Functional analysis reveals calcium-sensing receptor gene regulating cell-cell junction in renal tubular epithelial cells.

PURPOSE: Calcium-sensing receptor (CASR) influences the expression pattern of multiple genes in renal tubular epithelial cells. The objective of this inquiry was to explore the molecular mechanisms of CASR in renal tubular epithelial cells and nephrolithiasis. METHODS: HK-2 cells were transfected with lentiviruses carrying either CASR (named CASR) or an empty vector negative control (named NC), as well as shRNA intended to target CASR (named shCASR) or its corresponding negative control (named shNC). CCK-8 assay was used to detect the effect of CASR on the proliferation of HK-2 cells. RNA-Sequencing was applied to explore potential pathways regulated by CASR in HK-2 cells. RESULTS: PCR and western blot results showed that CASR expression was significantly increased in CASR cells and was decreased in shCASR cells when compared to their corresponding negative control, respectively. CCK-8 assay revealed that CASR inhibited the proliferation of HK-2 cells. RNA-Sequencing results suggested that the shCASR HK-2 cells exhibited a significant up-regulation of 345 genes and a down-regulation of 366 genes. These differentially expressed genes (DEGs) were related to cell apoptosis and cell development. In CASR HK-2 cells, 1103 DEGs primarily functioned in mitochondrial energy metabolism, and amino acid metabolism. With the Venn diagram, 4 DEGs (Clorf116, ENPP3, IL20RB, and CLDN2) were selected as the hub genes regulated by CASR. Enrichment analysis revealed that these hub genes were involved in cell-cell junction, and epithelial cell development. CONCLUSIONS: In summary, our investigation has the potential to offer novel perspectives on CASR regulating cell-cell junction in HK-2 cells.

Differences in the phenotypes and transcriptomic signatures of chimeric antigen receptor T lymphocytes manufactured via electroporation or lentiviral transfection.

Chimeric antigen receptor (CAR)-T cell therapy is an innovative treatment for CD19-expressing lymphomas. CAR-T cells are primarily manufactured via lentivirus transfection or transposon electroporation. While anti-tumor efficacy comparisons between the two methods have been conducted, there is a current dearth of studies investigating the phenotypes and transcriptome alterations induced in T cells by the two distinct manufacturing methods. Here, we established CAR-T signatures using fluorescent imaging, flow cytometry, and RNA-sequencing. A small fraction of CAR-T cells that were produced using the PiggyBac transposon (PB CAR-T cells) exhibited much higher expression of CAR than those produced using a lentivirus (Lenti CAR-T cells). PB and Lenti CAR-T cells contained more cytotoxic T cell subsets than control T cells, and Lenti CAR-T cells presented a more pronounced memory phenotype. RNA-sequencing further revealed vast disparities between the two CAR-T cell groups, with PB CAR-T cells exhibiting greater upregulation of cytokines, chemokines, and their receptors. Intriguingly, PB CAR-T cells singularly expressed IL-9 and fewer cytokine release syndrome-associated cytokines when activated by target cells. In addition, PB CAR-T cells exerted faster in vitro cytotoxicity against CD19-expressing K562 cells but similar in vivo anti-tumor efficacy with Lenti CAR-T. Taken together, these data provide insights into the phenotypic alterations induced by lentiviral transfection or transposon electroporation and will attract more attention to the clinical influence of different manufacturing procedures.

11S Glycinin Up-Regulated NLRP-3-Induced Pyroptosis by Triggering Reactive Oxygen Species in Porcine Intestinal Epithelial Cells.

