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Background and purpose: Polyethylenimine is a cationic polymer able to neutralise negative DNA charges and to condense large genes which makes it suitable for gene delivery in human cells. Because of its low cost, simplicity of use and moderate toxicity, there is still room for broader usage and experimental adjustments, especially in cell lines that are difficult to transfect. In the presented research, we used polyethylenimine for the delivery of plasmid DNA into triple-negative breast cancer cell line SUM159 and breast cancer stem cell model HMLE-Twist. Material and methods: Cultured cells were transfected with GFPexpressing plasmid using both polyethylenimine and Lipofectamine. Transfection efficiency was determined by flow cytometry measurements of the intensity of the green fluorescence, while viability was determined by measuring intensity of the red fluorescence after propidium iodide staining. Results: In SUM159 and HMLE-Twist cells we obtained transfection efficiency between 30-40% using polyethylenimine, while cytotoxicity was generally low to moderate. Polyethylenimine caused 10% of cell death in SUM159 and 20% in HMLE-Twist. Transfection efficiency of polyethylenimine was comparable and even higher than the efficiency of the Lipofectamine in both SUM159 and HMLE-Twist, but not significantly different. In mammary epithelia (control HMLE) we obtained only 20% transfection efficiency using both carriers. Conclusions: We demonstrated for the first time that polyethylenimine represents a suitable nanocarrier for gene delivery into breast cancer stem cell model. We successfully transfected both breast cancer stem cell model HMLE-Twist and triple-negative breast cancer line SUM159. Since polyethylenimine is inexpensive and easy to use, we recommend it for further exploitations of these cell lines in triple- negative breast cancer research.

Nucleic acid nanoparticles (NANPs) require a carrier to allow for their intracellular delivery to immune cells. Cytokine production, specifically type I and III interferons, allows for reliable monitoring of the carrier effect on NANP immunostimulation. Recent studies have shown that changes in the delivery platform (e.g., lipid-based carriers vs. dendrimers) can alter NANPs’ immunorecognition and downstream cytokine production in various immune cell populations. Herein, we used flow cytometry and measured cytokine induction to show how compositional variations in commercially available lipofectamine carriers impact the immunostimulatory properties of NANPs with different architectural characteristics.

To assess in vitro and in vivo tracking of iron oxide labeled stem cells transfected by lipofectamine using magnetic resonance imaging (MRI), rat dental pulp stem cells (DPSCs) were characterized, labeled with iron oxide nanoparticles, and then transfected with lipofectamine to facilitate the internalization of these nanoparticles. Cell proliferation, viability, differentiation, and apoptosis were investigated. Prussian blue staining and MRI were used to trace transfected labeled cells. DPSCs were a morphologically spindle shape, adherent to culture plates, and positive for adipogenic and osteogenic inductions. They expressed CD73 and CD90 markers and lacked CD34 and CD45. Iron oxide labeling and transfection with lipofectamine in DPSCs had no toxic impact on viability, proliferation, and differentiation, and did not induce any apoptosis. In vitro and in vivo internalization of iron oxide nanoparticles within DPSCs were confirmed by Prussian blue staining and MRI tracking. Prussian blue staining and MRI tracking in the absence of any toxic effects on cell viability, proliferation, differentiation, and apoptosis were safe and accurate to track DPSCs labeled with iron oxide and transfected with lipofectamine. MRI can be a useful imaging modality when treatment outcome is targeted.

Human mesenchymal stem cells (hMSCs) are primary cells with high clinical relevance that could be enhanced through genetic modification. However, gene delivery, particularly through nonviral routes, is inefficient. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological “priming” of hMSCs with clinically approved drugs can increase transfection in hMSCs by modulating transfection-induced cytotoxicity. However, even with priming, hMSC transfection remains inefficient for clinical applications. This work takes a complementary approach to addressing the challenges of transfecting hMSCs by systematically investigating key transfection parameters for their effect on transgene expression. Specifically, we investigated two promoters (cytomegalovirus [CMV] and elongation factor 1 alpha), four DNA vectors (plasmid, plasmid with no F1 origin, minicircle, and mini-intronic plasmid), two cationic carriers (Lipofectamine 3000 and Turbofect), and four donors of hMSCs from two tissues (adipose and bone marrow) for efficient hMSC transfection. Following systematic comparison of each variable, we identified adipose-derived hMSCs transfected with mini-intronic plasmids containing the CMV promoter delivered using Lipofectamine 3000 as the parameters that produced the highest transfection levels. The data presented in this work can guide the development of other hMSC transfection systems with the goal of producing clinically relevant, genetically modified hMSCs., Graphical abstract, Pannier and colleagues report a systematic comparison of key aspects of nonviral gene delivery systems as they pertain to human mesenchymal stem cells (hMSCs). They identify transfection parameters that can significantly modulate hMSC transfection and suggest that these parameters may need to be tuned in an application- and patient-specific manner.

