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The functional influence of microRNA (miRNA) backbone selection remains unclear with respect to multiplexing miRNA-based short hairpin RNAs (shRNAmiRs), due to a lack of comparative studies. To this end, a pair of shRNAmiR tetramers were designed in the present study that targeted four genes with a shared miR30a backbone (homo-BB) or four miRNA backbones (hetero-BB). A PBLT+ 293A cell line overexpressing four targets was established, which permitted simultaneous dissection of individual gene knockdown. Multi-target inhibition was confirmed by a decrease in positive cell populations of the relative gene and mean fluorescence intensities, with almost comparable activities of homo- and hetero-BB tetramers. Of note, this multi-inhibition was sustained over a 1-month period, with no notable difference, particularly in the late-phased inhibitory effects between homo- and hetero-BB tetra-shRNA miRs. These preliminary data may indicate little influence of scaffold substitution in the functionalities of multiplexed shRNAmiRs and little recombination-depleted risk of repetitively adopting the same miRNA backbone in this artificial in vitro system. More comparative studies are further required to explore extended repertoires of scaffold-paralleled multi-shRNAmiRs in more physiologically relevant models. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is associated with poor immunogenicity and lymphocytic infiltration, and immunotherapy effective against PCa remains unavailable. Pyroptosis, a novel immunotherapeutic modality for cancer, promotes systemic immune responses leading to immunogenic cell death in solid tumors. This paper describes the preparation and analysis of PSMA scFv -EV N-GSDMD ; this genetically engineered recombinant extracellular vesicle (EV) expresses a single-chain variable antibody fragment (scFv) with high affinity for prostate-specific membrane antigen (PSMA) on their surfaces and is loaded with the N-terminal domain of gasdermin D (GSDMD). Both in vitro and in vivo, PSMA scFv -EV N-GSDMD effectively targeted PSMA-positive PCa cells and induced pyroptosis through the carrier properties of EVs and the specificity of PSMA scFv . In the 22RV1 and PSMA-transfected RM-1-inoculated PCa mouse models, PSMA scFv -EV N-GSDMD efficiently inhibited tumor growth and promoted tumor immune responses. In conclusion, PSMA scFv -EV N-GSDMD can convert the immunosuppressive "cold" tumor microenvironment of PCa into an immunogenic "hot" tumor microenvironment., Competing Interests: Declaration of competing interest The authors have declared that no competing interest exists., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective To evaluate the toxicology of targeting human epidermal growth factor receptor-2 chimeric antigen receptor T (HER2-CAR-T) cells and to provide a safety basis for the clinical evaluation of HER2-CAR-T cell therapy. Methods The recombinant lentiviral vector was used to generate HER2-CAR-T cells. Soft agar colony formation assay was used to observe the colony formation of HER2-CAR-T cells, and the colony formation rate was statistically analyzed. The HER2-CAR-T cell suspension was co-incubated with rabbit red blood cell suspension, and the hemolysis of red blood cells was evaluated by direct observation and microplate reader detection. The HER2-CAR-T cell preparation was injected into the ear vein of male New Zealand rabbits, and the stimulating effect of HER2-CAR-T cells on the blood vessels of the animals was observed by staining of tissue sections. The vesicular stomatitis virus envelope glycoprotein (VSV-G) gene of pMD 2.G vector was used as the target sequence, and the safety of the lentiviral vector was verified by real-time fluorescence quantitative PCR. The heart, liver, lung, and kidney of mice receiving HER2-CAR-T cell infusion were collected, and the lesions were observed by HE staining. Results The HER2-CAR-T cells were successfully prepared. These cells did not exhibit soft agar colony formation ability in vitro, and the HER2-CAR-T cell preparation did not cause hemolysis in New Zealand rabbit red blood cells. After the infusion of HER2-CAR-T cells into the ear vein of New Zealand rabbits, no obvious vascular stimulation response was found, and no specific amplification of VSV-G was detected. No obvious lesions were found in the heart, liver, lung and kidney tissues of the treatment group. Conclusion The prepared HER2-CAR-T cells have reliable safety.

RNF2, also known as RING1b or RING2, is identified as the catalytic subunit of polycomb repressive complex 1 (PRC1), which mediates the mono-ubiquitination of histone H2A. RNF2 has been proved to have oncogenic function in many kinds of cancers, but the function of RNF2 in prostate cancer (PCa) has not been evaluated. Here we show that PCa tissues showed higher RNF2 expression than the benign prostatic hyperplasia (BPH) tissues. Knockdown of RNF2 in PCa cells resulted in cell cycle arrest, increased apoptosis and inhibited cell proliferation, and the growth of RNF2 knockdown PCa xenografts were obviously inhibited in nude mice. Gene microarray analysis was performed and tumor suppressor gene TXNIP was found to be significantly increased in RNF2 knockdown cells. Simultaneously knockdown of RNF2 and TXNIP can partially rescue the arrested cell cycle, increased apoptosis and inhibited cell proliferation in RNF2 single knockdown cells. Furthermore, ChIP assay result showed that RNF2 enriched at the TXNIP promoter, and the enrichment of RNF2 and ubiquitination of H2A in TXNIP promoter was obviously inhibited in RNF2 knockdown cells. In conclusion, our results demonstrate that RNF2 functions as an oncogene in PCa and RNF2 may regulate the progression of PCa through the inhibition of TXNIP.