11S glycinin is a major soybean antigenic protein, which induces human and animal allergies. It has been reported to induce intestinal porcine epithelial (IPEC-J2) cell apoptosis, but the role of pyroptosis in 11S glycinin allergies remains unknown. In this study, IPEC-J2 cells were used as an in vitro physiological model to explore the mechanism of 11S glycinin-induced pyroptosis. The cells were incubated with 0, 1, 5, and 10 mg·ml-1 11S glycinin for 24 h. Our results revealed that 11S glycinin significantly inhibited cell proliferation, induced DNA damage, generated active oxygen, decreased mitochondrial membrane potential, and increased the NOD-like receptor protein 3 (NLRP-3) expression of IPEC-J2 cells in a dose-dependent manner. Further, IPEC-J2 cells were transfected with designed sh-NLRP-3 lentivirus to silence NLRP-3. The results showed that 11S glycinin up-regulated the silenced NLRP-3 gene and increased the expression levels of apoptosis-related spot-like protein (ASC), caspase-1, the cleaved gasdermin D, and interleukin-1ß. The IPEC-J2 cells showed pyrolysis morphology. Moreover, we revealed that N-acetyl-L-cysteine can significantly inhibit the production of reactive oxygen species and reduce the expression levels of NLRP-3 and the cleaved gasdermin D. Taken together, 11S glycinin up-regulated NLRP-3-induced pyroptosis by triggering reactive oxygen species in IPEC-J2 cells.

Exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation after traumatic brain injury.

Exosomes derived from bone marrow mesenchymal stem cells can inhibit neuroinflammation through regulating microglial phenotypes and promoting nerve injury repair. However, the underlying molecular mechanism remains unclear. In this study, we investigated the mechanism by which exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation. Our in vitro co-culture experiments showed that bone marrow mesenchymal stem cells and their exosomes promoted the polarization of activated BV2 microglia to their anti-inflammatory phenotype, inhibited the expression of proinflammatory cytokines, and increased the expression of anti-inflammatory cytokines. Our in vivo experiments showed that tail vein injection of exosomes reduced cell apoptosis in cortical tissue of mouse models of traumatic brain injury, inhibited neuroinflammation, and promoted the transformation of microglia to the anti-inflammatory phenotype. We screened some microRNAs related to neuroinflammation using microRNA sequencing and found that microRNA-181b seemed to be actively involved in the process. Finally, we regulated the expression of miR181b in the brain tissue of mouse models of traumatic brain injury using lentiviral transfection. We found that miR181b overexpression effectively reduced apoptosis and neuroinflamatory response after traumatic brain injury and promoted the transformation of microglia to the anti-inflammatory phenotype. The interleukin 10/STAT3 pathway was activated during this process. These findings suggest that the inhibitory effects of exosomes derived from bone marrow mesenchymal stem cells on neuroinflamation after traumatic brain injury may be realized by the action of miR181b on the interleukin 10/STAT3 pathway.

Macrophage-Mediated Delivery of Fe3O4-Nanoparticles: A Generalized Strategy to Deliver Iron to Tumor Microenvironment.

BACKGROUND: Iron is used to alter macrophage phenotypes and induce tumor cell death. Iron oxide nanoparticles can induce macrophage polarization into the M1 phenotype, which inhibits tumor growth and can dissociate into iron ions in macrophages. OBJECTIVE: In this study, we proposed to construct high expression of Ferroportin1 macrophages as carriers to deliver Fe3O4-nanoparticles and iron directly to tumor sites. METHODS: Three sizes of Fe3O4-nanoparticles with gradient concentrations were used. The migration ability of iron-carrying macrophages was confirmed by an in vitro migration experiment and monocyte chemoattractant protein-1 detection. The release of iron from macrophages was confirmed by determining their levels in the cell culture supernatant, and we constructed a high expression of ferroportin strain of macrophage lines to increase intracellular iron efflux by increasing membrane transferrin expression. Fe3O4-NPs in Ana-1 cells were degraded in lysosomes, and the amount of iron released was correlated with the expression of ferroportin1. RESULTS: After Fe3O4-nanoparticles uptake by macrophages, not only polarized macrophages into M1 phenotype, but the nanoparticles also dissolved in the lysosome and iron were released out of the cell. FPN1 is the only known Fe transporter; we use a Lentiviral vector carrying the FPN1 gene transfected into macrophages, has successfully constructed Ana-1-FPN1 cells, and maintains high expression of FPN1. Ana-1-FPN1 cells increase intracellular iron release. Fe3O4-nanoparticles loaded with engineered Ana-1 macrophages can act as a "reservoir" of iron. CONCLUSION: Our study provides proof of strategy for Fe3O4-NPs target delivery to the tumor microenvironment. Moreover, increase of intracellular iron efflux by overexpression of FPN1, cell carriers can act as a reservoir for iron, providing the basis for targeted delivery of Fe3O4-NPs and iron ions in vivo.