Cryopreservation of spermatogonial stem cells (SSCs) is a useful method for fertility preservation in preadolescent children suffering from cancer. However, SSCs may become damaged during cryopreservation due to the generation of reactive oxygen species (ROS). For this reason, various antioxidant agents have been used to protect SSCs from cryopreservation-induced damages. Recently, it has been reported that miR-30a-5p has antiapoptotic and antioxidant activity. The aim of this study was to assess the antiapoptotic and antioxidant effects of miR-30a-5p mimics in frozen-thawed SSCs. To this end, SSCs were isolated from male BALB/C mice (3-6 days old) and cultivated for 14 days. After the detection of optimum concentration, a miR-30a-5p mimic or miR-30a-5p inhibitor with Lipofectamine was transfected into SSCs and, finally, the cell groups were frozen for 1 week. After thawing, different properties, including cell viability (using MTT), colonization of SSCs (number and diameter of colonies), ROS generation (using DCFH-DA assay), levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and gene expression of Bcl-2 and BAXBax (using quantitative real-time PCR), were investigated. The transfection of SSCs with miR-30a-5p mimics before the freezing-thawing process significantly increased the viability, number, and diameter of SSCs colonies. Also, the miR-30a-5p mimic decreased the levels of ROS production and MDA, but it increased the SOD levels. Moreover, the miR-30a-5p mimic decreased BAX and increased Bcl-2 expression in frozen-thawed SSCs. The transfection of SSCs with the miR-30a-5p mimic can increase cell viability and antioxidant defense, and it can decrease apoptosis during the freezing-thawing process. If SSC is able to produce spermatozoa after the transfection of miR-30a-5p and the freezing-thawing process, it can be suggested as a promising strategy for the cryopreservation of SSCs in prepubertal boys suffering from cancer.

Safe sample transport is of great importance for infectious diseases diagnostics. Various treatments and buffers are used to inactivate pathogens in diagnostic samples. At the same time, adequate sample preservation, particularly of nucleic acids, is essential to allow an accurate laboratory diagnosis. For viruses with single-stranded RNA genomes of positive polarity, such as foot-and-mouth disease virus (FMDV), however, naked full-length viral RNA can itself be infectious. In order to assess the risk of infection from inactivated FMDV samples, two animal experiments were performed. In the first trial, six cattle were injected with FMDV RNA (isolate A22/IRQ/24/64) into the tongue epithelium. All animals developed clinical disease within two days and FMDV was reisolated from serum and saliva samples. In the second trial, another group of six cattle was exposed to FMDV RNA by instilling it on the tongue and spraying it into the nose. The animals were observed for 10 days after exposure. All animals remained clinically unremarkable and virus isolation as well as FMDV genome detection in serum and saliva were negative. No transfection reagent was used for any of the animal inoculations. In conclusion, cattle can be infected by injection with naked FMDV RNA, but not by non-invasive exposure to the RNA. Inactivated FMDV samples that contain full-length viral RNA carry only a negligible risk of infecting animals.

Background and aims: Human interferon beta-1a (hIFNβ-1a) is a 22.5-kDa glycoprotein used to treat diseases such as multiple sclerosis (MS). Because of appropriate post-translation modifications, protein isolation, and lack of toxicity in Chinese hamster ovary (CHO) cells, we cloned hIFNβ-1a encoding sequence into these cells by recombinant DNA technology to achieve stable expression of this recombinant protein. Methods: The hIFNβ-1a encoding sequence was designed based on the CHO cells’ codon usage and the Gene Bank data, and then syntactically constructed in the pUC57 vector. After confirmation, the synthesized sequence was cloned into the pcDNA3.1 expression vector by using EcoRI and XhoI sites via Escherichia coli DH5α competent cells. Then, the recombinant vector pcDNA-hHIFNβ1a was linearized by BglII and transfected into the CHO cells using lipofectamine. The transfected cells were proliferated and screened by gentamicin. Certain concentrations of zinc sulfate, DMSO, and glycerol were used to enhance protein expression. Finally, the recombinant protein expression was qualitatively evaluated using different techniques. Results: The hIFNβ1a integrity was confirmed by DNA sequencing and specific software. The construction and sub-cloning of hIFNβ1a-pcDNA3.1 in E. coli were confirmed by colony-PCR with specific primers and restriction enzyme mapping. The screening of transfected CHO cells was performed using gentamicin. The protein expression was confirmed by RT-PCR, MTT assay, SDS-PAGE, and Western blot. Comparison of the optimized and control samples demonstrated that chemical treatment enhanced the protein expression. Conclusion: We achieved the stable clones of CHO cells expressing the active form of human interferon beta.