Objective: To investigate the effect of miR-365 on the proliferation and apoptosis in SOSP-9607 osteosarcoma cells., Methods: SOSP-9607 cells were transiently transfected with miR-365 mimic or miR-365 inhibitor which were artificially synthesized. The expression of miR-365 was detected by real-time PCR (qRT-PCR); the cell cycle and apoptosis was analyzed by flow cytometry; the cell proliferation was observed by MTT assay; and the level of KRAS was determined by qRT-PCR and Western blotting., Results: miR-365 mimic up-regulated the expression level of miR-365 in SOSP-9607 osteosarcoma cells. After miR-365 mimic transfection, the proliferation of SOSP-9607 cells was inhibited; the cell cycle was arrested in G1 phase; the apoptosis rate increased and the expression of KRAS was reduced in miR-365 mimic transfected cells. On the contrary, when miR-365 inhibitor was transfected into SOSP-9607 cells, the expression level of miR-365 was significantly reduced along with promoted cell proliferation, suppressed cell apoptosis and increased KRAS expression., Conclusion: miR-365 could inhibit the proliferation and promote the apoptosis in SOSP-9607 osteosarcoma cells probably by mediating the expression of KRAS.

Objective: To domesticate adherent Chinese hamster ovary (CHO) cells into suspension CHO cells (CHO-S) cultured in serum-free medium,and evaluate the application of the CHO-S cells in antibody expression., Methods: Adherent CHO cells were domesticated into CHO-S cells through suspension culture and gradually decreasing the serum concentration, and eventually the cells were cultured in serum-free medium. Based on the anti-prostate-specific membrane antigen ( PSMA) single chain antibody fragment ( Sc Fv) gene sequence obtained from a phage display library, the genes of heavy and light chains were designed, synthesized and cloned into the pc DNA3. 1 vector,and the products were named pc DNA3. 1-HC and pc DNA3. 1-LC respectively. The plasmids were transiently transfected at the ratio of light and heavy chain 3 ∶ 1 into CHO-S cells using Free Style MAX transfection reagent, and the supernatants were harvested at day 7 after transfection. SDS-PAGE and Western blotting were used to detect the antibody expression,and flow cytometry was applied to evaluate its binding activity to PSMA positive cells., Results: The adherent CHO cells were successfully domesticated into CHO-S cells. Expression plasmids for anti-PSMA antibody heavy chain and light chain were successfully constructed,and anti-PSMA antibody could be secretively expressed in CHO-S cells. Flow cytometry showed that the expressed antibody could specifically bind to PSMA positive cells., Conclusion: CHO-S cel s were successful y domesticated from adherent CHO cells, and could be used for antibody expression. This study provided a useful tool for further antibody expression, purification and function study.

Objective: To investigate the expression level of protein tyrosine phosphatase non-receptor type 14 (PTPN14), and analyze the relationship between PTPN14 and clinical pathological features and prognosis of patients with cholangiocarcinoma., Methods: Expression of PTPN14 protein was detected by immunohistochemistry (IHC) in 57 cholangiocarcinoma tissues and corresponding adjacent normal tissues. The relationship between PTPN14 protein level and the clinical-pathological features of cholangiocarcinoma was analyzed using IBM SPSS 20.0 statistical software. The relationship between PTPN14 protein expression and 5-year overall survival of cholangiocarcinoma patients was investigated by survival curves., Results: IHC revealed that positive rates of PTPN14 protein were 49.1% and 75.4% in cholangiocarcinoma tissues and adjacent tissues, respectively. The expression of PTPN14 protein was significantly associated with TNM I, II, and differentiation degree of cholangiocarcinoma patients, but not significantly associated with age and gender of cholangiocarcinoma patients. The 5-year overall survival rate was higher in the PTPN14-positive patients than the PTPN14-negative ones., Conclusion: PTPN14 was down-regulated in cholangiocarcinoma, and negatively correlated with better clinical-pathological features and 5-year overall survival rate of cholangiocarcinoma.

