Co-transfection Group Research Articles

MiR-328-3p overexpression attenuates the malignant proliferation and invasion of liver cancer via targeting Endoplasmic Reticulum Metallo Protease 1 to inhibit AKT phosphorylation.

BackgroundLiver cancer is one of the most common cancers worldwide. microRNAs (miRNAs) have been recognized as minimally invasive prognostic markers for distinct types of cancer. This study evaluates the mitigation role of miR-328-3p on liver cancer in vitro and in vivo.MethodsLiver cancer cell line Huh-7 and HepG2 were used for in vitro experiments. Compared with the control group, miR-328-3p overexpression inhibited the proliferation, invasion, and promoted apoptosis of Huh-7 cells. miR-328-3p and endoplasmic reticulum metalloprotease 1 (ERMP1) had an excellent targeting relationship. Compared with the pcDNA-ERMP1 transfection group, the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in miR-328-3p mimic and pcDNA-ERMP1 co-transfection group were significantly decreased. Animal models were set up using four-week-old immunodeficient BABL/c female nude mice. Huh-7 cells transfected with lentivirus holding miR-328-3p or empty vector were injected into the right dorsal side of BABL/c nude mice, respectively. Tumor volume was measured every five days. After one month, animals were sacrificed, xenograft tumors were dissected and weighed for RT-PCR and immunohistochemical assays.ResultsCompared with control group, miR-328-3p overexpression significantly inhibited tumor weight (0.46±0.07 vs. 0.11±0.05 g, P<0.05) and tumor volume (1876±321 vs. 543±168 mm3, P<0.05) after thirty days. miR-328-3p overexpression significantly downregulated the percentage of Ki67 positive cells, N-cadherin positive cells and vimentin positive cells.ConclusionsThese findings suggested that miR-328-3p could be a new treatment or a novel marker for liver cancer prognosis.

Association between matrix Gla protein and ulcerative colitis according to DNA microarray data

BackgroundMatrix Gla protein (MGP) is a secreted protein contributed to the immunomodulatory functions of mesenchymal stromal cells. Microarray profiling found a significantly higher expression level of the extracellular matrix gene MGP in patients with ulcerative colitis (UC). However, little is known about the role of MGP in UC and its upstream signaling regulation. This study aimed to identify the expression of MGP in UC and its upstream regulator mechanism.MethodsColonic mucosa biopsies were obtained from patients with UC and healthy controls. DNA microarray profiling was used to explore underlying genes correlating with UC development. Mice were fed with water containing different concentrations of dextran sodium sulfate (DSS) to induce an experimental colitis model. Colonic tissues were collected and evaluated using immunohistochemistry, immunoblot, real-time polymerase chain reaction, and chromatin immunoprecipitation assay. Bioinformatics analysis was performed to identify candidate MGP gene-promoter sequence and transcription-initiation sites. Luciferase-reporter gene assay was conducted to examine the potential transcription factor of MGP gene expression. ResultsThe expression of MGP was significantly increased in colonic tissues from UC patients and DSS-induced colitis models, and was positively correlated with disease severity. Bioinformatics analysis showed a conserved binding site for Egr-1 in the upstream region of human MGP gene. The significantly higher level of Egr-1 gene expression was found in UC patients than in healthy controls. The activity of luciferase was significantly enhanced in the Egr-1 expression plasmid co-transfected group than in the control group and was further inhibited when co-transfected with the Egr-1 binding-site mutated MGP promoter.ConclusionsUp-regulated expression of MGP was found in UC patients and DSS-induced colitis. The expression of MGP can be regulated by Egr-1.

Mechanism of microRNA-182 targeting and regulating forkhead box transcription factor 2 to promote the invasion, metastasis and angiogenesis of hepatocellular carcinoma