The present study was carried out to silence the transcription factor genes ZCT1, ZCT2 and ZCT3 via lipofectamine based antisense LNA GapmeRs transfection into the protoplasts of established photomixotrophic cell suspensions. The photomixotrophic cell suspensions with a threshold of 0.5% sucrose were raised and established using two-tiered CO2 providing flasks kept under high light intensity. The photomixotrophic cell suspensions showed morphologically different thick-walled cells under scanning electron microscopic analysis in comparison to the simple thin-walled parenchymatous control cell suspensions. The LC–MS analysis registered the vindoline production (0.0004 ± 0.0001 mg/g dry wt.) in photomixotrophic cell suspensions which was found to be absent in control cell suspensions. The protoplasts were isolated from the photomixotrophic cell suspensions and subjected to antisense LNA GapmeRs silencing. Three lines, viz. Z1A, Z2C and Z3G were obtained where complete silencing of ZCT1, ZCT2 and ZCT3 genes, respectively, was observed. The Z3G line was found to show maximum production of vindoline (0.038 ± 0.001 mg/g dry wt.), catharanthine (0.165 ± 0.008 mg/g dry wt.) and vinblastine (0.0036 ± 0.0003 mg/g dry wt.). This was supported by the multifold increment in the gene expression of TDC, SLS, STR, SGD, d4h, dat, CrT16H and Crprx. The present work indicates the master regulation of ZCT3 knockdown among all three ZCTs transcription factors in C. roseus to enhance the terpenoid indole alkaloids production. The successful silencing of transcription repressor genes has been achieved in C. roseus plant system by using photomixotrophic cell cultures through GapmeR based silencing. The present study is a step towards metabolic engineering of the TIAs pathway using protoplast transformation in C. roseus.

Objectives The aim of the current study was to identify the long noncoding RNAs (lncRNAs) ANRIL function and molecular pathways underlying hepatocellular carcinoma progression. Methods ANRIL knockdown with specific siRNA, and transfected into HepG2 cells according to the protocol of Lipofectamine 2000. Cell proliferation, apoptosis, migration and metastasis were assessed with MTT assay, flow cytometry and wound healing assay, respectively. Moreover, the expression level of ANRIL, apoptosis-related genes, and the Wnt pathway-associated genes were assessed by real time-PCR and Western blot assay. Key findings Knocking down of ANRIL led to alleviated cell growth and increased cell apoptosis of HepG2 cells through markedly increased expression levels of Bax and Bad. In contrast, dramatically diminished the expressions of anti-apoptotic factors including Bid and Bcl-2 in comparison to the scrambled control group (si-NC). Furthermore, ANRIL silencing resulted in an inactivated Wnt/β-catenin pathway by suppressing key genes associated with this pathway. Conclusions Taken together, these findings imply new insights into the regulatory network of the Wnt pathway through lncRNA ANRIL that indicate ANRIL may be a therapeutic factor potential for hepatocellular carcinoma.

Hepatitis B (HB) is a viral infectious disease that seriously endangers human health, and since there are no radical drugs to counter this, effective and safe therapies urgently need to be developed. HB virus (HBV) mainly infects hepatocytes (HCs), while the drugs are easily phagocytosed by Kupffer cells (KCs). In this study, the glutathione concentration difference between HCs and KCs was examined and utilized in an ideal drug-release strategy. Here, galactosylated chitosan-oligosaccharide-SS-octadecylamine (Gal-CSSO) was prepared to accurately deliver 10-23 DNAzyme DrzBC (blocking HBeAg expression) or DrzBS (blocking HBsAg expression) in targeted HB therapy. In vitro Gal-CSSO systems exhibited low cytotoxicity, endosomal escape, and glutathione responsiveness. The HBeAg and HBsAg secretion of HepG2.2.15 was significantly decreased by Gal-CSSO systems, and the maximum inhibition rates were 1.82-fold and 2.38-fold greater than those of commercial Lipofectamine 2000 (Lipo2000) systems. In vivo Gal-CSSO systems exhibited HC targeting and HC microenvironmental responsiveness without noticeable hepatotoxicity or systemic toxicity. The HBeAg and HBsAg titers of the HBV-infected mice were evidently decreased by Gal-CSSO systems, and the inhibition rates were 1.52-fold and 1.22-fold greater than those of Lipo2000 systems. This study presents a kind of glycolipid-like polymer micelles that promise efficient and safe gene therapy of HB., Graphical abstract, Wu, Hu, and colleagues developed a kind of glycolipid-like polymer micelles to accurately deliver DNAzyme in targeted hepatitis B therapy to block viral gene expression. This drug delivery system exhibited hepatocyte targeting and microenvironmentally responsive cargo release without hepatotoxicity or systemic toxicity, suggesting a potential gene therapy for hepatitis B.

