The effect of SNPs in lncRNA as ceRNA on the risk and prognosis of hepatocellular carcinoma.

BACKGROUND: Most susceptible loci of hepatocellular carcinoma (HCC) identified by genome-wide association studies (GWAS) are located in non-coding regions, and the mechanism of action remains unclear. The objective of this study was to explore the association of single nucleotide polymorphisms (SNPs) on long non-coding RNAs (lncRNAs) that affect competing endogenous RNAs (ceRNA) regulation mechanism with the risk and prognosis of HCC. METHODS: Based on a set of bioinformatics strategies, eight lncRNA genes that affect HCC through the mechanism of lncRNA-mediated ceRNA were systematically screened, and 15 SNPs that affect microRNA (miRNA) binding in these lncRNA genes were annotated. Genotyping was performed in 800 HCC cases and 801 healthy controls to examine associations of these SNPs with HCC in a northeastern Chinese Han population. RESULTS: The GG, GC and GG + GC genotypes of HOTAIR rs7958904 were associated with a 0.65, 0.59 and 0.63-fold decreased HCC risk, respectively. In addition, HCC patients with PVT1 rs3931282 AA + GA genotypes were less prone to develop late-stage cancers in a stratified analysis of clinical characteristics. When stratified by clinical biochemical indexes, rs1134492 and rs10589312 in PVT1 and rs84557 in EGFR-AS1 showed significant associations with aspartate aminotransferase (AST), alanine aminotransferase (ALT) or AST/ALT ratio in HCC patients. Furthermore, we constructed potential ceRNA regulatory axes that might be affected by five positive SNPs to explain the causes of these genetic associations. CONCLUSIONS: HOTAIR rs7958904, PVT1 rs3931282, rs1134492 and rs10589312, and EGFR-AS1 rs84557 might be predictors for HCC risk or prognosis. Our results provide new insights into how SNPs on lncRNA-mediated ceRNAs confer interindividual differences to occurrence and progression of HCC.

The protective effect of pericytes on vascular permeability after hemorrhagic shock and their relationship with Cx43.

Vascular hyperpermeability is a complication of hemorrhagic shock. Pericytes (PCs) are a group of mural cells surrounded by microvessels that are located on the basolateral side of the endothelium. Previous studies have shown that damage to PCs contributes to the occurrence of many diseases such as diabetic retinopathy and myocardial infarction. Whether PCs can protect the vascular barrier function following hemorrhagic shock and the underlying mechanisms are unknown. A hemorrhagic shock rat model, Cx43 vascular endothelial cell (VEC)-specific knockdown mice, and VECs were used to investigate the role of PCs in vascular barrier function and their relationship with Cx43. The results showed that following hemorrhagic shock, the number of PCs in the microvessels was significantly decreased and was negatively associated with an increase in pulmonary and mesenteric vascular permeability. Exogenous infusion of PCs (106 cells per rat) colonized the microvessels and improved pulmonary and mesenteric vascular barrier function. Upregulation of Cx43 in PCs significantly increased the number of PCs colonizing the pulmonary vessels. In contrast, downregulation of Cx43 expression in PCs or knockout of Cx43 in VECs (Cx43 KO mice) significantly reduced PC colonization in pulmonary vessels in vivo and reduced direct contact formation between PCs and VECs in vitro. It has been suggested that PCs have an important protective effect on vascular barrier function in pulmonary and peripheral vessels following hemorrhagic shock. Cx43 plays an important role in the colonization of exogenous PCs in the microvessels. This finding provides a potential new shock treatment measure.

The protective effects of pericyte-derived microvesicles on vascular endothelial functions via CTGF delivery in sepsis.

BACKGROUND: It is well known that sepsis is a prevalent severe disease caused by infection and the treatment strategies are limited. Recently pericyte-derived microvesicles (PMVs) were confirmed to be therapeutic in many diseases, whether PMVs can protect vascular endothelial cell (VEC) injury is unknown. METHODS: Pericytes were extracted from the retina of newly weaned rats, and PMVs were collected after starvation and characterized by flow-cytometry and transmission electron microscopy. First, the effect of PMVs on pulmonary vascular function in septic rats was measured via intravenous administration with HE staining, immunofluorescence, and Elisa analysis. Then, PMVs were co-incubated with VECs in the presence of lipopolysaccharide (LPS), and observed the protective effect of PMVs on VECs. Next, the proteomic analysis and further Gene Ontology (GO) enrichment analysis were performed to analyze the therapeutic mechanism of PMVs, and the angiogenesis-related protein CTGF was highly expressed in PMVs. Finally, by CTGF upregulation and downregulation in PMV, the role of PMV-carried CTGF was investigated. RESULTS: PMVs restored the proliferation and angiogenesis ability of pulmonary VECs, and alleviated pulmonary vascular leakage in septic rats and LPS-stimulated VECs. Further study showed that PMVs delivered CTGF to VECs, and subsequently activated ERK1/2, and increased the phosphorylation of STAT3, thereby improving the function of VECs. The further study found CD44 mediated the absorption and internalization of PMVs to VECs, the anti-CD44 antibody inhibited the protective effect of PMVs. CONCLUSIONS: PMVs may delivery CTGF to VECs, and promote the proliferation and angiogenesis ability by activating the CTGF-ERK1/2-STAT3 axis, thereby protecting pulmonary vascular function in sepsis. The therapeutic effect of PMVs was highly related to CD44-mediated absorption. Video Abstract.

