exposed to normoxia (20% O2) and hypoxia (1% O2)/serum deprivation (H/SD) conditions for 24 h. Cell proliferation, apoptosis and vascular densities were assessed. Bioinformatics and dual luciferase reporter assay were performed. Relevant biomarkers were detected in different experimental groups. Overexpression of lncRNA-H19 improved survival and angiogenic capacity of MSCs under both normoxia and H/SD conditions, whereas its knockdown impaired cell viability and their angiogenic potential. MicroRNA-199a-5p (miR-199a-5p) targeted and downregulated vascular endothelial growth factor A (VEGFA). MiR-199a-5p was a target of lncRNA-H19. LncRNA-H19 transfection led to a decreased level of miR-199a-5p, accompanied with an elevated expression of VEGFA. However, both miR-199a-5p and VEGFA presented inverse alterations in the condition of lncRNA-H19 knockdown. LncRNA-H19 enhanced MSCs survival and their angiogenic potential in vitro. It could directly upregulate VEGFA expression by inhibiting miR-199a-5p as a competing endogenous RNA. This mechanism contributes to a better understanding of MSCs biological activities and provides new insights for cell therapy based on MSCs transplantation.

Introduction: Mesenchymal stem cells (MSCs) transplantation has been demonstrated to be an effective strategy for the treatment of cardiovascular disease. However, the low survival rate of MSCs at local diseased tissue reduces the therapeutic efficacy. We therefore investigated the influence of MicroRNA-378 (miR-378) transfection on MSCs survival and vascularization under hypoxic-ischemic condition in vitro. Methods: MSCs were isolated from bone marrow of Sprague–Dawley rats and cultured in vitro. The third passage of MSCs were divided into the miR-378 group and control group. For the miR-378 group, cells were transfected with miR-378 mimic. Both groups experienced exposure to hypoxia (1% O₂) and serum deprivation for 24 hours, using normoxia (20% O₂) as a negative control during the process. After 24 hours of reoxygenation (20% O₂), cell proliferation and apoptosis were evaluated. Expressions of apoptosis and angiogenesis related genes were detected. Both groups were further co-cultured with human umbilical vein endothelial cells to promote vascular differentiation for another 6 hours. Vascular density was assessed thereafter. Results: Compared with the control group, MSCs transfected with miR-378 showed more rapid growth. Their proliferation rates were much higher at 72 h and 96 h under hypoxic condition (257.33% versus 246.67%, P <0.01; 406.84% versus 365.39%, P <0.05). Cell apoptosis percentage in the miR-378 group was significantly declined under normoxic and hypoxic condition (0.30 ± 0.10% versus 0.50 ± 0.10%, P <0.05; 0.60 ± 0.40% versus 1.70 ± 0.20%, P <0.01). The miR-378 group formed a larger number of vascular branches on matrigel. BCL2 level was decreased accompanied with an upregulated expression of BAX in the two experimental groups under the hypoxic environment. BAX expression was reduced in the miR-378 group under the hypoxic environment. In the miR-378 group, there was a decreased expression of tumor necrosis factor-α on protein level and a reduction of TUSC-2 under normoxic environment. Their expressions were both downregulated under hypoxic environment. For the angiogenesis related genes, enhanced expressions of vascular endothelial growth factorα, platelet derived growth factor-β and transforming growth factor-β1 could be detected both in normoxic and hypoxic-ischemic conditions. Conclusion: MiR-378 transfection could effectively promote MSCs survival and vascularization under hypoxic-ischemic condition in vitro.

