Objective To establish a stable strain of H9c2 cardiomyocytes overexpressing Cx40 and preliminarily investigate the effect of lentiviral vector-mediated Cx40 protein overexpression on the proliferation of H9c2 cells and its related mechanisms. Methods The Cx40 gene fragment was cloned from H9c2 cells by PCR and linked with lentivirus vector pLVX-IRES-Puro to obtain the recombinant plasmid pLVX-Flag-Cx40. Recombinant lentiviral particles carrying Flag-Cx40 were obtained by cotransfection with packaging plasmids into HEK293T cells. A stable expression strain (H9c2-Flag-Cx40 cell) was screened from infected H9c2 cells by purinomycin. The expression of Cx40 protein was detected by Western blot analysis, and the effect of Cx40 on H9c2 cells proliferation was determined by CCK-8 assay; cell cycle changes were measured by flow cytometry; the expression of the cell cycle protein cyclin D1 was detected by qRT-PCR and Western blot analysis. Co-immunoprecipitation (Co-IP) immunoprecipitation and Western blot analysis were used to identify the binding of Cx40 and Yes associated protein (YAP) in H9c2 cells; cytoplasmic and cytosolic proteins were isolated to detect the effect of Cx40 on the localization of YAP using Western blot analysis. Results Sequencing results showed that the recombinant pLVX-Flag-Cx40 expression vector was successfully established. A stable transfected cell line containing recombinant Flag-Cx40 lentivirus (H9c2-Flag-Cx40 cell) was successfully constructed from H9c2 cells. Compared with the control group, overexpression of Cx40 significantly reduced the proliferation of H9c2 cells, arrested the cell cycle at G0/G1 and reduced cyclin D1 expression. A significant increase in YAP expression was observed in the cytoplasm of the H9c2-Flag-Cx40 stable cell line, while the expression in the nucleus was significantly reduced. Cx40 bound to YAP in the cytoplasm and prevented it from entering the nucleus to play the role of transcriptional coactivation. Conclusion Overexpression of Cx40 induces cell-cycle arrest at G0/G1 phase and inhibits the proliferation in H9c2 cells.
Rats ; Humans ; Animals ; Cyclin D1/genetics* ; Transfection ; Myocytes, Cardiac ; HEK293 Cells ; Cell Cycle Checkpoints/genetics* ; Cell Proliferation/genetics* ; Lentivirus/genetics* ; Genetic Vectors/genetics* ; Gap Junction alpha-5 Protein

Metabolic reprogramming is a common phenomenon in cancer, with aerobic glycolysis being one of its important characteristics. Hypoxia-inducible factor-1α (HIF1Α) is thought to play an important role in aerobic glycolysis. Meanwhile, naringin is a natural flavanone glycoside derived from grapefruits and many other citrus fruits. In this work, we identified glycolytic genes related to HIF1Α by analyzing the colon cancer database. The analysis of extracellular acidification rate and cell function verified the regulatory effects of HIF1Α overexpression on glycolysis, and the proliferation and migration of colon cancer cells. Moreover, naringin was used as an inhibitor of colon cancer cells to illustrate its effect on HIF1Α function. The results showed that the HIF1Α and enolase 2 (ENO2) levels in colon cancer tissues were highly correlated, and their high expression indicated a poor prognosis for colon cancer patients. Mechanistically, HIF1Α directly binds to the DNA promoter region and upregulates the transcription of ENO2; ectopic expression of ENO2 increased aerobic glycolysis in colon cancer cells. Most importantly, we found that the appropriate concentration of naringin inhibited the transcriptional activity of HIF1Α, which in turn decreased aerobic glycolysis in colon cancer cells. Generally, naringin reduces glycolysis in colon cancer cells by reducing the transcriptional activity of HIF1Α and the proliferation and invasion of colon cancer cells. This study helps to elucidate the relationship between colon cancer progression and glucose metabolism, and demonstrates the efficacy of naringin in the treatment of colon cancer.
Glycolysis ; Colonic Neoplasms/metabolism* ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Phosphopyruvate Hydratase/metabolism* ; Flavanones/pharmacology* ; Cell Line, Tumor ; Databases, Genetic ; Cell Proliferation/drug effects* ; Transfection ; Warburg Effect, Oncologic

