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The Myh3 (myosin heavy chain 3) gene is a marker gene of muscle cell differentiation and regulates the utilization of energy in muscle cells, but whether it affects the glycolysis process of muscle cells in different states is rarely reported. In this paper, the expression patterns of Myh3 and glycolysis-related genes Pkm (M-type pyruvate kinse), Prkag3 (protein kinase adenosine monophosphate-activated γ3-subunit), and Gsk3β (glycogen synthase kinase-3β) were studied by the qRT-PCR (quantitative-Real-Time-PCR) method using C2C12 cells at different stages of myoblast and adipogenic differentiation as models. It was found that in the process of myoblast differentiation of C2C12 cells, the relative expression trends of Myh3 and glycolysis genes Prkag3 and Pkm were basically the same, and the relative expression levels first increased, reached the peak on the second day of differentiation, and then decreased; glycogen synthase the expression trend of the inhibitory gene Gsk3β was relatively stable. In the process of adipogenic differentiation of C2C12 cells, the relative expression trend of Myh3 and glycolysis genes Prkag3 and Pkm remained basically the same, and the relative expression gradually increased, reaching the highest value on the 8th day of differentiation; glycogen synthase inhibitory gene Gsk3β expression remained stable. In the myogenic differentiation state of C2C12 cells, qRT-PCR and Western blotting were used to detect the effects of interfering Myh3 on the mRNA and protein expressions of glycolysis-related genes Pkm, Prkag3, and Gsk3β. The results showed that after interfering with Myh3, the mRNA expressions of glycolysis genes Pkm and Prkag3 were significantly decreased (P 0.05). The protein levels of Myh3 and Pkm were significantly lower than those in the blank group and NC group. Under the adipogenic differentiation state of C2C12 cells, after interfering with Myh3, the mRNA levels of glycogen synthase inhibitor Gsk3β and glycolysis gene Prkag3 were significantly increased (P<0.01), and the mRNA level of glycolysis gene Pkm was decreased; the protein levels of Myh3 and Pkm in the Myh3 interference group were also lower than those in the blank group and NC group. Based on the above studies, there are significant differences in the levels of glycolysis in C2C12 cells in the myogenic and adipogenic states, and the expression patterns of Myh3 and glycolysis genes are similar. Further results showed that Myh3 suppression could inhibit the glycolysis of C2C12 cells in the myogenic state without affecting the glycogen synthesis. Unlike in the myogenic state, interfering expression of Myh3 in the adipogenic state of C2C12 cells inhibited both glycogen synthesis and glycolysis.
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@#Objective: To investigate the expression of tetraspanins-29 (Tspan29) in breast cancer tissues and cell lines and to explore the effect of Tspan29 knockdown on proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of breast cancer MCF-7 and MDA-MB-231 cells. Methods:Atotal of20pairsofbreast cancer tissues and corresponding para-cancerous tissues resected in Minhang Branch of Cancer HospitalAffiliated to Fudan University from June 2017 to February 2018 were collected for this study; in addition, breast cancer celllinesMCF-7,MDA-MB-231andhumanbreastepithelialMDA-kb2cellswerealsocollected.ThemRNAand protein expressions of Tspan29 in above mentioned tissues and cell lines were detected by Real-time quantitative (qPCR) and Western blotting. The expression of Tspan29 in MCF-7 and MDA-MB-231 cells was interfered by siRNA. qPCR was used to detect the mRNA and protein expressions of Tspan29. PCR microarray was used to examine the expressions of EMT-related genes in MCF-7 cells. CCK-8 assayandTranswellwereusedtodetectcellproliferation, migration and invasion of MCF-7 and MDA-MB-231 cells. Results: The mRNA and protein expressions of Tspan29 in breast cancer tissues were significantly higher than that in para-cancerous tissues (all P<0.01); and the mRNA and protein expressions of Tspan29 in MCF-7 and MDA-MB-231 cells were significantly higher than that in MDA-kb2 cells (P<0.01). After being interfered with siTspan29, the mRNA and protein expressions of Tspan29 were significantly down-regulated in MCF-7 cells (all P<0.05); the proliferation, invasion and migration of MCF-7 and MDA-MB-231 cells were significantly inhibited (all P<0.05); and among the EMT-related genes, two were significantly up-regulated while 7 were down-regulated. Conclusion: Tspan29 is significantly up-regulated in breast cancer tissues and cell lines, and knockdown of Tspan29 significantly inhibits the proliferation, invasion and migration of breast cancer cells. ··
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Objective To investigate the effects of sex hormone-binding globulin (SHBG)gene in the apoptosis of human trophoblastic cells.Methods The siRNA specific-targeting SHBG gene was transfected into human trophoblastic cells and they were divided into six groups:trophoblasts without transfection in normal control groups(group Ⅰ);transfect liposome in blank control groups(group Ⅱ);transfect nonspecific siRNA in negative control groups(group Ⅲ);transfect SHBG siRNA-Ⅰ,SHBG siRNA-Ⅱ,SHBG siRNA-Ⅲ respectively in trans-fection group(group Ⅳ,Ⅴ,Ⅵ).Hoechst 33258 dying method was used to detect cell apoptosis.SHBG and Caspase-3 mRNA profiling and the level of SHBG and caspase-3 protein were detected by real-time PCR and Western blot.Results There was no statistical significant difference in the gene expression and protein level of SHBG and caspase-3 in group Ⅰ,Ⅱ and Ⅲ (P >0.05).In Ⅳ,Ⅴ and Ⅵ group,there was no statistical significant difference in the expression level of SHBG and caspase 3 (P >0.05).Compared with group Ⅰ,Ⅱ and Ⅲ,the a-mount of SHBG gene expression decreased obviously,the caspase-3 mRNA and protein level increased obviously and the trophoblast cell ap-optosis increased markedly (P <0.05).Conclusion Through siRNA interference technology can reduce SHBG gene expression in human trophoblastic cells,and it can lead to excessive apoptosis of human trophoblasts cells.