Mesenchymal stem cell-derived microvesicles improve intestinal barrier function by restoring mitochondrial dynamic balance in sepsis rats.

BACKGROUND: Sepsis is a major cause of death in ICU, and intestinal barrier dysfunction is its important complication, while the treatment is limited. Recently, mesenchymal stem cell-derived microvesicles (MMVs) attract much attention as a strategy of cell-free treatment; whether MMVs are therapeutic in sepsis induced-intestinal barrier dysfunction is obscure. METHODS: In this study, cecal ligation and puncture-induced sepsis rats and lipopolysaccharide-stimulated intestinal epithelial cells to investigate the effect of MMVs on intestinal barrier dysfunction. MMVs were harvested from mesenchymal stem cells and were injected into sepsis rats, and the intestinal barrier function was measured. Afterward, MMVs were incubated with intestinal epithelial cells, and the effect of MMVs on mitochondrial dynamic balance was measured. Then the expression of mfn1, mfn2, OPA1, and PGC-1α in MMVs were measured by western blot. By upregulation and downregulation of mfn2 and PGC-1α, the role of MMVs in mitochondrial dynamic balance was investigated. Finally, the role of MMV-carried mitochondria in mitochondrial dynamic balance was investigated. RESULTS: MMVs restored the intestinal barrier function by improving mitochondrial dynamic balance and metabolism of mitochondria. Further study revealed that MMVs delivered mfn2 and PGC-1α to intestinal epithelial cells, and promoted mitochondrial fusion and biogenesis, thereby improving mitochondrial dynamic balance. Furthermore, MMVs delivered functional mitochondria to intestinal epithelial cells and enhanced energy metabolism directly. CONCLUSION: MMVs can deliver mfn2, PGC-1α, and functional mitochondria to intestinal epithelial cells, synergistically improve mitochondrial dynamic balance of target cells after sepsis, and restore the mitochondrial function and intestinal barrier function. The study illustrated that MMVs might be a promising strategy for the treatment of sepsis.

Research advances in pericyte function and their roles in diseases.

Pericyte, a kind of pluripotent cell, may regulate the irrigation flow and permeability of microcirculation. Pericytes are similar to the smooth muscle cells, which express several kinds of contractile proteins and have contractility. The dysfunction of pericytes is related to many microvascular diseases, including hypoxia, hypertension, diabetic retinopathy, fibrosis, inflammation, Alzheimer's disease, multiple sclerosis, and tumor formation. For a long time, their existence and function have been neglected. The distribution, structure, biomarker, related signaling pathways as well as the roles of pericytes on vascular diseases will be introduced in this review.

Molecular cloning and expression of glycoprotein IIb and IIIa.

OBJECTIVE: To amplify the cDNA genes of GPIIb, GPIIIa, then construct the eukaryotic expression carriers of GPIIb and GPIIIa respectively, finally establish CHO cell lines stably expressing GPIIb and GPIIIa. METHODS: Human erythroleukemia (HEL) cells were cultured for total RNA extraction. RT-PCR was accomplished using the specific GPIIb, GPIIIa primers designed according to Genbank by Primer 5, then each of cDNAs were obtained. The expressive vector pcDNA3.1(+) and PCR products were cut by NheI and HindIII, and then the fragements were directly cloned to pcDNA3.1(+) because of having the same adhesive ends. Then pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were transfected into CHO cells respectively by Lipofectamine 2000. The cell lines expressing GPIIb, GPIIIa were screened by G418. Then the Chinese hamster ovary (CHO) cell lines were examed through flow cytometry (FCM) and RT-PCR to detect the expression of GPIIb, GPIIIa in CHO cells. RESULTS: The cDNAs of GPIIb and GPIIIa were amplidied by RT-PCR, and the pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were constructed respectively. By sequencing and double digestion, pcDNA3.1(+)IIb and pcDNA3.1(+)IIIa were all correct. Expression of GPIIb and GPIIIa were detected on transfected CHO cells by FCM and RT-PCR. CONCLUSIONS: (1) Succeeded in constructing pcDNA3.1(+)IIb, pcDNA3.1(+)IIIa. (2) Succeeded in getting the cell lines expressing GPIIb, GPIIIa.