BACKGROUND: Long non-coding RNA testis-specific transcript, Y-linked 15 (TTTY15) is oncogenic in prostate cancer, however its expression and function in colorectal cancer remain largely unknown. METHODS: Paired colorectal cancer samples/normal tissues were collected, and the expression levels of TTTY15, miR-29a-3p and disheveled segment polarity protein 3 (DVL3) were examined by quantitative real-time polymerase chain reaction (qRT-PCR); TTTY15 shRNA and overexpression plasmids were transfected into HT29 and HCT-116 cell lines using lipofectamine reagent, respectively; the proliferation and colony formation were detected by CCK-8 assay and plate colony formation assay; qRT-PCR and Western blot were used to analyze the changes of miR-29a-3p and DVL3; dual-luciferase reporter gene assay was used to determine the regulatory relationships between miR-29a-3p and TTTY15, miR-29a-3p and DVL3. RESULTS: TTTY15 was significantly up-regulated in cancerous tissues of colorectal cancer samples, positively correlated with the expression of DVL3, while negatively correlated with the expression of miR-29a-3p. After TTTY15 shRNAs were transfected into colorectal cancer cells, the proliferation and metastasis of cancer cells were significantly inhibited, while TTTY15 overexpression had opposite biological effects. TTTY15 shRNA could reduce the expression of DVL3 on both mRNA and protein levels, and the luciferase activity of TTTY15 sequence was also inhibited by miR-29a-3p. DVL3 was also validated as a target gene of miR-29a-3p, and it could be repressed by miR-29a-3p mimics or TTTY15 shRNA. CONCLUSION: TTTY15 is abnormally upregulated in colorectal cancer tissues, and it can modulate the proliferation and metastasis of colorectal cancer cells. It functions as the ceRNA to regulate the expression of DVL3 by sponging miR-29a-3p.

AIM: We aimed to determine the possible correlation between voltage‑gated potassium channels and micro RNAs in breast cancer and metastatic breast cancer cells. METHOD: Kv1.3 and Kv10.1 channels were inhibited by specific siRNAs using a lipofectamine-based transfection in MCF-7 and MDA-MB-231 cells. After transfection, total RNA was isolated, and then miR-126 and miR-126* expressions were observed using RT-PCR. RESULTS: There was a negative correlation between Kv channels and miRNAs according to the characteristics of the breast cancer cells. The inhibition was observed not only in Kv1.3 but also in Kv10.1 in MCF-7 cells, and miR-126 and miR-126* expressions were downregulated compared to the control group (p < 0.001). The inhibition of these channels in MDA-MB-231 cells caused an upregulation of miR-126 and miR-126* expressions (p < 0.001). CONCLUSION: The miR-126 and miR-126* expressions differed according to benign and malign breast cancer cell lines. Furthermore, we found that miR-126/126* may interact with Kv1.3 and Kv10.1 voltage-gated potassium channels. Our study suggests and indicates the relationship between Kv channels and miRNAs in breast cancer cells (Tab. 1, Fig. 2, Ref. 51).

Summary Objective miR-122 stimulates proliferation of growth plate chondrocytes whereas miR-451 stimulates terminal differentiation and matrix turnover. Here, we examined the potential of these microRNA as regulators of articular chondrocytes using an in vitro model of osteoarthritis. Methods miR-122 and miR-451 presence in rat articular cartilage was assessed using the anterior cruciate ligament transection model of OA. In vitro testing used first passage rat articular chondrocytes (rArCs) that were transfected with lipofectamine (Lipo) and miR-122 or miR-451 for 24-h, then treated with 10 ng/mL IL-1β in order to mimic an osteoarthritic environment. Conditioned media were collected and MMP13, PGE2 and OA-related cytokines were measured. Matrix vesicles were collected from cell layer lysates using ultra-centrifugation. Cells were treated with miR-122 or miR-451 inhibitors to verify miR-specific effects. Results Both miR-122 and miR-451 were increased in the OA articular cartilage compared to healthy tissue; rArCs expressed both microRNAs in MVs. miR-122 prevented IL-1β-dependent increases in MMP-13 and PGE2, whereas miR-451 significantly increased the IL-1β effect. Multiplex data indicated that miR-122 reduced the stimulatory effect of IL-1β on IL-1α, IL-2, Il-4, IL-6, GM-CSF, MIP-1A, RANTES and VEGF. In contrast, IL-2, IL-4, IL-6, GM-CSF, and MIP-1A were increased by miR-451 while VEGF was decreased. Inhibiting miR-122 exacerbated the response to IL-1β indicating endogenous levels of miR-122 were present. There were no differences in MMP-13 or PGE2 with miR-451 Locked Nucleic Acid (LNA) inhibitor treatment. Conclusions Both miRs were elevated in OA in a rat bilateral anterior cruciate ligament transection (ACLT) model. miR-122 prevented, while miR-451 exacerbated the effects of IL-1β on rArCs.