STEAP2 is down-regulated in breast cancer tissue and suppresses PI3K/AKT signaling and breast cancer cell invasion in vitro and in vivo.

The six-transmembrane epithelial antigen of prostate 2 (STEAP2) protein was identified in advanced prostate cancer, and is highly over-expressed in various types of cancer. This study aimed to investigate the prognostic value and the function of STEAP2 in breast cancer. STEAP2 mRNA and protein expressions in breast normal and cancer tissues, breast cancer cell lines (MCF-7, BT-549, BT-474, MDA-MB-361, HCC1937, and MDA-MB-468) and normal mammary epithelial cell lines (HBL-100 and MCF-10A) were evaluated by immunohistochemistry, real time RT-qPCR and western blotting. The expression of STEAP2 in breast cancer tissues and its value of evaluating the prognosis of breast cancer patients was validated in the Public Databases (Oncomine and Kaplan-Meier plotter database). Lentiviral vectors with STEAP2 cDNA and shRNA were constructed and used to infect breast cancer cell lines and normal mammary epithelial cell line to investigate the effects of STEAP2 up- and down- regulation on the biological behavior of breast cells. The low expression of STEAP2 was detected in breast cancer tissues, which was associated with malignant phenotype and poor prognosis of breast cancer. The public databases analyses were consistent with our findings. STEAP2 up-regulation hindered cellular proliferation, invasion and metastasis abilities by inhibiting EMT process and suppressing PI3K/AKT/mTOR signaling pathway. On the other hand, STEAP2 down-regulation could promote cell proliferation and invasion by inducing EMT and activating the PI3K/AKT/mTOR signaling pathway. Collectively, STEAP2 acted as an anti-oncogene in breast cancer development, which suggested a new research objective for the future studies.

Role of trefoil factor3 / phosphatidylinositl 3-kinase / protein kinase B / nuclear factor κB in the proliferation and apoptosis of human papillary thyroid carcinoma cell line TPC-1 / 解剖学报

Objective To investigate the effects of trefoil factor 3 (TFF3) on the proliferation and apoptosis of human thyroid papillary carcinoma cell line (TPC-1) and its molecular mechanism. Methods The lentiviral expression vector of overexpression and knockdown of TFF3 gene were constructed, 293T cell was packaged to produce lentiviral particles, virus solution was collected and transfected into TPC-1 cells, enhanced cell TFF3-TPC-1 and enhanced control group ConTFF3-TPC-1; silencing cell shRNA-TFF3-TPC-1 and silencing control cells shCon -TPC-1. Western blotting, and Real-time PCR were used to detect the expression of TFF3 protein and mRNA of four groups. Growth curve and colony formation assay were used to detect the proliferation. Flow cytometry was used to analyze the apoptosis level of the four groups; Western blotting and immunocytochemistry were used to detect apoptosis-related protein and pathway protein phosphatidylinositol 3-kinase/ protein kinase B (PI3K/ Akt), nuclear factor-κB (NF-κB) expression. Results 1. Overexpression and inhibition of expression of TFF3 stable cell TFF3-TPC-1 and shRNA-TFF3-TPC-1 were constructed suscessfully. 2. The proliferation and cloning ability of TFF3-TPC-1 cells were significantly higher than those of ConTFF3-TPC-1 cells(P<0. 05 or P< 0. 01), the proliferation and cloning ability of shRNA-TFF3-TPC-1 cells were significantly lower than those of shCon-TPC-1 cells(P<0. 01); 3. The apoptosis rate of TFF3-TPC-1 cells was lower than that of ConTFF3-TPC-1 (0. 75%±0. 08% vs 5. 62%±0. 3%, P<0. 01),and the apoptosis rate of shRNA-TFF3-TPC-1 was higher than that of shConTPC-1 (22. 2% ± 1. 2% vs 5. 34% ± 0. 4%, P<0. 01); 4. After silencing TFF3 gene, the expressions of Bax, cytochrome C (Cyt-C), cleaved-Caspase-9, cleaved-Caspase-3 were up-regulated, and the expressions of Bcl-2, Akt, p-Akt and NF-κB-P65 were down-regulated (P<0. 05 or P<0. 01). Conclusion TFF3 may regulate the proliferation and apoptosis of TPC-1 cells by affecting the PI3K/ Akt/ NF-κB signaling pathway.