Objective To investigate the expression of miRNA-182 in hepatocellular carcinoma (HCC) cell lines and tissues, to verify the targeted regulation relationship between miRNA-182 and forkhead box transcription factor 2 (FOXF2) and to explore whether miRNA-182 can promote the invasion, metastasis and angiogenesis of HCC by targeting FOXF2. Methods From January to October 2017, a total of 41 patients with primary HCC admitting to Cancer Hospital Affiliated to Zhengzhou University and receiving operation were enrolled. The expression of miRNA-182 in tumor tissues and normal paracancerous tissues was detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). The effects of overexpression of miRNR-182 and FOXF2 on invasion and metastasis of HCC were detected by Transwell transfer experiment and the Boyden invasion experiment. The effects of miRNA-182 and FOXF2 on angiogenesis of HCC were determinded by in vitro angiogenesis experiments. The targeted regulation relationship between miRNA-182 and FOXF2 was investigated by dual luciferase reporter gene. The expression of FOXF2 at mRNA and protein level after miRNA-182 and miRNA-182 scramble transfection was detected by qRT-PCR and Western blotting method. T test was performed for statistical analysis. Results The expression in miRNA-182 in HCC tissue was higher than that in normal tissues adjacent to cancer (5.41±1.72 vs. 1.80±0.76), and the difference was statistically significant (t=-5.764, P<0.01). The results of Transwell transfer experiment and the Boyden invasion experiment showed that the number of cells passing through the membrane of the small chamber of miRNA-182 over-expression group was higher than that of miRNA-182 scramble group (85.65±4.86 vs. 31.43±2.41, 55.34±5.66 vs. 19.13±2.12), the number of cells passing through the membrane of the small chamber of miRNA-182+ FOXF2 over expression group was less than that of simple miRNA-182 over expression group (11.21±3.16 vs. 31.43±2.41, 8.67±2.83 vs. 19.13±2.12), and the differences were statistically significant (t=15.110, 9.220, 5.443 and 4.410; all P<0.05). The activity of luciferase of wild FOXF2 and miRNA-182 cotransfection group was lower than that of miRNA-182 scramble group (0.43±0.10 vs. 0.97±0.05), and the difference was statistically significant (t=8.042, P=0.012). However the activity of luciferase of mutated FOXF2 and miRNA-182 cotransfection group was higher than that of miRNA-182 scramble group (0.97±0.47 vs. 1.06±0.52), and the difference was not statistically significant (t=0.934, P=0.402). The results of in vitro angiogenesis experiments demonstrated that the number of constituent vessels in miRNA-182 over expression group was higher than that in miRNA-182 scramble group (14.27±1.77 vs. 5.91±1.42), and after FOXF2 over-expressing plasmids cotransfected, the angiogenesis ability restored (1.78±0.71 vs. 14.27±1.77), and the differences were statistically significant (t=6.530 and 4.570, both P<0.05). The results of Western blotting indicated that the expression level of FOXF2 in miRNA-182 transfection group was lower than that of miRNA-182 scramble group (1.01±0.13 vs. 4.32±0.46), and the difference was statistically significant (t=7.420, P=0.012). The results of qRT-PCR showed that the expression level of FOXF2 mRNA of miRNA-182 transfection group was lower than that in miRNA-182 scramble group (0.591±0.284 vs. 1.534±0.345), and the difference was statistically significant (t=3.465, P=0.014). Conclusion miRNA-182 can promote the invasion and metastasis of HCC cell line by targeting regulation of FOXF2. Key words: Carcinoma, hepatocellular; miRNA-182; FOXF2; Invasion and metastasis; Angiogenesis

Study of miR-26a inhibiting epithelial-mesenchymal transition and invasion of Tu686 cell line through SMAD1.

This study aims to investigate the effect of miR-26a on the epithelial-mesenchymal transition (EMT) and invasion of Tu686 cell line through SMAD1. Tu686 Squamous cell carcinoma of head and neck (SCCHN) cell strains were divided into the miR-26a group, miR-NC group, co-transfection group and blank control group. Among them, the miR-26a group only transfected miR-26a mimics, the miR-NC group only transfected miR-26a negative control, the co-transfection group transfected miR-26a mimics and pcDNA3.1-SMAD1 plasmid. The qRT-PCR method was used for the detection of the expressions of miR-26a and SMAD1 in each group of cells, transwell assay for the detection of the invasion ability of each group of cells and Western blot for detecting the expression level of SMAD1 and the expressions of EMT-related proteins E-cadherin and N-cadherin. The relative expression of miR-26a in the miR-26a group was significantly higher than that in the miR-NC group and blank control group, and the relative expression in the co-transfection group was significantly higher than that in the miR-NC and blank control groups. The relative expression of SMAD1 in the miR-26a group was significantly lower than that in the miR-NC and blank control groups, and the relative expression in the co-transfection group was lower than that in the miR-NC and blank control groups, and higher than that in the miR-26a group (all p<0.05). There was no significant difference between the miR-NC group and the blank control group (p>0.05). miR-26a may reduce the expression level of SMAD1, affect the expression of EMT-related proteins, inhibit the EMT function of Tu686 cells of squamous cell carcinoma of head and neck, and inhibit the invasion of them.