Objective: To investigate the effect of RING finger protein 2 (RNF2) knockdown on the biological characteristics and radiosensitivity in glioma U87 cells., Methods: Plasmids containing shRNA targeting RNF2 were transfected into U87 cells. Real-time quantitative PCR (qRT-PCR) and Western blotting were respectively applied to detect the mRNA and protein level of RNF2. MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were measured by flow cytometry combined with annexin V-FITC/PI staining in the control and RNF2 knockdown cells. Apoptosis was also detected after X-ray radiation., Results: Both shRNAs efficiently inhibited RNF2 expression in U87 cells. Cell proliferation was obviously depressed in RNF2 knockdown cells. The percentage of cells decreased in S phase (shRNA-NC: 27.31 ± 1.35; shRNF2-1: 16.72 ± 2.90; shRNF2-3: 10.35 ± 1.33) and increased in G1 phase (shRNA-NC: 56.13 ± 1.80; shRNF2-1: 76.32 ± 3.11; shRNF2-3: 80.45 ± 2.83). More cell apoptosis was observed in RNF2 knockdown cells. After X ray radiation, the apoptosis rate was significantly raised in RNF2 knockdown cells (shRNA-NC: 20.88 ± 0.64; shRNF2-1: 39.69 ± 0.57; shRNF2-3: 47.82 ± 0.45)., Conclusion: Knockdown of RNF2 can inhibit cell proliferation, induce cell cycle arrest and promote apoptosis in U87 cells. RNF2 knockdown can obviously increase the sensitivity of U87 cells to X ray radiation.

Objective: To investigate the impact of microRNA-93 on the biological behaviors of A172 glioma cells by observing the changes of cell proliferation, cell cycle and apoptosis., Methods: Real-time quantitative PCR (qRT-PCR) was applied to detect the expression of microRNA-93 in 2 samples of human normal brain tissues, 10 samples of glioma tissues and 5 glioma cell lines. Artificially synthesized microRNA-93 mimic was transiently transfected into A172 glioma cells, and then the expression of microRNA-93 was tested by qRT-PCR. MTT assay was used to detect the cell proliferation of A172 glioma cells; apoptosis and cell cycle of A172 glioma cells were measured by flow cytometry., Results: MicroRNA-93 was over-expressed in glioma tissues and glioma cell lines as compared with normal samples. The transient transfection of microRNA-93 mimic into A172 glioma cells significantly increased the expression of microRNA-93 in A172 glioma cells, promoted cell proliferation, raised the cell proportion in S phase, reduced the cell proportion in G1 phase, and inhibited cell apoptosis., Conclusion: MicroRNA-93 was aberrantly over-expressed in glioma tissues and cell lines. Transient transfection of microRNA-93 mimic led to increased proliferation, G1-to-S cell cycle progression and reduced apoptosis in A172 glioma cells, indicating that micro-RNA-93 might be a new target for the diagnosis and treatment of glioma.

Objective: To explore the roles of vacuum sealing drainage (VSD) in controlling infection and promoting healing on the experimental pigs with blast injury in the abdomen and exposed internal organs., Methods: All animals with full-thickness abdominal wall defect were randomly divided into experimental group (VSD group) and control group (saline gauze group). Debridement was performed 6 hours after wounding. VSD devices (-125 mmHg) were imbedded on animals in the experimental group, while in the control group gauzes with saline solution were used to cover the wound and conventional treatment of dressing change was done. Specimens of muscle tissue in the wound were collected respectively from the two groups to make bacteria quantification 6 hours before the treatment and on the 1st, 3rd, 5th, and 7th day of treatment. Specimens of abdominal drainage fluid were collected respectively on the 1st, 3rd, 5th, and 7th day of treatment to detect inflammatory cytokines (TNF-α, IL-1, IL-6) using ELISA kit. Specimens of the skin and muscle tissues were collected respectively from the two groups on the 7th day to detect target genes (VEGF, bFGF, EGF, and MMP-9) using qRT-PCR., Results: The bacteria counts (CFU/g) in the VSD group on the 1st, 3rd, 5th, and 7th day of treatment were significantly less than those in the control group at the corresponding time points, and the differences between the two groups were statistically significant (P<0.01). There were no distinct differences between the two groups in the expressions of TNF-α, IL-1 and IL-6 in the abdominal drainage fluid of pig on the 1st day of treatment. The expressions of TNF-α, IL-1 and IL-6 on the 3rd, 5th, and 7th day of treatment in the VSD group were significantly lower than those in the control group at the corresponding time points (P<0.01). The expressions of VEGF, EGF and bFGF in the skin and soft tissues in the VSD group on the 7th day was higher than those in the control group (P<0.01), while the expression of MMP-9 showed no statistical significant difference between the two groups (P>0.05)., Conclusion: VSD can effectively control the amount of bacteria in the wound, and reduce the expressions of pro-inflammatory factors such as TNF-α, IL-1 and IL-6 in drainage fluid. VSD also promotes the expressions of growth factors in the wound.

Objective: To design and package shRNA expressing lentiviral particles targeting B7-H1, and evaluate their inhibitory effect on B7-H1 expression in U251 cells., Methods: Three shRNAs targeting B7-H1 was designed and the sense and antisense primers were produced by chemical synthesis. After annealing, they were linked into restriction enzyme digested pLKO.1 vector. Confirmed by DNA sequencing, the lentiviral particles were packaged and applied to infect U251 cells. qRT-PCR and Western blotting were used to detect the B7-H1 mRNA and protein levels respectively., Results: qRT-PCR and Western blotting showed that two of the three shRNAs effectively knocked-down B7-H1 expression in U251 cells., Conclusion: The packaged lentiviral particles can specifically inhibit B7-H1 expression, which will be helpful for further functional study on B7-H1.