Background Ultrasound-targeted microbubble destruction (UTMD) is a novel adjuvant tumor therapeutic method by enhancing exogenous gene transfection to target tissues. This study aims to investigate the role of microRNA-492 (miR-492) in non-small cell lung cancer (NSCLC) and further analyze the effects of UTMD-mediated miR-492 inhibitor on tumorigenesis. Methods The expression of miR-492 was detected by qRT-PCR. Co-transfection of microbubbles and miR-492 inhibitor with Lipofectamine 3000 was performed to achieve UTMD-mediated miR-492 inhibition in NSCLC cells. CCK-8 and Transwell assay were used to determine NSCLC cell proliferation, and the migration and invasion. Result High expression of miR-492 was associated with poor prognosis in NSCLC patients. miR-492 inhibitor suppressed tumor cell proliferation, migration and invasion, and UTMD not only increased the transfection efficiency of miR-492 inhibitor, but also enhance the inhibitory effects on cell biological behaviors. Conclusion The results showed that the expression level of miR-492 was up-regulated in NSCLC tissue samples and cells. Silencing of miR-492 inhibited NSCLC cell proliferation, migration and invasion, and UTMD-mediated miR-492 inhibitor could promote more significant inhibition, which indicated that UTMD-mediated miR-492 inhibitor might provide a novel strategy for the treatment of NSCLC.KEY MESSAGESmiR-492 inhibitor inhibited cell proliferation, migration and invasion.UTMD-mediated miR-492 inhibitor can promote more significant inhibition.UTMD-mediated miR-492 inhibitor provide a new strategy for NSCLC.

MicroRNA (miR)-518-3p has been shown to function as a tumor suppressor. This study was conducted to investigate the effects of miR-518-3p in colorectal cancer (CRC). The miR-518-3p mimic, mimic negative control (NC), miR-518-3p inhibitor, inhibitor-NC, ShRNA-TRIP4, and ShRNA-NC vectors were transfected into SW480 cells using Lipofectamine 2000. Cell viability was detected using CCK-8. Colony formation, cell invasiveness, and cell migration were assessed by plate colony formation, Transwell assays, and wound healing assays, respectively. Relative mRNA and protein levels were detected using RT–qPCR and Western blot, respectively. The target gene thyroid hormone receptor interactor 4 (TRIP4) of miR-518-3p was identified and further verified using dual-luciferase reporter assay. Compared with normal tissues, levels of miR-518-3p were decreased and TRIP4 was significantly increased in the tissues from patients with CRC. Following transfection with a miR-518-3p mimic or ShRNA-TRIP4, cell viability decreased in a time-dependent manner, and colony formation rate, wound closure rate, and the number of invasive cells were much lower for the transfected cells than in the corresponding NC and control groups. miR-518-3p overexpression or silencing of TRIP4 significantly down-regulated the expression of MMP-2 and MMP-9. Knockdown of miR-518-3p had the opposite effects, and TRIP4 was identified as a target of miR-518-3p. The inhibitory effects of miR-518-3p on the progressions of CRC are associated with TRIP4.

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Introduction A disintegrin and metallopeptidase with thrombospondin motifs (ADAMTSs), whose expression is dysregulated in various cancers, is implicated in cancer development. Herein, we aimed to investigate the functional role of ADAMTS8 in breast cancer (BC) and explore the underlying mechanisms. Methods The protein expression of ADAMTS8 in BC cell lines and tumor tissues from BC patients was quantified by Western blot. ADAMTS8 overexpression was induced by transfection with pEZ-M90-ADAMTS8 plasmid using lipofectamine 2000. To generate ADAMTS8 stable knockdown cells, MDA-MB-231 cells were transfected with psi-H1-ADAMTS8siRNA plasmids. Cell counting kit-8 (CCK-8) assay, wound-healing assay, transwell assay and flow cytometry assay were employed to analyze the effects of ADAMTS8 on the proliferation, migration, invasion and apoptosis of BC cells. Chemosensitivity also was assessed using CCK-8 assay. The expressions of β-catenin, MMP-7 and c-Myc were measured by Western blot. Results Our results showed that ADAMTS8 expression was significantly lower in BC tissues than that in adjacent non-tumor tissues. Overexpression of ADAMTS8 in MDA-MB-453 cells could inhibit the cell proliferation, migration and invasion and promote apoptosis. ADAMTS8 knockdown displayed the reverse effect in MDA-MB-231 cells. Consistently, in vivo data showed that ADAMTS8 overexpression led to a reduction in tumor growth. In addition, chemosensitivity testing in MDA-MB-453 cells transfected with pEZ-M90-ADAMTS8 plasmid indicated that cisplatin inhibited cell growth dramatically. Furthermore, attenuated β-catenin, MMP-7 and c-Myc level was detected after ADAMTS8 overexpression. Conclusion These results indicate that increased ADAMTS8 expression could modify the progression of BC by inhibiting cell proliferation and invasion while promoting the apoptosis of BC cells. Thus, ADAMTS8 represents a potential therapeutic target for BC therapy.