Mechanism of regulation of invasion and migration of osteosarcoma cells by fibroblast growth factor receptor 2 mediated by tiny microRNA-494

Objective To investigate the mechanism of microRNA (miRNA, miR)-494 mediated fibroblast growth factor receptor 2 (FGFR2) regulation on invasion and migration of osteosarcoma cells. Methods The miR-494 level of osteosarcoma 143B cells was up-regulated or down-regulated by transient transfection. Methyl thiazol tetrazolium (MTT) assay was used to detect cell proliferation. Transwell was used to detect cell invasiveness. The migration ability of the cells was detected by scratch test, and the expression level of matrix metalloproteinase (MMP)-2 and MMP-9 protein was detected by Western blotting. The target gene of miR-494 was predicted by bioinformatics software Targetscan, and the direct target interaction relationship was verified by double luciferase reporter test. A nude mouse model of osteosarcoma was established in situ, and treated with miR-494 agomir. The effect of up-regulation of miR-494 on tumor growth in nude mice was observed. Results The proliferation ability of 143B cell miR-494 was significantly inhibited at all time points, and the cell proliferation ability of osteosarcoma 143B cells was promoted after the down-regulation of miR-494 cells. After transient transfection of 24 h, the number of cross membrane in the miR-494 up-regulation group was less than that of the control group, but the number of cross membrane in the miR-494 down-regulation group was more than that of the control group. The migration ability of the cells in each group was detected by scratch test. The results showed that the cell migration rate in the miR-494 up-regulation group was less than that of the control group, and the cell migration rate in the miR-494 down-regulation group was higher than that of the control group. It is suggested that up-regulation of miR-494 can inhibit cell migration, while down-regulation of miR-494 can promote cell migration. The expression level of MMP-2 and MMP-9 protein in miR-494 up-regulation group was lower than that in control group, while the expression level of MMP-2 and MMP-9 protein in miR-494 down-regulation group was higher than that in control group. The luciferase activity in FGFR2 wild type and miR-494 mimic co-transfection group was significantly lower than that in the control group. Up-regulation of miR-494 could inhibit the mRNA level of FGFR2 in osteosarcoma 143B cells, while down-regulation of miR-494 could promote mRNA level of 143B cell FGFR2 in osteosarcoma. The tumor volume in miR-494 agomir (494-agomir) drug delivery group in nude mice was smaller than that in control group on the 14th, 21st and 28th day after administration. Conclusion The miR-494 can inhibit the proliferation, invasion and migration of osteosarcoma 143B cells, and its mechanism is closely related to the regulation of target gene FGFR2. Key words: Osteosarcoma; MicroRNA; Invasion; Metastasis; Fibroblast growth factor receptor 2

Effect and mechanism of microRNA-126-3p on inflammatory response after spinal cord injury in rats

Objective To investigate the effect and mechanism of microRNA (miRNA, miR)-126-3p on inflammatory response after spinal cord injury in rats. Methods The SD rat model of spinal cord injury was established. The relative expression of miR-126 in the spinal cord tissues of 3, 7 and 14 d after spinal cord injury in rats was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The effect of agomir-126 injection on the motor function of rats with acute spinal cord injury was evaluated by Basso-Beattie-Bresnahan (BBB) scoring method, and the online target gene was used to predict the database Targetscan. The target gene of miR-126-3p was predicted and the target gene of agomir-126 was verified by double luciferase reporter gene. After transfecting NEURO-2A cells to 150 nmol/L concentration miR-126-3p mimics for 24 hours, Western blotting was used to detect the change of the expression level of interleukin (IL)-7 protein, and Western blotting was used to detect the agomir-126 administration group. In the negative control group (agomir-NC), the expression level of inflammation related factors in spinal cord injured rats was changed. Results The expression level of miR-126 in spinal cord tissue of model group (2.13±0.44) in 3 days was lower than that of control group (3.69±0.59, t=2.769, P=0.021). The expression level of miR-126 in spinal cord tissue of model group (1.85±0.42) in 7 days was lower than that of control group (3.77±0.88, t=2.485, P=0.026). The expression level of miR-126 in spinal cord tissue of model group (1.33±0.28) in 14 days was lower than that of control group (3.82±0.92, t=3.388, P=0.019). The BBB score of 3 days in the agomir-126 group (4.690±0.530) was higher than that of the control group (3.650±0.350, t=3.661, P=0.006); The BBB score of 7 days in the agomir-126 group was higher than that of the control group (3.690±0.650, t=5.452, P=0.001), and agomir-126 administration group 14 days (6.720±0.770) was higher than that of the control group (3.770±0.470, t=7.320, P=0.001). The luciferase activity ratio of IL-7-WT-3’UTR and miR-126-3p mimic co-transfection group (6.92±0.556) was significantly lower than that of the control group (13.69±0.925, t=10.870, P=0.001). The expression of IL-7 protein in miR-126 mimic transfection group (0.23±0.14) NEURO-2A cells was lower than that in the control group (0.86±0.17, t=4.955, P=0.009). The expression of IL-7 protein in the spinal cord of the drug group (0.369±0.093) SCI rats was lower than that of the control group (0.956±0.124, t=6.559, P=0.003), and the expression of IL-1 beta protein in the spinal cord of the administration group (0.572±0.085) SCI rats was lower than that of the control group (0.764±0.086, t=2.750, P=0.026), and the spinal cord of the drug group (0.133±0.009) SCI rat ridges The expression of TGF- beta protein was lower than that in the control group (0.257±0.028, t=7.303, P=0.002). Conclusion The expression of miR-126-3p in spinal cord injury is reduced, and up regulation of miR-126-3p can protect the spinal cord injury. The mechanism may be related to the inflammatory response of the spinal cord injury mediated by the key target gene IL-7. Key words: Spinal cord injury; MicroRNA-126; Interleukin-7; Inflammatory response

