MicroRNA Hsa-Let-7b Regulates the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting CTHRC1

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells’ (MSCs’) biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.

Macrophage-derived Apoptotic Vesicles Regulate Fate Commitment of Mesenchymal Stem Cells via miR155

Background In tissue engineering, mesenchymal stem cells (MSCs) are common seed cells because of abundant sources, strong proliferation ability and immunomodulatory function. Numerous researches have demonstrated that MSC-macrophage crosstalk played a key role in the tissue engineering. Macrophages could regulate the differentiation of MSCs via different molecular mechanisms, including extracellular vesicles. Apoptotic macrophages could generate large amounts of apoptotic vesicles (apoVs), whereas the functions of macrophage-derived apoVs remain largely unknown. There was no research to clarify the role of macrophage-derived apoVs in MSC fate choices. In this study, we aimed to characterize macrophage-derived apoVs, and investigate the roles of macrophage-derived apoVs in the fate commitment of MSCs. Methods We characterized macrophage-derived apoVs, and investigated their role in MSC osteogenesis and adipogenesis in vitro and in vivo. Furthermore, we performed microRNA loss- and gain- of function experiments and western blot to determine the molecular mechanism. Results We found that macrophage-derived apoVs inhibited osteogenesis and promoted adipogenesis in vitro and in vivo. In mechanism, apoVs regulated osteogenesis and adipogenesis of MSCs by delivering microRNA155 (miR155). Conclusions Macrophage-derived apoVs could regulate the osteogenesis and adipogenesis of MSCs through delivering miR155, which provided novel insights for MSC-mediated tissue engineering.

Transcriptome Analysis of EnJSRV-Env on the Inhibition of Cell Proliferation

Background Endogenous retroviruses (ERVs) exert important biological roles, such as mammalian placental development and suppression of the infection of exogenous retrovirus. In addition, ERVs could also inhibit the proliferation of cell. The envelope-protein (Env) of endogenous Jaagsiekte sheep retroviruses (enJSRVs) possesses fusogenic activity, which promoted the formation of nonproliferative multinucleated syncytiotrophoblasts. Methods The proliferation of HeLa cells was detected by MTT. Six samples were extracted for RNA-seq transcriptome analysis. Quantitative real-time PCR (qRT-PCR) and western blotting were employed for the validation of interested target. Results enJSRV-Env transfection inhibited the proliferation ability of Hela cells and 170 differentially expressed genes (DEGs) were obtained by RNA-seq analysis. Among these, 5 DEGs (BHLHE41, CCN1, DLX2, DUSP6 and SH2D5) were validated by qRT-PCR, which closely related with proliferation. Western blotting analysis showed that the expression of DUSP6 and p-ERK1/2 was decreased, which suggested that ERK1/2 signaling pathway may be involved in enJSRV-Env transfection. Conclusions enJSRV-Env transfection inhibited the proliferation of Hela cells, probably via DUSP6 and ERK1/2 signaling pathway.

Downregulation of long noncoding RNA T1NCR inhibits cell proliferation, invasion, and epithelial-mesenchymal transition of hepatocellular carcinoma

Accumulating reports have identified that long non-coding RNAs (IncRNAs) function as key regulators of tumor initiation and progression. The aim of the current study was to determine the clinical significance and functional role of TINCR in hepatocellular carcinoma (HCC). In the present study, the level of IncRNA TINCR expression was significantly upregulated in HCC tissues compared to adjacent normal tissues. Higher levels of IncRNA TINCR expression were significantly correlated with tumor size and vascular invasion of HCC patients. LncRNA TINCR knockdown inhibited cell proliferation ability, increased the proportion of G1 phase cells, reduced the proportion of S phase cells, and suppressed cell invasion of HCC in vitro. Additionally, IncRNA TINCR knockdown inhibited the HCC cell epithelial-mesenchymal transition (EMT) phenomenon by upregulating E-cadherin and reducing N-cadherin expression. We demonstrated that knockdown of IncRNA reduced tumor growth in vivo. Thus, these results indicated that IncRNA TINCR exhibits a tumor oncogenic role in HCC and inhibition of IncRNA TINCR might serve as a therapeutic target for HCC.

