Lentiviral Expression Research Articles

Identification and ultrasensitive photoelectrochemical detection of LncNR_040117: a biomarker of recurrent miscarriage and antiphospholipid antibody syndrome in platelet-derived microparticles

The abnormal expression of long non-coding RNAs (LncRNAs) in platelet-derived microparticles (PMPs) is closely related to immune disorders and may lead to antiphospholipid antibody syndrome and recurrent miscarriage. To understand the association between the LncRNAs in PMPs and RM/APS, the differences in the expression of LncRNAs in RM/APS patients and healthy controls were analyzed. Microarray analysis and RT-qPCR detection proved that RM/APS patient exhibited high levels of LncNR_040117 expression. The lentiviral silent expression transfection of HTR-8/SVneo cells indicated that LncNR_040117 downregulation decreased the activity of HTR-8/SVneo cells and inhibited the MAPK signaling pathway, further confirming the biomarker proficiency of LncNR_040117 for RM/APS. After that, we proposed a β-In2S3@g-C3N4 nanoheterojunction-based photoelectrochemical (PEC) biosensor to achieve the ultrasensitive detection of LncNR_040117. The nanoheterojunction aids in the effective separation of photogenerated carriers and significantly improve the photocurrent response of the biosensor. The conjugation of LncNR_040117 onto the PEC biosensing platform increased the steric hindrance between electrolyte and electrode, subsequently decreasing the photocurrent signal. The PEC biosensor showed a wide detection range of 0.1–106 fM and a low limit of detection of 0.025 fM. For clinical sample testing, the results of the PEC and RT-qPCR were highly consistent. Overall, LncNR_040117 in PMPs was identified as an effective biomarker for RM/APS and could be accurately detected by the proposed PEC biosensor, which is expected to provide a reliable diagnostic platform for RM/APS.

Establishment of a fetal cow (Bos Borus) skin fibroblasts cell line with immortalized characterization through human telomerase reverse transcriptase (hTERT) ectopic expression

The ectopic introduction of the human telomerase reverse transcriptase (hTERT) is an effective way to establish an immortalized cell line. Here, hTERT was obtained by RT-PCR, and the eukaryotic expression plasmid and lentivirus shuttle plasmid of hTERT was successfully constructed by the homologous recombination method. The stable expression of hTERT in fetal cow skin fibroblasts (CSF) was established using the lentivirus package system. The hTERT-CSF proliferate and have immortalized characteristics. Meanwhile, the chromosome analysis identified that the number and structure of the hTERT-CSF genome maintain stable. The indirect immunofluorescence, western blot, and flow cytometry showed that the hTERT gene had been successfully integrated into the primary genome of bovine skin and stably expressed. The viral infection experiment first identifies the hTERT-CSF as a vulnerable cell model responding to the Lumpy skin disease virus (LSDV). Establishing hTERT-CSF provides an important cell model for basic and applied research, clinical application, and vaccine development. It provides an essential reference for the future’s rapid establishment of other immortalized cell lines.

Function of AXL and molecular mechanisms in regulation of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics. NPC is mostly found in south China and Southeast Asia, and its treatment mainly depends on radiotherapy and chemotherapy. However, NPC is usually found in the late stage, and local recurrence and distant metastasis are common, leading to poor prognosis. The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation, migration, invasion, and other processes, which are associated with poor prognosis of tumors. This study aims to detect the expression of AXL in NPC cell lines and tissues, and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC. The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799, GSE12452, and GSE53819 data sets based on Gene Expression Omnibus (GEO) database. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma (HNSC). The indicators of prognosis included overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI). Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines. Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues. Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector, and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector. Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines. The effects of AXL knockdown or overexpression on proliferation, migration, and invasion of NPC cells were detected by CCK-8, plate colony formation, and Transwell assays, and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay. The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database. The PROMO online database was used to predict AXL transcription factors with 0% fault tolerance, and the JASPAR online database was used to predict the binding sites of ETS1 to AXL. Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and mRNA expression. The AXL upstream 2.0 kb promoter region was divided into 8 fragments, each of which was 250 bp in length. Primers were designed for 8 fragments. The binding of ETS1 to AXL promoter region was detected by chromatin immuno-precipitation (ChIP) assay to determine the direct regulatory relationship between ETS1 and AXL. Rescue assay was used to determine whether ETS1 affected the proliferation, migration, and invasion of NPC cells through AXL. Bioinformatics analysis showed that AXL was highly expressed in NPC tissues (P<0.05), and AXL expression was positively correlated with OS, DFI, DSS, and PFI in HNSC patients. Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues. Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments. The results of CCK-8, plate colony formation, Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation, migration, invasion of NPC cells and tumor growth (all P<0.05), and the up-regulation of AXL significantly promoted the proliferation, migration, and invasion of NPC cells (all P<0.05).As predicted by PROMO and JASPAR online databases, ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region. Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and mRNA expression levels. ChIP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression. Rescue assay showed that AXL rescued the effects of ETS1 on proliferation, migration and invasion of NPC cells (P<0.05). AXL is highly expressed in NPC cell lines and tissues, which can promote the malignant progression of NPC, and its expression is regulated by transcription factor ETS1.