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80–90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young’s were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration.

The effect and mechanism of microRNA-83a targeting on differentiation of bone marrow mesenchymal stem cells in vitro

Objective To study the anti-proliferation and inducing apoptosis effects of microRNA (miRNA, miR)-83a on bone marrow mesenchymal stem cells. Methods The experiment was divided into 3 groups: group A (PEF miR-83a and PSV2neo co-transfection group), group B (non-transfection group), and group C (PEF and PSV2neo co-transfection group). Group A served as the experimental group, and groups B and C group as the control groups. Transfection according to instructions, effects of application of expression detected miR-83a transcription of bone marrow mesenchymal stem cells lines in vitro antiproliferative effect of and biological function; by detecting the expression level of miR-83a immunohistochemical method and real-time quantitative polymerase chain reaction (Real-time PCR) technology in cells, Real-time PCR was measured after transfection, bone marrow mesenchymal stem cells lines Ras, Raf, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK): mRNA expression changes, and through the methyl thiazol tetrazolium (MTT) method, the scratch test, hematoxylin and eosin (HE) staining, microscope observation and comparison of miR-83a transfection on the migration ability of cell proliferation, apoptosis and morphological changes. To analyse by using SPSS 14.0 statistical software. Results Real-time PCR and immunohistochemistry confirmed in miR-83a transfected cells with stable expression; Assingland staining results: after seventh days culture, the cells of the induction differentiation group were full of brown specific staining granules, and no specific staining granules were found in the undifferentiated differentiation group. Real-time PCR results: The expression of Ras, Raf and Akt mRNA in group A were 0.97±0.27, 0.54±0.18 and 0.99±0.31, in group B were 1.68±0.30, 1.58±0.25 and 2.06±0.42, in group C were 1.68±0.30, 1.58±0.25 and 1.89±0.31. Compared with group B and C, the levels of Ras, Raf and Akt mRNA in group A were significantly lower (t=1.531 and 1.838, P=0.016 and 0.011). MTT assay showed that the apoptotic rates of the cells were (23.49±4.64)%, (45.73±8.72)% and (59.25±13.16)% after 24, 48 and 72 h, respectively. There was significant difference between the two groups (χ2= 5.203 and 2.146, P =0.013 and 0.045). The scratch test showed that miR-83a transfected cell migration ability is lower than the normal cells. The effect of miR-83a on lung cancer cell 24 h, morphological observation of lung cancer cell apoptosis or necrosis. Conclusion Transfection of miR-83a can inhibit proliferation and promote apoptosis effect on pituitary adenoma cells in vitro cells, which is likely to play a role through inhibition of epidermal growth factor receptor (EGFR) and EGFRvⅢ signal transduction pathway in the RAS-RAF-mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)-Akt of two important signaling pathways activated. Key words: Bone marrow mesenchymal stem cells; MicroRNA-83a; Proliferation; Apoptosis; Biological function

Function and mechanism of TRIM22 targeting eIF4E in the process of NB4 cells differentiation