Positive Effects of Heme Oxygenase Upregulation on Adiposity and Vascular Dysfunction: Gene Targeting vs. Pharmacologic Therapy.

OBJECTIVE: Heme oxygenase (HO-1) plays a critical role in adipogenesis and it is important to understand its function in obesity. Many studies have shown that upregulation of HO-1 can affect the biologic parameters in obesity-mediated diabetes, hypertension and vascular endothelial cell function. Thus, we aimed to explore the hypothesis that upregulation of HO-1, using a pharmacologic approach as well as gene targeting, would improve both adiposity and endothelial cell dysfunction by direct targeting of endothelial cells. Our second aim was to compare the short-term effect of a HO-1 inducer, cobalt-protoporphrin IX (CoPP), with the long-term effects of gene targeted therapy on vascular and adipocyte stem cells in obese mice. METHOD: We examined the effect of CoPP on fat pre-adipocytes and mesenchymal stem cells (MSC) in mice fed a high-fat diet (HFD). We also used a lentiviral construct that expressed heme oxygenase (HO-1) that was under the control of an endothelium specific promoter, vascular endothelium cadherin (VECAD) heme oxygenase (VECAD-HO-1). We targeted endothelial cells using vascular endothelium cadherin/green fluorescent protein fusion construct (VECAD-GFP) as the control. Conditioned media (CM) from endothelial cells (EC) was added to fat derived adipocytes. Additionally, we treated renal interlobar arteries with phenylephrine and dosed cumulative increments of acetylcholine both with and without exposure to CoPP. We did the same vascular reactivity experiments with VECAD-HO-1 lentiviral construct compared to the control. RESULTS: CoPP improved vascular reactivity and decreased adipogenesis compared to the control. MSCs exposed to CM from EC transfected with VECAD-HO-1 showed decreased adipogenesis, smaller lipid droplet size and decreased PPAR-γ, C/EBP and increased Wnt 10b compared to the control. HO-1 upregulation had a direct effect on reducing adipogenesis. This effect was blocked by tin mesoporphrin (SnMP). EC treated with VECAD-HO-1 expressed lower levels of ICAM and VCAM compared to the control, suggesting improved EC function. This also improved ACH induced vascular reactivity. These effects were also reversed by SnMP. The effect of viral transfection was much more specific and sustained than the effects of pharmacologic therapy, CoPP. CONCLUSION: This study demonstrates that a pharmacological inducer of HO-1 such as CoPP improves endothelial cell function while dampening adipogenesis, but long-term HO-1 expression by direct targeting of endothelial cells by gene transfer therapy may offer a more specific and ideal solution. This was evidenced by smaller healthier adipocytes that had improved insulin sensitivity, suggesting increased adiponectin levels. HO-1 upregulation reestablished the "crosstalk" between perivascular adipose tissue and the vascular system that was lost in the chronic inflammatory state of obesity. This study demonstrates that gene targeting of EC may well be the future direction in treating obesity induced EC dysfunction, with the finding that targeting the vasculature had a direct and sustained effect on adipogenesis.