Aspergillus fumigatus Fumagillin Contributes to Host Cell Damage

The activity of fumagillin, a mycotoxin produced by Aspergillus fumigatus, has not been studied in depth. In this study, we used a commercial fumagillin on cultures of two cell types (A549 pneumocytes and RAW 264.7 macrophages). This toxin joins its target, MetAP2 protein, inside cells and, as a result, significantly reduces the electron chain activity, the migration, and the proliferation ability on the A549 cells, or affects the viability and proliferation ability of the RAW 264.7 macrophages. However, the toxin stimulates the germination and double branch hypha production of fungal cultures, pointing out an intrinsic resistant mechanism to fumagillin of fungal strains. In this study, we also used a fumagillin non-producer A. fumigatus strain (∆fmaA) as well as its complemented strain (∆fmaA::fmaA) and we tested the fumagillin secretion of the fungal strains using an Ultra High-Performance Liquid Chromatography (UHPLC) method. Furthermore, fumagillin seems to protect the fungus against phagocytosis in vitro, and during in vivo studies using infection of immunosuppressed mice, a lower fungal burden in the lungs of mice infected with the ∆fmaA mutant was demonstrated.

Isolation And Culture of Rat Intestinal Mucosal Microvascular Endothelial Cells Using Immunomagnetic Beads And Their Marker Expression

Microvascular endothelial cells (MVECs) have been an important tool in many research fields, while their purification remained challenging. This study aimed to establish a method for isolating rat intestinal mucosal MVECs using immunomagnetic beads, and characterize their proliferation ability and marker expression. Rat jejunum mucosas were scraped and digested by collagenase type II to obtain primary cell culture by the differential adhesion method. Magnetic beads were coated with anti-CD31 antibodies and incubated with the single-cell suspension of the primary cell culture. The CD31+ cells were separated using an automatic magnetic separation system, and their proliferation ability were assayed at different passages. The expression of factor VIII (FVIII), CD31, and CD34 was detected by immunofluorescence staining. Results indicated that rat intestinal mucosal MVECs grew into a contact-inhibited cobblestone-like monolayer after about 6 days, whose proliferation ability had no significant decline at passage 20. Three markers were generally expressed in the cells, and the fluorescence intensity of FVIII was higher after magnetic separation than before, and those of CD31 and CD34 remained similar. In conclusion, highly pure MVECs can be isolated from the primary cell culture of rat intestinal mucosas using magnetic beads coated with anti-CD31 antibodies, and magnetic separation may influence the expression of FVIII.

Effects of Nanosecond Pulsed Electric Fields in Cell Vitality, Apoptosis, and Proliferation of TPC-1 Cells

Objective. To evaluate the effects of nanosecond pulsed electric fields (nsPEFs) with different pulse durations in cell vitality, apoptosis, and proliferation of TPC-1 cells, optimize pulse parameters and expand the application range of nsPEFs. Methods. The pulse duration of 0, 300 ns, 500 ns, and 900 ns is generated with nsPEF generator. CCK-8 was used to investigate the effect of nsPEFs on the viability of TPC-1 cells. Flow cytometry was used to evaluate the apoptosis of TPC-1 after pulse treatment. The effect of nsPEFs on the proliferation ability of TPC-1 cells was detected by 5-ethy-nyl-2 ′ -deoxyuridine. The morphological changes of TPC-1 cells after pulse treatment were observed by transmission electron microscopy. Results. NsPEFs with 900 ns pulse duration can significantly affect the viability of TPC-1 cells and inhibit the proliferation ability of TPC-1 cells. In addition, nsPEFs can also induce apoptosis of TPC-1 cells. Conclusion. NsPEFs with longer pulse duration can significantly affect the biological behavior of TPC-1 cells, such as cell viability and proliferation ability, and can also induce cell apoptosis, thereby inhibiting cell growth.

Recombinant TSG-6 protein inhibits the growth of capsule fibroblasts in frozen shoulder via suppressing the TGF-β/Smad2 signal pathway