Objective To investigate the function of tripartite motif protein 22 (TRIM22) and the interaction with eukaryotic translation initiation factor-4E (eIF4E) in the differentiation of NB4 cells, one kind of acute promyelocytic leukemia cells, which elucidates the mechanism of TRIM22 targeting to regulate eIF4E. Methods The model of NB4 cells inducing differentiation was established in vitro. The expression changes of gene and protein of TRIM22 and eIF4E were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. In addition, the effect on cell function and protein expression level of eIF4E after adopting electroporation technology to depress or over-express TRIM22 was detected by CCK-8 and flow cytometry. Finally, the interaction of TRIM22 and eIF4E was verified by using co-immunoprecipitation (CO-IP). Results The mRNA relative expression level of TRIM22 was gradually increasing from 1.01±0.15 to 30.98±2.79 (F=280.700, P=0.000), and the protein relative expression level was gradually increasing from 0.22±0.03 to 0.51±0.05 (F=51.430, P=0.000) after the all-trans-retinoic acid (ATRA) induction for NB4 cell. However, the mRNA relative expression level of eIF4E was gradually decreasing from 1.01±0.09 to 0.47±0.06 (F=20.520, P=0.000), with the same trend, the protein relative expression level was gradually decreasing from 0.97±0.02 to 0.64±0.09 (F=14.700, P=0.001). The expression level of PE-CD11b in the TRIM22 over-expression group with ATRA detected by flow cytometry [(78.80±2.00)%] was higher than that in the transfection group of empty vetor with ATRA [(58.70±2.70)%] (t=9.535, P=0.000) and the cotransfection group with ATRA [(61.60±3.80)%] (t=8.187, P=0.000). Meanwhile, the protein level of eIF4E changed reversely after over-expressing the gene level of TRIM22 (t=4.985, P=0.007). The CO-IP experiment was used to verify the interaction of TRIM22 and eIF4E. Conclusion TRIM22 is able to promote the cell differentiation during the process of NB4 cells differentiation. Furthermore, eIF4E is a target of TRIM22 for binding with, which plays an important role in depressing the expression of eIF4E. Key words: Leukemia; Cell differentiation; Eukaryotic initiation factor-4E; Tripartite motif protein 22

Decrease of miR-622 expression suppresses migration and invasion by targeting regulation of DYRK2 in colorectal cancer cells.

BackgroundMore and more evidence indicates that microRNAs are present and involved in many tumor-related diseases. The function of microRNA-622 (miR-622) in colorectal cancer (CRC) remains controversial. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) has been reported as a tumor suppressor gene in different cancers. The detailed regulation mechanism of DYRK2 in CRC remains unclear.MethodsmiR-622 and DYRK2 expression levels were detected at tissue and cellular level respectively by using real time polymerase chain reaction (PCR), Western blot, and immunohistochemical staining. Pearson’s correlation analysis was used to evaluate the correlation between miR-622 and DYRK2. Transwell assay was applied to measure the effect of miR-622 on migration and invasion of SW1116 and SW480. We used dual luciferase reporter assay to confirm the targeted binding effect of miR-622 and DYRK2 3′-untranslated region (3′UTR). An antisense experiment was executed to further confirm the role miR-622 had played with regard to migration and invasion by targeting regulation of DYRK2 pathway in CRC cells.ResultsIn our research, we found that the expression of miR-622 was elevated in CRC tissues and cell lines compared to that of nonCRC tissues and the normal human colon epithelial cell line NCM460. Correspondingly, the expression of DYRK2 in CRC tissues and cell lines showed a contrary tendency. The different expression level of DYRK2 was closely correlated with clinicopathological characteristics of CRC patients. We demonstrated that down-regulation of miR-622 could inhibit the ability of migration and invasion of CRC cell lines SW1116 and SW480. Also, we confirmed that DYRK2 was negatively regulated by miR-622 via a specific targeted binding site within the 3′UTR. We finally verified that the migration and invasion ability of CRC cells in the conducted DYRK2 3′UTR defect plasmid transfection group were lower compared to miR-622 and cotransfection group.ConclusionThe findings of this study indicate that a decrease of miR-622 expression could suppress migration and invasion by targeting regulation of DYRK2 and miR-622/DYRK2 could be a potential molecular treating target of CRC.

Effect of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7.

To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7. Hepatocellular carcinoma Huh7 was cultured invitro and lipidosome was used to transfect miR-520c-3p and miR-132, respectively or together. The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry, and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR. Compared with the control group, the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly, and the apoptosis ratio increased distinctly (P<0.05). Besides, the effect of the co-transfection group was better than that of the single transfection group. The protein levels of GPC3 (Glypican-3) and YAP (Yes-associated protein), the target genes transfected only by miR-520c-3p and miR-132, respectively, reduced obviously (P<0.05), which was similar with the co-infected cells, but cells transfected by miR-132 only showed a decrease of YAP. The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP, enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.