VCAM-1 improves migration and invasion of human glioma cell lines / 基础医学与临床

Objective To investigate the effects of VCAM-1 on migration and invasion of glioma cell lines . Methods The techniques of lentivirus pSGU6/GFP/Neo-based VCAM-1 shRNA and EF1 a-GFP/puro-based VCAM-1 expression vector, the scratch wound healing migration and transwell invasion assays , and the Western blot and cell staining were applied to observe the effects of VCAM-1 expression levels on migration and invasion of glioma cell line cells.There are four groups in T98G cells including control, vector, scramble and shRNA-VCAM-1 groups and three groups in U251 cells covering control, vector and VCAM-1 overexpressed groups ( n=6 per group) .Results The stabled glioma cell lines of T98 G cells with down-regulated VCAM-1 and U251 cells with VCAM-1 overexpression were established by using lentivirus-based VCAM-1 shRNA and expression vector.The ability of scratch wound healing (migration activity) decreased significantly (P<0.01) in T98G cells with lower VCAM-1 expression levels, while the migration activity was obviously improved in U251 cells with overexpressed VCAM-1 ( P <0.05 ) .Similarly, the invasion ability was significantly inhibited ( P <0.05) in T98G cells with silenced VCAM-1, as well as VCAM-1 overexpression could enhance the invasion ability of U251 cells ( P<0.01 ) .Conclusions VCAM-1 improves the migration activity and invasion ability of human glioma cell line cells.

Effects of p38-2 G4 high-expression on the proliferation and erythriod differentiateon of murine erythroleukemia cells / 解剖学报

Objective To explore the effect of mouse proliferation-associated protein 2G4 (p38-2G4) high-expression on the proliferation and erythriod differentiation of murine erythroleukemia ( MEL ) cells.Methods To establish the recombinant lentivirus vector p 38-2G4-pLJM1, the p38-2G4-pLJM1 was cotransfected into HEK293T cells to obtain lentivirus with pCMV-VSV-G and pCMV-dR8.2.Lentivirus were infected into MEL cells to establish the stably p 38-2G4 high-expressed MEL cells.Western blotting was used to analyse the high-expression efficiency.MTT assay and benzidine staining were applied to detect the cell viability and hemoglobin synthesis of the stable cell line in presence /absence of inducers.Results Western blotting showed that the p38-2G4 high-expression stable cell strain had a higher expression of p38-2G4 as compared to the control group ( MEL) ( P 0.05), but the hemoglobin synthesis had been reduced as compared to the control group (P<0.05).Conclusion p38-2G4 high-expression does not affect the cell viability of MEL cells , but inhibits the erythriod differentiation of MEL cells in three independent experiments .

Treatment of multiple organ dysfunction syndrome by administration of mesenchymai stem cells / 国际外科学杂志

Objective To study the bone marrow-derived mesenchymal stem cell(MSC) potential as seed cells for treatment of multiple organ dysfunction syndrome(MODS) in rabbit model, lay fundamental for clin-ical utilization of the MSC for treatment of MODS. Methods MODS rabbit model was established by hemor-rhagic shock combined with endotoxin, isolation and characterization of rabbit bone marrow mesenchymal stem cells labeled MSC with GFP by lentivirus transfection, administration of MSC by ear margin vein injec-tion, MSC integration determined by PCR and pathological section, the MSCs effect on MODS rabbits evalu-ated by physical observation. Results Compared with controls, ransplanted MSCs were found in liver, lung, kidney, MODS rabbits were physically improved obviously. Conclusions MSCs are able to integrate into host without any rejection reaction, and capable of ameliorating MODS evidently.