Abstract 2329: Variable HPV E6 levels among oropharyngeal cancers govern therapy response via the PGC1/ERR axis

Abstract BACKGROUND: Elevated mitochondrial antioxidant defenses contribute to progression and therapy resistance of some poor prognosis cancer types. By contrast, HPV+ oropharyngeal squamous cell carcinomas (OPSCCs) are generally sensitive to oxidative damage from radiation plus cisplatin, which are often curative. It is unknown whether the subset of HPV+ OPSCCs with poor outcomes exploit antioxidant capacity provided by high mitochondrial mass to evade therapy. It is also unclear whether the wide range of HPV oncoprotein expression among them contributes to diversity in mitochondrial mass and treatment outcomes. AIMS: (1) evaluate HPV+ OPSCCs for associations between mitochondrial mass and clinical outcome (2) elucidate relationships among HPV oncoprotein levels, mitochondrial mass, and therapy response (3) delineate mechanisms by which variable HPV oncoprotein levels govern therapy response. METHODS/RESULTS: High expression of genes involved in oxidative metabolism was associated with decreased survival in three patient cohorts. In PDX and cell line models, cisplatin resistance positively correlated with mitochondrial mass (MTCO1/B2M), oxidative metabolism (basal OCR), and antioxidant capacity (NADPH/NADP+). Among the HPV oncoproteins, only full length E6 (fl-E6) was linked to these features, showing negative correlation with mitochondrial mass in the cell lines, PDXs, and patient tumors. Fl-E6 levels also positively correlated with cisplatin response in the PDXs and cell lines. To test whether fl-E6 can mediate such effects, lentiviral fl-E6 expression in HPV+ cancer cell lines with low endogenous fl-E6 was used to increase levels to the high end of the range seen in human tumors. Doing so reduced MTCO1/B2M, basal OCR, and NADPH/NADP+ while sensitizing to radiation and cisplatin in vitro and in vivo. The same effects on mitochondrial biogenesis were maintained in nTERT/E7 keratinocytes. Using luciferase reporter assays, fl-E6 was shown to repress the PGC-1/ERR axis for mitochondrial biogenesis by attenuating p53-dependent PGC1α promoter activity. The prediction of PGC1/ERR axis activation in tumors with low fl-E6 was confirmed in PDXs and patient cohorts, where high ERRα was negatively prognostic. CONCLUSIONS: These results reveal a novel ability of E6-mediated p53 downregulation to contribute to treatment sensitivity by depleting mitochondrial antioxidant capacity. They also provide evidence that differing fl-E6 levels across HPV+ cancers variably repress the PGC1/ERR pathway, leading to diversity in mitochondrial mass that impacts therapy response. Therapy-refractory features may prove identifiable using expression-based biomarkers in the PGC1/ERR axis and be mitigated through targetable nodes in this pathway. In addition, HPV+ OPSCCs may find competitive advantage in down-regulating fl-E6 upon acquiring alternate drivers to compensate decrease in its oncogenic functions. Citation Format: Malay K. Sannigrahi, Pavithra Rajagopalan, Ling Lai, Xinyi Liu, Varun Sahu, Hiroshi Nakagawa, Robert Brody, Iain M. Morgan, Bradford E. Windle, Xiaowei Wang, Phyllis A. Gimotty, Daniel P. Kelly, Elizabeth A. White, Devraj Basu. Variable HPV E6 levels among oropharyngeal cancers govern therapy response via the PGC1/ERR axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2329.