Effect of metastasis suppressor 1 gene expression on migration and apoptosis of human gastric cancer cell BIU87 by regulating the hepatocyte growth factor/c-Met signaling pathway

Objective To investigate the effect of metastasis suppressor 1 (MTSS1) gene on the biological function of bladder cancer BIU87 cells by regulating the hepatocyte growth factor (HGF)/c-Met signaling pathway. Methods Cultured gastric cancer BIU87 cells and passaged cells were divided into three groups: group A (PEF MTSS1 and PSV2neo cotransfection group); group B: (shNC and PSV2neo co transfection group); group C (non transfection group). Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR) were used to detect MTSS1, c-Met, E-cadherin and N-cadherin protein and mRNA expression levels. Flow cytometry and scratch test were performed to examine the migration, proliferation and apoptosis of BIU87 cells transfected with PEF MTSS1. Results (1) Western blotting and Real-time PCR results showed that as compared with groups B and C, MTSS1 and c-Met protein and mRNA expression levels were significantly down-regulated in group A (P 0.05). (4) Flow cytometry showed that cell apoptosis rate in groups A, B and C was respectively (26.73±3.72)%, (4.28±0.68)% and (2.73±0.41)%. The apoptosis rate in group A was significantly higher than in group C (P<0.01), and there was no significant difference between groups B and C. Conclusion The transfection of MTSS1 gene can promote the proliferation and inhibit the apoptosis of gastric cancer cell line BIU87 cells probably by regulating the HGF/c-Met signal transduction pathway. Key words: Bladder cancer; Metastasis suppressor 1 gene; Hepatocyte growth factor/c-Met signaling pathway

Short hairpin RNA silencing of TGF-βRII and FZD-7 synergistically suppresses proliferation and metastasis of hepatocellular carcinoma cells.

Transforming growth factor-β (TGF-β) is a multifunctional regulator of cell growth, apoptosis, differentiation and migration. The Wnt/β-catenin signaling pathway has been implicated in a wide spectrum of diseases, including numerous cancers and degenerative disease. The aim of the present study was to investigate if simultaneous blocking of TGF-β and Wnt/β-catenin signaling pathways exerts synergistic anti-tumor effects on hepatocellular carcinoma (HCC) cells. Short hairpin (sh) RNA eukaryotic expression vectors, specific to TGF-β receptor II (RII) and Frizzled receptor (FZD)-7, were constructed by gene recombination. The expression vectors were transfected into human HCC HepG2 and Huh-7 cells using Lipofectamine 2000 to investigate the synergistic effects between TGF-β and Wnt/β-catenin signaling pathways on HCC cell proliferation, invasion and migration and the cell-cycle distribution. Western blot analysis was used to identify the expression of β-catenin, c-Myc and cyclin D1 in transfected cells to investigate the underlying mechanisms that cause TGF-β and Wnt/β-catenin signaling in HCC cells. shTGF-βRII-c and shFZD-7–2 were selected as the most efficient plasmids. A cell growth assay and colony-forming assay consistently demonstrated that the proliferative activity of the co-transfected group was significantly decreased compared to the single-transfected group. A wound healing invasion and migration assay demonstrated that co-transfection of shTGF-βRII-c and shFZD-7-2 decreased the invasion and migration abilities of the cells compared with either single-transfected group. In addition, the present study demonstrated that the observed reduction in cell proliferation was due to the cells arresting at the G1 phase of the cell cycle, and the downregulation of β-catenin, c-Myc and cyclin D1 impaired the proliferative and invasive abilities of the HCC cells. The present results demonstrate that simultaneous blocking of TGF-β and Wnt/β-catenin signaling by targeting TGF-βRII and FZD-7 may inhibit the proliferation and metastasis of HCC cells more effectively compared with blocking either the TGF-β or Wnt/β-catenin pathway.

Regulation of vitellogenin (vtg1) and estrogen receptor (er) gene expression in zebrafish (Danio rerio) following the administration of Cd²⁺ and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

We evaluated the individual and joint estrogenic effects of cadmium (Cd(2+)) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the zebrafish liver (ZFL) cell line, zebrafish embryo, larvae and the liver of adult zebrafish. The mRNA expression of vtg1 was inhibited by Cd(2+), but unaffected by TCDD in ZFL cells. Similar changes in the mRNA levels of ERα, ERβ1, ERβ2 and GPER (G protein coupled estrogen receptor) in ZFL cells were also observed. Deletion mutants of vtg1 gene promoters were constructed to investigate transcriptional regulation, and we found that all of the constructs failed to respond to TCDD or Cd(2+). However, after co-transfection with a vtg1 promoter-luciferase construct to the ERα, ERβ1, ERβ2 and GPER expression vectors, decreased luciferase activity was observed in the ERα co-transfection group after treatment with Cd(2+), suggesting that ERα participates in vtg1 transcriptional regulation and is affected by Cd(2+). Differences in the regulation of the mRNA levels of these genes were also observed between different developmental stages and between the livers of male and female zebrafish.

Effects of nerve growth factor and basic fibroblast growth factor dual gene modification on rat bone marrow mesenchymal stem cell differentiation into neuron-like cells in vitro.