Background & Objective: Acute lymphoblastic leukemia (ALL) is a malignant disease that arises from various mutations in B or T-lymphoid progenitors. MicroRNAs (miRNAs) regulate gene expression by binding to the 3' untranslated region of protein-coding genes. Dysregulation of miRNA expression may result in the development of cancerous phenotypes. Therefore, for the first time in this field, the present study aims to investigate the effect of overexpression of miR-506 in Jurkat (acute T cell leukemia) cell line. Methods: In this study, Jurkat cell lines were cultured in RPMI-1640 medium. Next, miR-506 was transfected with concentrations of 50 and 100 nM with Lipofectamine 2000. The accuracy of the transfection was confirmed by the transfection of siRNA conjugated with FITC. 48 h after transfection, the cells were prepared for other tests (flow cytometry, MTT assay, and RNA extraction). The expression level of miR-506 in the cells was analyzed using the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Finally, SPSS 21 software was used for the data analysis. Results: According to our results, the viability of cells in concentrations of 50 and 100 nM was significantly higher than the control group. By overexpression of miR-506, the expressions of pro-apoptotic genes (p53, p21) and anti-apoptotic gene B-cell lymphoma-2 (BCL-2) are decreased and increased, respectively. Conclusion: This study showed that miR-506 may function as an oncogenic miRNA in the T- ALL cell line. In conclusion, overexpression of miR-506 leads to an increase in viable cancer cells.

Purpose The aim of this study was to study the roles and potential mechanism of LINC00520 in the progression of lung cancer. Methods The expression of LINC00520 and miR-3175 in lung cancer tissues and cells was detected by qRT-PCR. The relationship between LINC00520 level and disease stage was also calculated. Kaplan-Meier survival curve was drawn to observe the survival difference between high and low expression patients. Lipofectamine 2000 was used to transfect siLINC00520, miR-3175 inhibitor and their controls in lung cancer cells. CCK8 and colony formation assay were processed for cell proliferation. Transwell assay was undertaken for migration and invasion of lung cancer cells. MiRDB predicts the combination of LINC00520 and miR-3175. Luciferase and RNA pulldown assay were applied to verify the binding site. Correlation analysis of miR-3175 and LINC00520 expression in lung cancer tissues was shown. Results LINC00520 was highly expressed in lung cancer tissues and cells. Patients at III+IV stage were always with higher LINC00520 level than patients at I+II stage. Patients with high expression of lncRNA LINC00520 have short survival time (hazard ratio=1.7). Knockdown of LINC00520 inhibited proliferation, invasion and migration of lung cancer cells. LINC00520 targeted and negatively regulated miR-3175 (r=-0.528; P

Purpose: In type 2 diabetes, palmitic acid could damage vessels and induce insulin resistance. This present in vitro study evaluates the possible role of Spatholobus suberectus (FSS) extract in diabetes.Methods: Human HUVECc cells were treated with palmitic acid, palmitic acid and Spatholobus suberectus extract. MALAT1 overexpression plasmid (pcDNA-MALAT1) and blank vector were transfected into the cells using lipofectamine 2000. RT-qPCR assay was used to evaluated the expression changes of lncRNA, VEGFR2 and VEGFA in the cells as well as Epithelial-Mesenchymal Transition (EMT) biomarkers and apoptosis. CCK-8 was used to detect cell viabilities of HUVECs. Expressions of proteins in VEGF signaling pathway were analyzed using Western Blot.Results: LncRNA MALAT1 had high expression in diabetes-like cells and suppressed proliferation and EMT but promoted apoptosis. The SS extract promoted proliferation and EMT and repressed apoptosis in diabetes-like HUVECs cells. The promotion of apoptosis by LncRNA MALAT1, inhibition of apoptosis and regulated functions of diabetes-like HUVECs cells by SS extract occurred via the VEGF signaling pathwayConclusion: SS extract might contribute to survival of cells by inhibiting MALAT1 via VEGF signaling pathway in vitro, suggesting FSF might be a potential therapeutic agent in the treatment of diabetes. Keywords: flavone of Spatholobus suberectus, diabetes, vascular endothelial cell, LncRNA MALAT1