Mesenchymal stem cells (MSCs) have limited potential of cardiogenic differentiation. In this study, we investigated the influence of long noncoding RNA Braveheart (lncRNA-Bvht) on cardiogenic differentiation of MSCs in vitro. MSCs were obtained from C57BL/6 mice and cultured in vitro. Cells were divided into three groups: blank control, null vector control, and lncRNA-Bvht. All three groups experienced exposure to hypoxia (1% O2) and serum deprivation for 24 h, and 24 h of reoxygenation (20% O2). Cardiogenic differentiation was induced using 5-AZA for another 24 h. Normoxia (20% O2) was applied as a negative control during the whole process. Cardiogenic differentiation was assessed, and expressions of cardiac-specific transcription factors and epithelial-mesenchymal transition (EMT)-associated biomarkers were detected. Anti-mesoderm posterior1 (Mesp1) siRNA was transfected in order to block its expression, and relevant downstream molecules were examined. Compared with the blank control and null vector control groups, the lncRNA-Bvht group presented a higher percentage of differentiated cells of the cardiogenic phenotype in vitro both under the normal condition and after hypoxia/re-oxygenation. There was an increased level of cTnT and α-SA, and cardiac-specific transcription factors including Nkx2.5, Gata4, Gata6, and Isl-1 were significantly upregulated (P < 0.01). Expressions of EMT-associated genes including Snail, Twist and N-cadherin were much higher (P < 0.01). Mesp1 exhibited a distinct augmentation following lncRNA-Bvht transfection. Expressions of relevant cardiac-specific transcription factors and EMT-associated genes all presented a converse alteration in the condition of Mesp1 inhibition prior to lncRNA-Bvht transfection. lncRNA-Bvht could efficiently promote MSCs transdifferentation into cells with the cardiogenic phenotype in vitro. It might function via enhancing the expressions of cardiac-specific transcription factors and EMT-associated genes. Mesp1 could be a pivotal intermediary in the procedure.

Background: Mesenchymal stem cells (MSCs) have exerted their brilliant potential to promote heart repair following myocardial infarction. However, low survival rate of MSCs after transplantation due to harsh conditions with hypoxic and ischemic stress limits their therapeutic efficiency in treating cardiac dysfunction. ELABELA (ELA) serves as a peptide hormone which has been proved to facilitate cell growth, survival, and pluripotency in human embryonic stem cells. Although ELA works as an endogenous ligand of a G protein-coupled receptor APJ (Apelin receptor, APLNR), whether APJ is an essential signal for the function of ELA remains elusive. The effect of ELA on apoptosis of MSCs is still vague. Objective: We studied the role of ELABELA (ELA) treatment on the anti-apoptosis of MSCs in hypoxic/ischemic (H/I) conditions which mimic the impaired myocardial microenvironment and explored the possible mechanisms in vitro. Methods: MSCs were obtained from donated rats weighing between 80~120 g. MSCs were exposed to serum-free and hypoxic (1% O₂) environments for 24 h, which mimics hypoxic/ischemic damage in vivo, using serum-containing normoxic conditions (20% O₂) as a negative control. MSCs that were exposed to H/I injury with ELA processing were treated by 5 μM of ELA. Cell viability and apoptosis of MSCs were evaluated by CCK8 and flow cytometry, respectively. Mitochondrial function of MSCs was also assessed according to mitochondrial membrane potential (MMP) and ATP content. The protein expression of key kinases of the PI3K/AKT and ERK1/2 signaling pathways involving t-AKT, p-AKT, t-ERK1/2, and p-ERK1/2, as well as apoptosis-related protein expression of Bcl-2, Bax, and cleaved Caspase 3, were monitored by Western blot. Results: We found that ELA treatment of H/I-induced MSCs improved overall cell viability, enhanced Bcl/Bax expression, and decreased Caspase 3 activity. ELA inhibited H/I-induced mitochondrial dysfunction by increasing ATP concentration and suppressing the loss of mitochondrial transmembrane potential. However, this anti-apoptotic property of ELA was restrained in APJ-silenced MSCs. Additionally, ELA treatment induced the phosphorylation of AKT and ERK, while the blockade of PI3K/AKT and ERK1/2 pathways with respective inhibitors, LY294002 and U0126, suppressed the action of ELA. Conclusion: ELA positively affected on the survival of MSCs and exhibited anti-apoptotic characteristics when exposed to hypoxic/ischemic condition in vitro. Also, the function of ELA was correlated with the APJ receptor, reduced mitochondrial damage, and activation of the PI3K/AKT and ERK1/2 signal axes.