Recent studies regarding regenerative medicine have focused on bone marrow mesenchymal stem cells (BMSCs), which have the potential to undergo neural differentiation, and may be transfected with specific genes. BMSCs can differentiate into neuron-like cells in certain neurotropic circumstances in vitro. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are often used to induce neural differentiation in BMSCs in vitro. However, previous studies regarding their combined actions are insufficient. The present study is the first, to the best of our knowledge, to thoroughly assess the enhancement of neural differentiation of BMSCs following transfection with bFGF and NGF. Sprague-Dawley (SD) rat BMSCs were separated through whole bone marrow adherence, and were then passaged to the third generation. The cells were subsequently divided into five groups: The control group, which consisted of untransfected BMSCs; the plv-blank-transfected BMSCs group; the plv-bFGF-trans-fected BMSCs group; the plv-NGF-transfected BMSCs group; and the plv-NGF-bFGF co-transfected BMSCs group. Cell neural differentiation was characterized in terms of stem cell molecular expression, and the neuronal morphology and expression of neural-like molecules was detected in each of the groups. A total of 72 h post-transfection, the expression levels of neuron-specific enolase, glial fibrillary acidic protein, and nestin protein, were higher in the co-transfected group, as compared with the other groups, the expression levels of β-tubulin III were also increased in the co-transfected cells, thus suggesting the maturation of differentiated neuron-like cells. Furthermore, higher neuronal proliferation was observed in the co-transfected group, as compared with the other groups at passages 2, 4, 6 and 8. Western blotting demonstrated that the transfected groups exhibited a simultaneous increase in phosphorylation of the AKT and extracellular signal-regulated kinases (ERK) signaling pathway. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of transfected BMSCs.

Effects of blocking two sites of transforming growth factor-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts

To explore the effects of blocking two sites of TGF-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts. Two lentivirus vectors encoding soluble TGF-β receptor II (sTβRII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTβRII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-β1 for 72 h; sTβRII group, transfected with lenti-sTβRII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-β1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test). (1) HFF-1 cells transfected with lenti-sTβRII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTβRII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTβRII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTβRII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05). In human skin fibroblasts, blockage of two sites of TGF-β/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-β1 to a greater extent than that of blocking one single site.

ShRNAs targeting high mobility group box-1 lead to inhibition of E-selectin expression via homeobox A9 in human umbilical vein endothelial cells

To approach the regulatory mechanism of high mobility group box-1 ( HMGB1 ) on the expression of E-selectin in human umbilical vein endothelial cell ( HUVEC ). Homeobox A9 ( HOXA9 ) siRNA was transfected to HUVEC at logarithmic phase, real-time fluorescence quantitative polymerase chain reaction ( real-time qPCR ) and Western Blot were used to determine the HOXA9 mRNA expression and protein expressions; a blank control group and a nonsilence negative control group were set. HUVEC stable transfected with pRNA-u6.1/Neo-HMGB1 shRNA plasmids ( HUVEC with low-expression HMGB1 ) was obtained, and HOXA9 and E-selectin mRNA expressions were determined with real-time qPCR; a nonsilence transfection group served as the negative control. The HOXA9 siRNA was transfected to HUVEC with low-expression HMGB1 as co-transfection group, and the E-selectin expressions was determined with real-time qPCR; a HMGB1 shRNA group and a HOXA9 nonsilence group served as control. (1) HOXA9 mRNA ( 2(-Δ ΔCT) ) and protein expression ( integral A value ) in blank control group were 1.094±0.115 and 1.031±0.060. Compared with nonsilence transfection group, HOXA9 siRNA transfection group could significantly reduced mRNA and protein expression of HOXA9 [ HOXA9 mRNA ( 2(-Δ ΔCT) ): 0.257±0.030 vs. 1.035±0.091, t = 14.010, P = 0.002; HOXA9 protein ( integral A value ): 0.278±0.042 vs. 0.975±0.014, t = 27.310, P = 0.002 ]. (2) Compared with nonsilence transfection group, HMGB1 shRNA transfection could up-regulate HOXA9 mRNA expression in HUVEC ( 2(-Δ ΔCT) : 2.519±0.278 vs. 0.856±0.063, t = 10.100, P = 0.001 ), also could down-regulate E-selectin mRNA expression ( 0.311±0.046 vs. 1.080±0.201, t = 7.415, P = 0.000 ). (3) Compared with HOXA9 nonsilence group and HMGB1 shRNA group, HMGB1 shRNA and HOXA9 siRNA co-transfected HUVEC cells could significantly elevate E-selectin mRNA expression ( 2(-Δ ΔCT) : 3.445±0.428 vs. 1.085±0.212, 1.004±0.104, t(1) = 8.507, t(2) = 9.603, both P < 0.001 ). HMGB1 may regulate E-selectin expression through the HOXA9 regulation in HUVEC.