Diabetic chronic wound, characterized by prolonged inflammation and impaired angiogenesis, has become one of the most serious challenges in clinic and pose a significant healthcare burden worldwide. Although a great variety of wound dressings have been developed, few of encouraged achievements were obtained so far. In this study, the gene-activated strategy was applied to enhance sustained expression of vascular endothelial growth factor (VEGF) and achieve better healing outcomes by regulating inflammation and promoting angiogenesis. The gene-activated bilayer dermal equivalents (Ga-BDEs), which has good biocompatibility, were fabricated by loading the nano-sized complexes of Lipofectamine 2000/plasmid DNA-encoding VEGF into a collagen-chitosan scaffold/silicone membrane bilayer dermal equivalent. The DNA complexes were released in a sustained manner and showed the effective transfection capacities to up-regulate the expression of VEGF in vitro. To overcome cutaneous contraction of rodents and mimic the wound healing mechanisms of the human, a reformative rat model of full-thickness diabetic chronic wound was adopted. Under the treatment of Ga-BDEs, speeding wound healing was observed, which is accompanied by the accelerated infiltration and phenotype shift of macrophages and enhanced angiogenesis in early and late healing phases, respectively. These proved that Ga-BDEs possess the functions of immunomodulation and pro-angiogenesis simultaneously. Subsequently, the better regeneration outcomes, including deposition of oriented collagen and fast reepithelialization, were achieved. All these results indicated that, being different from traditional pro-angiogenic concept, the up-regulated expression of VEGF by Ga-BDEs in a sustained manner shows versatile potentials for promoting the healing of diabetic chronic wounds., Graphical abstract Image 1, Highlights • Gene-activated strategy overcomes short half-life period of growth factors. • Releasing DNA complexes in sustained manner and effective transfection capacities. • Without cutaneous contraction, reformative full-thickness diabetes chronic wound. • VEGF versatile potentials: immunoregulation, pro-angiogenesis in different phases. • One intervenor covered two main pathogenic factors: inflammation and angiogenesis.

Background Long noncoding RNA taurine upregulated 1 (TUG1) has been reported to play an important role in human cancers. However, little is known about the role of TUG1 in drug resistance and its mechanism in tongue squamous cell carcinoma (TSCC). Methods Twenty-one cisplatin-sensitive or resistant TSCC patients were enrolled in this study. Cisplatin-resistant cells (SCC25/CDDP and CAL27/CDDP) were used for experiments in vitro. Transfection was performed using Lipofectamine 2000 transfection reagent. The levels of TUG1, microRNA-133b (miR-133b) and cysteine-X-cysteine chemokine receptor 4 (CXCR4) were measured by quantitative real-time polymerase chain reaction or western blot. The cisplatin resistance was investigated by cell viability, transwell invasion and apoptosis assays. The interactions among TUG1, miR-133b and CXCR4 were evaluated by luciferase reporter assay and RNA immunoprecipitation. Murine xenograft model was established using the stably transfected CAL27/CDDP cells. Results TUG1 expression was elevated in cisplatin-resistant TSCC tissues and cells compared with that in sensitive group and its knockdown inhibited cisplatin resistance to SCC25/CDDP and CAL27/CDDP cells. miR-133b was targeted via TUG1 and its overexpression suppressed cisplatin resistance. Moreover, CXCR4 was a target of miR-133b. CXCR4 silence repressed cisplatin resistance, which was reversed by miR-133b knockdown. The level of CXCR4 protein was decreased by inhibition of TUG1 and recuperated by miR-133b knockdown. Besides, interference of TUG1 attenuated tumor growth by regulating miR-133b and CXCR4 in vivo. Conclusion Downregulation of TUG1 impeded cisplatin resistance in TSCC-resistant cells by mediating miR-133b and CXCR4, indicating TUG1 as a promising target for TSCC chemotherapy.

Objective Recent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications. Materials and methods CMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 μL/g). Results Western blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours. Conclusion The human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.