Abstract 2179: Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma

Abstract Background: BCAT1 (Branched Chain Amino Transferase 1) has been shown to be upregulated in human IDH (isocitrate dehydrogenase) wild-type glioblastoma (GBM) and BCAT1 knockdown leads to reduced cell proliferation in vitro and in vivo1. Here we investigated the role of BCAT1 in human patient-derived glioblastoma cells and the corresponding orthotopically implanted xenografts to understand how upregulation of the enzyme leads to a more aggressive phenotype. Methods: The expression of BCAT1 was investigated in a panel of patient derived IDH wild-type glioblastoma cells and BCAT1 expression modulation was achieved using lentiviral expression of a doxycycline-inducible shRNA, to knockdown BCAT1 and expression of a BCAT1 cDNA to overexpress BCAT1. Effects of BCAT1 knockdown and overexpression were investigated in vitro in cell culture and in vivo, following intracranial implantation of mice with the relevant cells. Results: Variable levels of BCAT1 expression were identified in different patient-derived cell lines. RNA sequencing analysis of BCAT1 knockdown in BCAT1 expressing glioblastoma cells revealed that knockdown led to significant downregulation of HIF 1α (Hypoxia Inducible Factor 1α) and its targets, which was confirmed by Western Blot analysis of Carbonic Anhydrase IX (CAIX) and Hexokinase II (HKII) in cell and tumor lysates. The opposite was observed following BCAT1 overexpression. BCAT1 knockdown also led to higher DNA demethylation levels. Treatment with membrane permeant dimethyl-alpha ketoglutarate mimicked the effect of BCAT1 knockdown. These data suggest that decreased cell proliferation following BCAT1 knockdown in BCAT1 expressing cells is mediated by accumulation of alpha-ketoglutarate, a substrate of BCAT1. Cells that had no BCAT1 expression showed a similar growth profile and overexpression had no effect on proliferation, suggesting that these cells are not dependent on BCAT1 expression. Conclusion: We conclude that in a subtype of GBM characterized by upregulated BCAT1, BCAT1 expression results in lowered levels of alpha-ketoglutarate, leading to reduced prolyl hydroxylase activity and stabilization of HIF 1α and therefore a pseudo-hypoxic state, resulting in a transcriptome that favors a high proliferation rate. This makes BCAT1 a potential therapeutic target, for tumors that show BCAT1 upregulation.1. Tönjes M, Barbus S, Park YJ, et al. BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1. Nat Med. 2013;19(7):901-908. doi:10.1038/nm.3217 Citation Format: Maria Fala, Susana Ros, Ashley Sawle, Kevin M. Brindle. Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2179.

Use of anti-GD2 (Dinutuximab) as a target for CAR-T cells immunotherapy in neuroblastoma

Treating solid tumours with chimeric antigen receptors-modified T cells (CAR-T cells) has shown limited efficacy due to the lack of cancer-type specific antigens. Dinutuximab is a monoclonal antibody that recognizes a sphingolipid, disialoganglioside GD2, which has limited expression in normal tissues but is overexpressed in paediatric tumours, mainly neuroblastoma (NB) and diffuse intrinsic pontine glioma (DIPG) and other histone H3 K27M (H3K27M) mutated diffuse midline gliomas. Dinutuximab, an anti-GD2 antibody, is currently standard of care in the treatment of NB. We are contemplating the possibility of using Dinutuximab as target element for antiNB-CAR-T cells. Our group have explored the feasibility of producing CAR-T cells from peripheral blood (PB), post-apheresis CD45RA+ fraction and cord blood (CB) using similar protocols of T cell transduction and expansion ability as a way to favour the persistence of CAR-T cells in the host. Afterward, we performed a deeply characterization of phenotype and functionality of anti-FITC-CAR-T cells derived from different sources, and the cytotoxic effect against anti-GD2-FITC- NB labelled cell line. T cells purified from PB, CB and 45RA fraction after apheresis were cultured in vitro with cytokines IL-7+IL-15+IL- 21 to maintain early memory phenotypes as Naïve T cells (TN). A second generation lentiviral expression vector (LV) was used to generate second generation CARs (FITC-L-BBz CAR). After 48h of activation in culture with antiCD3/CD28 and cytokines, T cells were transduced with viral supernatants. Next, we performed a characterization of phenotype of anti-FITC-CAR-T cells by flow cytometry studying how the culture conditions enhanced a TN phenotype expressing CD45RA+ and CCR7+. Cytotoxic function of CAR-T cells was analysed in cocultures with LAN-1 NB cell line labelled with Dinutuximab conjugated with FITC. Preliminary results showed that culture condition with IL-7/15/21 maintained more primitive phenotypes in 45RA (71% TN CD4+ and 66% TN CD8+) and CB (60% of TN CD4+ and 77% of TN CD8+) derived T cells than PB T cells (only a 20% of TN CD4+ and TN CD8+). PB and CB-derived CAR-T cells showed higher cytolitic function than 45RA fraction, resulting in a 30% of dead cells in both cases in a 24h-culture assay. Both, PB and CB derived CAR-T cells showed the best CAR-T activation capacity with 19,6% of cells from PB and 33,9% of cells from CB expressing early activation markers as CD25 and CD134. In vivo experiments are currently on-going to test the efficacy of CAR-T cells in combination with GD2 treatment. Our strategy may complement the current use of Dinutuximab in the treatment of NB through its combination with a targeted CAR-T cell approach.