RNAi Silencing of IL-1β and TNF-α in the Treatment of Post-traumatic Arthritis in Rabbits.

Post-traumatic arthritis is a secondary complication to severe joint trauma. With the disease progression, it may eventually lead to osteoarthritis in patients whose age is considerably younger than patients with traditional bone arthritis. The main objective of this study was to explore the feasibility of using lentiviral-mediated RNA interference silencing of IL-1β and TNF-α to treat post-traumatic arthritis in rabbits. About 48 New Zealand rabbits underwent bilateral knee joint surgery to stimulate traumatic arthritis. They were then randomly divided into four groups of 12 rabbits each. The histopathology of the cartilage was observed, and the changes were assessed by Mankin scoring. ELISA was used to detect the expression of IL-1β and TNF-α in the synovial fluid. (i) Compared with the control group, the transfection and co-transfected groups displayed reduced cartilage damage and speed of degeneration. The co-transfected group showed the greatest alleviation of symptoms. The Mankin score was statistically different (p<0.01). (ii) Compared with the control group, the expression of IL-1β or TNF-α was reduced in the respective transfection groups (p<0.01 in both groups) and IL-1β and TNF-α were reduced in the co-transfected group (p<0.01). The co-transfected group showed the lowest expression of the three experimental groups of both IL-1β and TNF-α (p<0.01). Lentivirus-mediated RNA interference can knock down the expression of IL-1β and TNF-α in joint fluids and, in a synergistic effect when two siRNAs are co-transfected, ease cartilage degeneration.

Simultaneous silencing of β-catenin and signal transducer and activator of transcription 3 synergistically induces apoptosis and inhibits cell proliferation in HepG2 liver cancer cells.

The tumorigenesis and maintenance of a cancer cells is dependent upon the collaboration of multiple signaling pathways. Signal transducer and activator of transcription 3 (STAT3) and β-catenin are at the center of multiple cancer-associated signaling pathways; therefore, simultaneously targeting STAT3 and β-catenin may be a potential cancer treatment, leading to induced lethality of cancer cells. In the present study, HepG2 liver cancer cells were transfected with small interfering RNA (siRNA) against β-catenin and STAT3 alone or in combination. The cell growth was assessed using an MTT assay and the levels of cell apoptosis were detected using flow cytometry. Protein levels of caspase-3, cleaved caspase-3, poly(ADP-ribose) polymerase (PARP) and cleaved PARP were determined using western blot analysis. Following siRNA transfection, β-catenin and STAT3 protein levels decreased at 72 h. HepG2 cell growth inhibition and early apoptosis in the β-catenin and STAT3 siRNA co-transfection group were significantly greater than those in the groups transfected with β-catenin or STAT3 siRNA alone. Decreased caspase-3 and PARP levels, as well as enhanced cleavage of caspase-3 and PARP were observed in the β-catenin and STAT3 co-transfection group. Simultaneous silencing of β-catenin and STAT3 using siRNAs resulted in an enhanced loss of cell viability and induction of apoptosis in HepG2 liver cancer cells, suggesting that these genes are promising targets for the further preclinical and clinical development of anti-cancer therapeutic strategies, which target several cancer signaling pathways simultaneously.

Adeno-associated virus-mediated BMP-7 and SOX9 in vitro co-transfection of human degenerative intervertebral disc cells.

Bone morphogenetic protein-7 (BMP-7) and SOX9 are important transcription factors in chondrogenesis. In this study, we examined the biological function of the adeno-associated virus (AAV)-mediated BMP-7 and SOX9 double gene in vitro co-transfection of nucleus pulposus cells of human degenerative intervertebral disc. Human intervertebral disc nucleus pulposus cells were cultured in vitro and subcultured for 5 generations. Using rAAV-BMP-7 and rAAV-SOX9 AAV2-type AAV viruses, the cells were divided into 4 groups: blank normal, BMP-7 transfection, SOX9 transfection, and BMP-7 and SOX9 co-transfection. After 48 h, expression of type II collagen and its mRNA in nucleus pulposus cells was determined. The expression of type II collagen in BMP-7 transfection, SOX9 transfection, and co-transfection groups was up-regulated to varying degrees compared to the blank control group. The type II collagen mRNA level expression in the co-transfection group was significantly higher than in other groups (P < 0.05). AAV-mediated BMP-7 and SOX9 in vitro co-transfection can promote the synthesis of type II collagen in nucleus pulposus cells in the human degenerative intervertebral disc. Double-gene therapy has a synergistic effect in the treatment of intervertebral disc degeneration.