Conventional electroporation approaches show limitations in the delivery of macromolecules in vitro and in vivo. These limitations include low efficiency, noticeable cell damage and nonuniform delivery of cells. Here, we present a simple 3D electroporation platform that enables massively parallel single-cell manipulation and the intracellular delivery of macromolecules and small molecules. A pyramid pit micropore array chip was fabricated based on a silicon wet-etching method. A controllable vacuum system was adopted to trap a single cell on each micropore. Using this chip, safe single-cell electroporation was performed at low voltage. Cargoes of various sizes ranging from oligonucleotides (molecular beacons, 22 bp) to plasmid DNA (CRISPR-Cas9 expression vectors, >9 kb) were delivered into targeted cells with a significantly higher transfection efficiency than that of multiple benchmark methods (e.g., commercial electroporation devices and Lipofectamine). The delivered dose of the chemotherapeutic drug could be controlled by adjusting the applied voltage. By using CRISPR-Cas9 transfection with this system, the p62 gene and CXCR7 gene were knocked out in tumor cells, which effectively inhibited their cellular activity. Overall, this vacuum-assisted micropore array platform provides a simple, efficient, high-throughput intracellular delivery method that may facilitate on-chip cell manipulation, intracellular investigation and cancer therapy. Applying an electric field to individually trapped cells allows for the efficient and safe delivery of drugs or genes, and the characterization and manipulation of cellular activity. Electroporation is a technique that increases the permeability of a cell’s membrane, to allow the infiltration of drugs or genes. In practice, traditional electroporation has issues with efficiency and cellular damage. The scientists of Lingqian Chang (Beihang University) and Mo Li (Peking University Third Hospital) led a team to develop an on-chip “3D” system that uses a vacuum to trap cells in individual micropores. By applying an electric field, the team were able to deliver cargo of different sizes, from drugs to genes, into the cells with greater efficiency and safety than benchmark methods. The dose of chemotherapy drugs was controlled through altering the voltage applied. The team were able to inhibit tumor activity by delivering CRISPR-Cas9 gene editing plasmid with their system. This “3D” system was showed the potential for on-chip manipulation, in situ intracellular interrogation and cancer therapy.

Chimeric antigen receptor (CAR) T cell therapy relies on the ex vivo manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for ex vivo mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14—4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14—4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.

In this study, we developed coordinative amphiphiles for use as novel non-viral DNA vectors. As a modification of a conventional cationic lipid structure, we replaced the cationic head with a zinc(II)-1,4,7,10-tetraazacyclododecane (Zn-cyclen) complex as a phosphate-directing group, and used biocompatible skeletons (α-tocopherol or cholesterol) as hydrophobic tails. The structure-activity relationship (SAR) was systematically investigated to study the effect of Zn-coordination on the gene transfection between cyclen-based traditional head-tail lipids and Zn(II)-cyclen coordinative amphiphiles. The results reveal that both Zn-free lipids and Zn-containing amphiphiles could condense DNA into nano-sized particles with appropriate size and zeta-potentials. Agarose gel retardation assay and MTS-based cell viability assays demonstrated that the Zn(II)-cyclen complex exhibited slightly lower DNA binding ability and much lower cytotoxicity compared to liposome analogues, respectively. Most importantly, in vitro transfection studies showed that the coordination of zinc(II) to cyclen may dramatically increase the transfection efficiency of the conventional cationic lipid, and α-tocopherol-containing coordinative amphiphile Zn-Cyc-Toc gives the best transfection efficiency, which was enhanced 24.4 times after coordination and was 6.1 times higher than commercial transfection reagent lipofectamine 2000. Mechanism studies confirmed that the DNA complex formed from Zn-Cyc-Toc might induce higher cellular uptake and better endosomal escape ability than the lipoplexes formed from Zn-free lipid Cyc-Toc. This study not only demonstrates that these coordinative amphiphiles might be promising non-viral gene vectors, but also presents a novel strategy to enhance the gene transfection efficiency and biocompatibility of cyclen-based cationic lipids.

A fusion protein comprising an antibody and a cationic peptide, such as arginine-9 (R9), is a candidate molecule for efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and R9 with the Q-tag sequence LLQG at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 was synthesized at a maximum binding efficiency of 85.1%, without changing the binding affinity of the nanobody for the antigen. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced an approximately 58.6% reduction in the amount of target protein due to RNAi in mRNA compared to lipofectamine.

Background Producing an appropriate number of engineered cells is considered as one of the influential factors in the successful treatments with chimeric antigen receptor (CAR) T cells. To this aim, the transduction rate of the viral vectors can play a significant role. In addition, improving transduction rates can affect the success rate of this treatment due to hard-transduced T lymphocytes. Results In this study, activated T cells were transduced using different transduction methods such as spinoculation, retronectin, polybrene, spinoculation + retronectin, and spinoculation + polybrene after selecting the most efficient transfection method to produce recombinant viral particles containing MUC1 CAR. PEI and lipofectamine with the amount of 73.72 and 72.53%, respectively, showed the highest transfection rates with respect to calcium phosphate (14.13%) for producing lentiviral particles. However, the cytotoxicity of transfection methods was not significantly different. Based on the results, spinoculation + retronectin leads to the highest transduction rates of T cells (63.19 ± 4.45%) relative to spinoculation + polybrene (34.6 ± 4.44%), polybrene (10.23 ± 0.79%), retronectin (10.37 ± 1.85%), and spinoculation (21.11 ± 1.55%). Further, the polybrene (40.02%) and spinoculation + polybrene (48.83% ± 4.83) increased cytotoxicity significantly compared to other groups. Conclusion Improving transduction conditions such as using spinoculation with retronectin can ameliorate the production of CAR-T cells by increasing the rate of transduction, as well as the success rate of treatment.