Hypoxia-inducible lentiviral gene expression in engineered human macrophages

BackgroundHuman immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression...

Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy

To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin. The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34+ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test. The human β-globin was expressed stably in the MEL cells, and CD34+ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34+ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant. Stable β-globin expression through the optimization of HS4 from CD34+ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

Vimentin promotes porcine circovirus type 2 (PCV2) replication in pig alveolar macrophage

Porcine circovirus type 2 (PCV2) is a swine pathogen that causes postweaning multisystemic wasting syndrome, associated with a number of other disease symptoms in pigs. It has caused high economic losses for pig industries in China and worldwide. Vimentin is a cytoskeletal protein involved in the lifecycle of numerous viruses. In this study, the role of Vimentin in PCV2 replication in pig alveolar macrophage (3D4/21) cells was studied. A 3D4/21 cell line overexpressing Vimentin was established using a lentivirus expression system, in which PCV2 replication was significantly enhanced. Meanwhile, in the Vimentin silenced 3D4/21 cells, PCV2 replication was significantly reduced. Furthermore, we found that PCV2 infection in 3D4/21 cells promoted Vimentin phosphorylation and rearranged Vimentin distribution in cells. By inhibition of CaMKII pathway, Vimentin phosphorylation was significantly suppressed and PCV2 replication was significantly inhibited. The results in this study indicated that in 3D4/21 cells, PCV2 promoted CaMKIIγ-medicated Vimentin phosphorylation and altered its cellular distributions to accomplish its own replication.

HRP-conjugated-nanobody-based cELISA for rapid and sensitive clinical detection of ASFV antibodies.

African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and wild boars and brings huge economic losses to world swine industry. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, and cost-effective detection and strict control and elimination strategies. Traditional serological testing methods are generally associated with high testing costs, complex operations, and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity, although the system is dependent on the immunization of Bactrian camels to obtain the specific VHH library of the target protein. The application of Nbs in the detection of ASFV antibodies has not yet been reported yet. Using a phage display technology, one Nb against the ASFV p54 protein that exhibited high specificity and affinity, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. There was no cross-reaction with healthy pig serum, porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and classical swine fever virus (CSFV) positive sera. The optimal cut-off value for the cELISA by ROC analysis was 52.5%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The results showed that the relative specificity of the cELISA was 98.97%, and the relative sensitivity of the cELISA was 93.3%, with the percent agreement between the two ELISA methods being 98.56%. In conclusion, a specific, sensitive, and repeatable cELISA was successfully developed based on the Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.Key points• We successfully screened a specific, high affinity nanobody against ASFV p54 protein.• We establish a method for continuous and stable expression of Nb-HRP fusion protein using a lentiviral packaging system.• We establish a nanobody cELISA detection method that can monitor an ASF infection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-11981-4.

Effect of miR-204 targeted regulation of DVL3 gene in silica-induced mouse lung epithelial cells

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells

Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.

Epithelial-to-mesenchymal transition promotes immune escape by inducing CD70 in non-small cell lung cancer

IntroductionEpithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. MethodsWe evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). ResultsWe uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. ConclusionOur results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

Lentiviral gene therapy prevents anti-human acid α-glucosidase antibody formation in murine Pompe disease

Enzyme replacement therapy (ERT) is the current standard treatment for Pompe disease, a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). ERT has shown to be lifesaving in patients with classic infantile Pompe disease. However, a major drawback is the development of neutralizing antibodies against ERT. Hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) provides a novel, potential lifelong therapy with a single intervention and may induce immune tolerance. Here, we investigated whether ERT can be safely applied as additional or alternative therapy following HSPC-LVGT in a murine model of Pompe disease. We found that lentiviral expression at subtherapeutic dose was sufficient to induce tolerance to the transgene product, as well as to subsequently administered ERT. Immune tolerance was established within 4–6 weeks after gene therapy. The mice tolerated ERT doses up to 100 mg/kg, allowing ERT to eliminate glycogen accumulation in cardiac and skeletal muscle and normalizing locomotor function. The presence of HSPC-derived cells expressing GAA in the thymus suggested the establishment of central immune tolerance. These findings demonstrate that lentiviral gene therapy in murine Pompe disease induced robust and long-term immune tolerance to GAA either expressed by a transgene or supplied as ERT.