Cardiac stem cells (CSCs) transplantation has been regarded as an optimal therapeutic approach for cardiovascular disease. However, inferior survival and low differentiation efficiency of these cells in the local infarct site reduce their therapeutic efficacy. In this study, we investigated the influence of hypoxia preconditioning (HP) on CSCs survival and cardiogenic differentiation in vitro and explored the relevant mechanism. CSCs were obtained from Sprague–Dawley rats and cells of the third passage were cultured in vitro and exposed to hypoxia (1% O2). Cells survival and apoptosis were evaluated by MTS assay and flow cytometry respectively. Cardiogenic differentiation was induced by using 5-azacytidine for another 24 h after the cells experienced HP. Normoxia (20% O2) was used as a negative control during the whole process. Cardiogenic differentiation was assessed 2 weeks after the induction. Relevant molecules were examined after HP and during the differentiation process. Anti-hypoxia-inducible factor-1α (HIF-1α) small interfering RNA (siRNA), anti-apelin siRNA, and anti-putative receptor protein related to the angiotensin receptor AT1 (APJ) siRNA were transfected in order to block their expression, and relevant downstream molecules were detected. Compared with the normoxia group, the hypoxia group presented more rapid growth at time points of 12 and 24 h (p < 0.01). Cells exhibited the highest proliferation rate at the time point of 24 h (p < 0.01). The cell apoptosis rate significantly declined after 24 h of hypoxia exposure (p < 0.01). Expression levels of HIF-1α, apelin, and APJ were all enhanced after HP. The percentage of apelin, α-SA, and cTnT positive cells was greatly increased in the HP group after 2 weeks of induction. The protein level of α-SA and cTnT was also significantly elevated at 7 and 14 days (p < 0.01). HIF-1α, apelin, and APJ were all increased at different time points during the cardiogenic differentiation process (p < 0.01). Knockdown of HIF-1α, apelin or APJ by siRNAs resulted in a significant reduction of α-SA and cTnT. HIF-1α blockage caused a remarkable decrease of apelin and APJ (p < 0.01). Expression levels of apelin and APJ were depressed after the inhibition of apelin (p < 0.01). HP could effectively promote CSCs survival and cardiogenic differentiation in vitro, and this procedure involved activation of the HIF-1α/apelin/APJ axis. This study provided a new perspective for exploring novel strategies to enhance CSCs transplantation efficiency.

Introduction: Hepatocellular carcinoma (HCC) is characterized by the complex pathogenesis, limited therapeutic methods, and poor prognosis. Endoplasmic reticulum stress (ERS) plays an important role in the development of HCC, therefore, we still need further study of molecular mechanism of HCC and ERS for early diagnosis and promising treatment targets. Method: The GEO datasets (GSE25097, GSE62232, and GSE65372) were integrated to identify differentially expressed genes related to HCC (ERSRGs). Random Forest (RF) and Support Vector Machine (SVM) machine learning techniques were applied to screen ERSRGs associated with endoplasmic reticulum stress, and an artificial neural network (ANN) diagnostic prediction model was constructed. The ESTIMATE algorithm was utilized to analyze the correlation between ERSRGs and the immune microenvironment. The potential therapeutic agents for ERSRGs were explored using the Drug Signature Database (DSigDB). The immunological landscape of the ERSRGs central gene PPP1R16A was assessed through single-cell sequencing and cell communication, and its biological function was validated using cytological experiments. Results: An ANN related to the ERS model was constructed based on SRPX, THBS4, CTH, PPP1R16A, CLGN, and THBS1. The area under the curve (AUC) of the model in the training set was 0.979, and the AUC values in three validation sets were 0.958, 0.936, and 0.970, respectively, indicating high reliability and effectiveness. Spearman correlation analysis suggests that the expression levels of ERSRGs are significantly correlated with immune cell infiltration and immune-related pathways, indicating their potential as important targets for immunotherapy. Mometasone was predicted to be the most promising treatment drug based on its highest binding score. Among the six ERSRGs, PPP1R16A had the highest mutation rate, predominantly copy number mutations, which may be the core gene of the ERSRGs model. Single-cell analysis and cell communication indicated that PPP1R16A is predominantly distributed in liver malignant parenchymal cells and may reshape the tumor microenvironment by enhancing macrophage migration inhibitory factor (MIF)/CD74 + CXCR4 signaling pathways. Functional experiments revealed that after siRNA knockdown, the expression of PPP1R16A was downregulated, which inhibited the proliferation, migration, and invasion capabilities of HCCLM3 and Hep3B cells in vitro. Conclusion: The consensus of various machine learning algorithms and artificial intelligence neural networks has established a novel predictive model for the diagnosis of liver cancer associated with ERS. This study offers a new direction for the diagnosis and treatment of HCC.