Background The tumor necrosis factor-stimulated gene-6 (TSG-6) has been confirmed to inhibit inflammation. It is now generally accepted that local inflammatory stimulation around shoulder capsule causes proliferative fibrosis. This study aims to investigate the mechanism of recombinant TSG-6 protein inhibiting the growth of capsule fibroblasts in frozen shoulder via the TGF-β/Smad2 signal pathway. Methods Human frozen shoulder capsule tissue was taken for primary and passage culture, and the 3rd generation fibroblasts from pathological frozen shoulder capsule were treated with different concentrations of recombinant TSG-6 protein, or with TGF-β1 agonist SRI-011381. Immunoconfocal analysis was used to identify the isolated fibroblasts, and MTT assay, colony formation assay, and flow cytometry were used to detect the viability, proliferation, and apoptosis rate of fibroblast. The contents of fibrosis and inflammation indexes COL1A1, TNF-α, IL-6, and IL-1β in the cell supernatant were detected using ELISA and then further examined by qRT-PCR. The expression of Bax, Bcl-2, and proteins related to TGF-β/Smad2 pathway were detected by Western Blot. Results Compared with the blank control group, fibroblasts intervened with TSG-6 (2 μg and 5 μg) showed significantly decreased viability and proliferation ability and enhanced cell apoptosis, concurrent with the reductions in Bcl-2 expression; COL1A1, TNF-α, IL-6, and IL-1β levels; and the expression of TGF-β1 and phosphorylated Smad22, and an increase in Bax expression, while SRI-011381 treatment would reverse the effect of recombinant TSG-6 protein. Conclusion Recombinant TSG-6 protein inhibited the growth of primary fibroblasts from human frozen shoulder capsule by suppressing the TGF-β/Smad2 signaling pathway.

Targeting miR-9 in Glioma Stem Cell-Derived Extracellular Vesicles: A Novel Diagnostic and Therapeutic Biomarker

Background Glioblastoma, the lethal brain tumor with undesirable prognosis, still has no effective strategies in early diagnosis and treatment. Extracellular vesicles (EVs) isolated from cerebrospinal fluid (CSF) are a potential liquid biopsy source. This study was performed to assess the miRNA expression profiles in EVs from CSF and tissue of GBM patients to identify significantly upregulated miRNAs and investigate the underlying neoplastic mechanisms.Methods EVs were measured by TEM and NTA assays. Differentially regulated miRNAs were measured using RNA sequencing in GBM CSF EVs and in GBM tissues compared with controls. RT-qPCR was employed to analyze certain miRNA and gene expression. Luciferase report assay was used in investigate downstream gene target of miR-9. The proliferation ability was detected by EdU and CCK-8 experiment while cell migration was measured by transwell and wound healing assay.Results The expression level of miR-9 was significantly higher in GBM CSF EVs and tissues than controls (p = 0.038). The area under curve for CSF EV miR-9 was 0.080 (95% CI: 0.583–1.000, p = 0.033). The expression of miR-9 was significantly higher in GSCs and GSC-derived EVs compared with glioblastoma cells. GSC culture medium, which contained the GSC-derives EVs, could promote GBM growth and migration after administration to GBM cells. Moreover, inhibition of miR-9 in GSCs showed the reverse anti-tumor effects. MiR-9 could bind to the 3’UTR region of DACT3 and suppress its expression. The miR-9/DACT3 axis might attribute to GBM malignant phenotype.Conclusion MiR-9 in CSF EVs might act as a novel diagnostic biomarker for GBM and targeting miR-9 by GSC-derived EVs may be a specific and efficient strategy for GBM biotherapy.

miR-340 Inhibits the Development of Hepatocellular Carcinoma by Down-Regulating Akt Signaling Pathway

Hepatocellular carcinoma (HCC) is a serious threat to human health. miR-340 participates in HCC pathogenesis, but its specific mechanism is not completely clear. Therefore, our study assessed the mechanism by how miR-340 involves in HCC. The cancer tissues and paracancerous tissues of HCC patients were collected. miR-340 mimics/NC and Akt siRNA were transfected into HepG2 cells followed by analysis of miR-304 and EMT-related molecules expression by Real-time PCR, cell invasion and migration by Transwell assay, cell proliferation ability by CCK8 assay as well as p-Akt and p-mTOR level by Western blot. miR-340 in HCC tissues was significantly downregulated compared to adjacent tissues (P <0.001). With increased pathological grade, miR-340 expression was decreased gradually. p-Akt and p-mTOR in HCC tissues was significantly upregulated and elevated gradually with increased pathological grade. p-Akt and p-mTOR was negatively associated with miR-340 (P <0.001). After overexpression of miR-340, HepG2 cell proliferation, invasion, migration and epithelialization were significantly inhibited, and p-Akt and p-mTOR was reduced. When Akt expression was interfered with siRNA, cell proliferation and epithelialization was further inhibited. miR-340 inhibits the development of hepatocellular carcinoma through Akt signaling pathway.