OBJECTIVE: To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia. METHODS: Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene. RESULTS: The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups. CONCLUSION After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.
Humans ; Transduction, Genetic ; Genetic Vectors ; Hemophilia A/genetics* ; Transfection ; Blood Coagulation Factors/genetics* ; Lentivirus/genetics*

Objective: To explore the effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1 (HMEC-1) in vitro and the potential molecular mechanism. Methods: The experimental research method was used. HMEC-1 was collected and divided into P311 adenovirus group and empty adenovirus group according to the random number table (the same grouping method below), which were transfected correspondingly for 48 h. The cell proliferation activity was detected using the cell counting kit 8 on 1, 3, and 5 days of culture. The residual scratch area of cells at post scratch hour 6 and 11 was detected by scratch test, and the percentage of the residual scratch area was calculated. The blood vessel formation of cells at 8 h of culture was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The protein expressions of vascular endothelial growth factor receptor 2 (VEGFR2), phosphorylated VEGFR2 (p-VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2) in cells were detected by Western blotting. HMEC-1 was collected and divided into P311 adenovirus+small interfering RNA (siRNA) negative control group, empty adenovirus+siRNA negative control group, P311 adenovirus+siRNA-VEGFR2 group, and empty adenovirus+siRNA-VEGFG2 group, which were treated correspondingly. The protein expressions of VEGFR2, p-VEGFR2, ERK1/2, and p-ERK1/2 in cells were detected by Western blotting at 24 h of transfection. The blood vessel formation of cells at 24 h of transfection was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. HMEC-1 was collected and divided into P311 adenovirus+dimethylsulfoxide (DMSO) group, empty adenovirus+DMSO group, P311 adenovirus+ERK1/2 inhibitor group, and empty adenovirus+ERK1/2 inhibitor group, which were treated correspondingly. The protein expressions of ERK1/2 and p-ERK1/2 in cells were detected by Western blotting at 2 h of treatment. The blood vessel formation of cells at 2 h of treatment was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The sample number at each time point in each group was 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, and least significant difference test. Results: Compared with that of empty adenovirus group, the proliferation activity of cells in P311 adenovirus group did not show significant difference on 1, 3, and 5 days of culture (with t values of -0.23, -1.30, and -1.52, respectively, P>0.05). The residual scratch area percentages of cells in P311 adenovirus group were significantly reduced at post scratch hour 6 and 11 compared with those of empty adenovirus group (with t values of -2.47 and -2.62, respectively, P<0.05). At 8 h of culture, compared with those of empty adenovirus group, the number of nodes and total length of the tubular structure of cells in P311 adenovirus group were significantly increased (with t values of 4.49 and 4.78, respectively, P<0.01). At 48 h of transfection, compared with those of empty adenovirus group, the protein expressions of VEGFR2 and ERK1/2 of cells in P311 adenovirus group showed no obvious changes (P>0.05), and the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus group were significantly increased (with t values of 17.27 and 16.08, P<0.01). At 24 h of transfection, the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA negative control group (P<0.01). The protein expressions of VEGFR2, p-VEGFR2, and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in P311 adenovirus+siRNA-VEGFR2 group (P<0.01). The protein expressions of VEGFR2 and p-ERK1/2 of cells in empty adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA-VEGFR2 group (P<0.05 or P<0.01). At 24 h of transfection, the number of nodes of the tubular structure in cells of P311 adenovirus+siRNA negative control group was 720±62, which was significantly more than 428±38 in empty adenovirus+siRNA negative control group and 364±57 in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+siRNA negative control group was (21 241±1 139) μm, which was significantly longer than (17 005±1 156) μm in empty adenovirus+siRNA negative control group and (13 494±2 465) μm in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+siRNA negative control group was significantly more than 310±75 in empty adenovirus+siRNA-VEGFR2 group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+siRNA negative control group was significantly longer than (11 600±2 776) μm in empty adenovirus+siRNA-VEGFR2 group (P<0.01). At 2 h of treatment, the protein expression of p-ERK1/2 of cells in P311 adenovirus+DMSO group was significantly higher than that in empty adenovirus+DMSO group and P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01), and the protein expression of p-ERK1/2 of cells in empty adenovirus+DMSO group was significantly higher than that in empty adenovirus+ERK1/2 inhibitor group (P<0.05). At 2 h of treatment, the number of nodes of the tubular structure in cells of P311 adenovirus+DMSO group was 726±72, which was significantly more than 421±39 in empty adenovirus+DMSO group and 365±41 in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+DMSO group was (20 318±1 433) μm, which was significantly longer than (16 846±1 464) μm in empty adenovirus+DMSO group and (15 114±1 950) μm in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+DMSO group was significantly more than 317±67 in empty adenovirus+ERK1/2 inhibitor group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+DMSO group was significantly longer than (13 188±2 306) μm in empty adenovirus+ERK1/2 inhibitor group (P<0.01). Conclusions: P311 can enhance the angiogenesis ability of HMEC-1 by activating the VEGFR2/ERK1/2 signaling pathway.
Adenoviridae/genetics* ; Cell Line ; Endothelial Cells ; Endothelium, Vascular ; Humans ; Neovascularization, Physiologic ; Nerve Tissue Proteins ; Oncogene Proteins ; Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor A

OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection

OBJECTIVE: To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs. METHODS: 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. RESULTS: 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). CONCLUSION 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.
Auranofin ; Cell Line, Tumor ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus/genetics* ; RNA, Messenger ; Transfection

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.
Adenoviridae/genetics* ; Cell Proliferation ; Estrogen Receptor alpha/metabolism* ; Recombinant Proteins ; Transfection

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.
Animals ; Epithelial Cells ; Genetic Vectors ; Lentivirus/metabolism* ; Lung ; Mice ; MicroRNAs/metabolism* ; Silicon Dioxide/toxicity* ; Transfection

OBJECTIVE: To explore the effects of the rat FVIII light chain (rLC) on the activity of human FVIII heavy chain hHC745 and hHC1690. METHODS: hHC745, hHC1690, human FVIII light chain (hLC) and rLC were cloned into adeno-associated virus serotype 8 (AAV8) expression vectors with CB promoter (ubiquitous expression), respectively, and transfected into 293T cells using a dual-chain strategy of co-expression of HC and LC. The cultured cell supernatant was collected at 48 hours after transfection. The plasmids (pAAV8-CB-hHC745, pAAV8-CB-hHC1690, pAAV8-CB-hLC and pAAV8-CB-rLC) were hydrodynamically injected into hemophilia A (HA) mice via lateral tail vein. Forty-eight hours after injection, the peripheral blood of the mice was collected through retroorbital venous plexus and the plasma was obtained by centrifugation. The activity of FVIII was detected by activated partial thromboplastin time (aPTT) assay, and the antigen expression level of FVIII was determined by enzyme-linked immunosorbent assay (ELISA). The specific activity of FVIII was calculated based on the activity and the antigen expression level. RESULTS: In 293T cells, the coagulation activity of FVIII was significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC. The FVIII activity of hHC745+rLC was about 4.6 times higher than that of hHC745+hLC, while hHC1690+rLC was about 2.9 times higher than that of hHC1690+hLC. The data from ELISA showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.1 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 2.8 times compared with hHC1690+hLC. In HA mice administrated with hydrodynamic injection, the FVIII activity of hHC745+rLC and hHC1960+rLC was significantly higher than that of hHC745+hLC and hHC1690+hLC at comparable expression level. The FVIII activity of hHC745+rLC was significantly higher than that of hHC745+hLC, increasing to about 5.1 times, while, that of hHC1690+rLC increasing to about 2.1 times than hHC1690+hLC. ELISA results also showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.2 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 1.8 times compared with hHC1690+hLC. In addition, the activity of hHC1690 combined with rLC was significantly higher than that of hHC745 combined with rLC. CONCLUSION rLC can significantly enhance the coagulation activity of FVIII when co-expressed with hHC of different length including hHC745 and hHC1690.
Rats ; Humans ; Mice ; Animals ; Dependovirus/genetics* ; Transfection

OBJECTIVE: To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes. METHODS: The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag. RESULTS: The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05). CONCLUSION We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.
Adenoviridae/metabolism* ; Alternative Splicing ; Animals ; Animals, Newborn ; Escherichia coli/metabolism* ; Genetic Vectors ; Myocytes, Cardiac/metabolism* ; Plasmids ; RNA Splicing Factors/metabolism* ; Rats ; Transfection