Metformin-induced downregulation of c-Met is a determinant of sensitivity in MDA-MB-468 breast cancer cells

Metformin, the widely used anti-diabetic drug, is emerging as a promising anti-cancer agent. However, response variation among different tumors remains a significant challenge. Hence, identification of the factors that determine metformin sensitivity is of greatest significance for its clinical implementation. In this study, we showed that MDA-MB-468 cells were most sensitive among the five breast cancer cell lines tested. We found that metformin-induced inhibition of MDA-MB-468 cells was correlated with downregulation of c-Met at both protein and mRNA levels. To understand the functional significance of c-Met downregulation in metformin-mediated tumor inhibition, we established control and c-Met overexpressing sublines of MDA-MB-468 cells (468/C and 468/Met) using lentiviral expression system. We demonstrated that overexpression of c-Met significantly attenuated metformin induced inhibition of MDA-MB-468 cells. Metformin-induced inhibition of ALDH1+ cells, which are enriched with cancer stem cells, was also abrogated in 468/Met cells as compared to 468/C cells. Signal transduction analysis of the paired cell lines indicated that c-Met-induced activation of STAT3 and AKT1, and upregulation of Gab1 are related to c-Met-modulated metformin responsiveness. These findings highlight c-Met as a potential key regulator of metformin-mediated inhibition of proliferation and stemness of breast cancer cells, indicating that c-Met overexpression may be a critical factor contributing to metformin resistance. The data also suggest that combination of metformin with c-Met inhibitors could be a useful strategy to improve metformin-mediated anti-cancer efficacies in breast cancer treatment.

Exosomal Dvl3 promoted the aggressive phenotypic transformation of RA-FLS via wnt pathway

Objective This study was performed to explore the function and mechanism of Dvl3 in RA-FLS by exosome intervention. Methods The expression pattern of Dvl3 was examined by IHC, WB, and qPCR. Modified exosomes obtained from culturing supernatant of RA-FLS infected with Dvl3 over expression (OE) lentivirus were administrated to the target RA-FLS. The ability of survival, migration, and the production of inflammatory factor influenced by exosomal Dvl3 were detected by CKK8 kits, Tunel, migration test, qPCR, and enzyme-linked immunosorbent assay (ELISA) respectively; Flow cytometry analysis was conducted to explorer the inflammatory moderate role of exosomes on CD4+ T cells. The possible downstream pathways of Dvl3 were screened by qPCR and WB and verified by double luciferase reporter experiment. Results The expression level of Dvl3 was significantly increased in RA and CIA. Exosomes from the OE group could significantly promote cell proliferation activity, migration/invasion ability. The augment of TNF-α, IL-1β, IL-17, and IL-21 was observed in exosomal Dvl3-OE group. Th1 and Th17 cells polarisation and cytokines related were both enhanced by Exosomal Dvl3. Over expression of Dvl3 was accompanied by the significant increase of β-catenin and RhoA activities. Conclusion This study discovered the high expression of Dvl3 of exosomes derived from RA patients which may possessed the ability to promote phenotypic transformation of RA-FLS through Wnt pathway.

Targeting Inhibition of Notch1 Signaling Pathway on the Study of Human Gastric Cancer Stem Cells with Chemosensitization.

Background Gastric cancer is the second most frequent cause of cancer death worldwide, although much geographical variation in incidence exists. Prevention and personalized treatment are regarded as the best options to reduce gastric cancer mortality rates (Hartgrink et al., 2009). Numerous studies have suggested that Notch1 and its ligands are overexpressed in gastric cancer, and its knockdown can inhibit the proliferation and survival of gastric cancer cells. Objective To investigate the effect of Notch1 on the stemness and drug sensitivity of human gastric cancer SGC-7901 cells. Methods Highly expressed Notch1 intracellular domain (NICD1) and Notch1-shRNA lentiviral expression vector were used to infect human gastric cancer SGC-7901 cells cultured in vitro, and western blot and immunofluorescence staining were used to identify highly expressed NICD and Notch1 silenced cells. The percentage of CD133+ cells was analyzed by flow cytometry, the expression of nestin and CFAP by immunofluorescence staining, the formation rate of tumor cell spheres and the tumorigenicity of SCID mice in vivo, and the regulation of cell stemness by Notch1. The sensitivity of each group of cells to the chemotherapeutic drugs teniposide (VM-26) and carmustine (BCNU) was also detected by the MTT method. Results The stemness phenotype of tumor cells with the increased NICD expression was enhanced, such as an increased proportion of CD133+ cells, enhanced nestin expression, decreased GFAP expression, increased tumor cell sphere formation rate and tumorigenic rate of SCID mice implantation, and decreased sensitivity to VM-26 and BCNU. In contrast, the stemness phenotype of tumor cells with downregulated Notch1 gene expression was significantly suppressed, while the sensitivity to VM-26 and BCNU was increased. Conclusion High Notch1 expression increased the stemness of SGC-7901 cells and decreased the sensitivity of SGC-7901 cells to chemotherapeutic drugs.

Construction and identification of a HEK293 cell line with stable TrxR1 overexpression

To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs. TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay. TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005). We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.

Correction of a chronic pulmonary disease through lentiviral vector-mediated protein expression

We developed a novel lentiviral vector, pseudotyped with the F and HN proteins from Sendai virus (rSIV.F/HN), that produces long-lasting, high-efficiency transduction of the respiratory epithelium. Here we addressed whether this platform technology can secrete sufficient levels of a therapeutic protein into the lungs to ameliorate a fatal pulmonary disease as an example of its translational capability. Pulmonary alveolar proteinosis (PAP) results from alveolar granulocyte-macrophage colony-stimulating factor (GM-CSF) insufficiency, resulting in abnormal surfactant homeostasis and consequent ventilatory problems. Lungs of GM-CSF knockout mice were transduced with a single dose of rSIV.F/HN-expressing murine GM-CSF (mGM-CSF; 1e5–92e7 transduction units [TU]/mouse); mGM-CSF expression was dose related and persisted for at least 11 months. PAP disease biomarkers were rapidly and persistently corrected, but we noted a narrow toxicity/efficacy window. rSIV.F/HN may be a useful platform technology to deliver therapeutic proteins for lung diseases requiring long-lasting and stable expression of secreted proteins.

MiR-203, fine-tunning neuroinflammation by juggling different components of NF\u2010\u03baB signaling

BackgroundmiR-203 was first indicated in maintaining skin homeostasis and innate immunity. Aberrant expression of miR-203 was found associated with pathological progressions of immune disorders, cancers, as well as neurodegenerations. Recently, increasing data on miR-203 in regulating neuroinflammation and neuronal apoptosis has raised extensive concern about the biological function of this microRNA.MethodsMouse model with ectopic miR-203 expression in the hippocampus was constructed by stereotactic injection of lentiviral expression vector of pre-miR-203. Association of miR-203 and mRNA of Akirin2, as well as the competition for miR-203 targeting between Akirin2 3ʹUTR and another recently characterized miR-203 target, 14-3-3θ, was verified using Dual-Luciferase Reporter Gene Assay and western blot. Microglia activation and pro-inflammatory cytokines expression in the hippocampus of mice overexpressing miR-203 was evaluated using immunohistochemistry analysis and western blot. Neuronal cell death was monitored using anti-caspase 8 in immunohistochemistry as well as TUNEL assay. Cognition of mice was assessed with a behavior test battery consisting of nesting behavior test, Barnes maze and fear conditioning test.ResultsAkirin2, an activator of NF‐κB signaling, was identified as a direct target of miR-203. By also targeting 14-3-3θ, a negative regulator of NF‐κB signaling, miR-203 displayed an overall pro-inflammatory role both in vitro and in vivo. Promoted nuclear translocation of NF‐κB and increased expression of proinflammatory cytokines were observed in cultured BV2 cells transfected with miR-203 mimics. Microglia activation and upregulation of NF‐κB, IL-1β and IL-6 were observed in mouse hippocampus with overexpression of miR-203. In addition, promoted neuronal cell death in the hippocampus and impaired neuronal activities resulted in cognitive dysfunction of mice with ectopic miR-203 expression in the hippocampus.ConclusionA pro-inflammatory and neurodisruptive role of miR-203 was addressed based on our data in this study. Given the identification of Akirin2 as a direct target of miR-203 and the competition with 14-3-3θ for miR-203 targeting, together with the findings of other signaling molecules in NF‐κB pathway as targets of miR-203, we proposed that miR-203 was a master modulator, fine-tunning neuroinflammation by juggling different components of NF‐κB signaling.