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Objective To investigate the effects of p38 gene sliencing on the proliferation, invasion, cell cycle and sensitivity to sunitinib of human renal carcinoma cell line 786-O. Methods We designed two sequence-specific small interfering RNA(siRNA531 and siRNA659) targeting p38 gene and transfected them into renal carcinoma cell line 786-0. 786-0 cells transfected with nonsense siRNA served asnegative control and those cultured with transfection medium served as blank control. The change of #38 gene expression was observed by RT-PCR and the expression of p38 protein was detected by Western blotting analysis. The proliferation, sensitivities to sunitinib, invasion capabilities, and the cell cycle of 786-0 cells were examined by CCK-8 assay, transwell chamber test and flow cytometry, respectively. Results RT-PCR and Western blotting analysis revealed that p38 expression in #38 siRNA group was significantly decreased compared with the controls. The cell proliferation rates were also significantly decreased 3-5 days after siRNA531 or siRNA659 transfection compared with the controls (P<0. 05, P<0. 01), and cells in the siRNA531 and siRNA659 groups become more sensitive to sunitinib compared with negative control group, with two IC50 values being significantly lower than that of the negative control group ([3. 2 ± 0. 3,, [1. 4±0. 1, μmol/mL vs [5. 4 ± 0. 2, μmol/mL; P<0. 05). In addition, analysis of cell cycle demonstrated a marked G50/G50 arrest of the 786-0 cells transfected with siRNA531 or siRNA659. We also noticed that 24 h after transfection, the cell invasion capabilities was significantly decreased in siRNA531 and siRNA659 compared with negative control (numbers of cells permeating septum: 56. 43 ± 6. 02, 34. 00 ± 8. 12 vs 76. 27 ± 5. 08; P<0. 01). Conclusion We have successfully suppressed #38 gene expression by specific siRNA, which can inhibit the proliferation and invasion of human renal carcinoma cell line 786-0 and increase its sensitivity to sunitinib, paving a way for future treatment and targeted drug resistance of renal cell carcinoma.
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Purpose To investigate the effects of chemotactic factor CCR4 on the abi1ity of pro1iferation,ce11 cyc1e,invasion,and mi-gration of human ga11b1adder cancer ce11. Methods Western b1ot was used to detect the expression 1eve1 of CCR4 in ga11b1adder carci-noma ce11s. Ga11b1adder carcinoma ce11s was infected by means of s1ow virus,the CCR4 gene si1encing was conducted using siRNA-CCR4 interference techno1ogy. Ga11b1adder carcinoma ce11s GBC-SD were divided into three groups( GBC-SD,GBC-SD/CCR4-RNAi and GBC-SD/contro1). CCL17,a 1igand of CCR4,was used to act on these three groups of ce11s. CCK8 method was used to detect the ce11 pro1iferation abi1ity of three groups. F1ow cytometry was used to test ce11 cyc1e. Tanswe11 assay was app1ied to detect ce11 migration and invasion abi1ity. Western b1ot was performed to detect the expression of its corresponding 1igands CCL17 and CCL22 proteins. Re-sults CCR4 gene si1ence did not inf1uence ce11 cyc1e and pro1iferation of ga11b1adder ce11 GBC-SD,but can significant1y inhibit GBC-SD ce11 invasion and movement abi1ity,CCR4 gene si1ence had no inf1uence on the expression of CCL17 and CCL22 gene in tumor ce11s. Conclusion Ga11b1adder carcinoma ce11s GBC-SD express chemokine receptor CCR4,chemokine receptor CCR4 can promote the invasion and metastasis of GBC-SD ce11s.
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Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) may be a precursor lesion of inifltrating pancreatic ductal adenocarcinoma. The mutation of the phenotypic impact of K-ras G12D alone, silencing of p53 and p16 could promote this process. The role of Smad4 in this progression was poorly understood. In our previous studies, we investigated that RNA interference silence of Smad4 to promote the PanIN cell malignant transformation. In the present study, we investigate. The further explores the siRNA interference of Smad4 expression on PanIN cells could lead to proliferation and metastasis in vitro and in vivo. Methods:Smad4 knock-down PanIN cells (PanIN-S) were established by stable transfection with lentiviral-mediated Smad4 RNA interference. In vitro,silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. A soft agar assay was used to assess the anchorage-independent growth ability of cells. Cell migration and invasion assays were performed using transwell chambers with or without Matrigel. In xenograft model experiments, PCNA, VEGF and MMP-9 staining was separately used to evaluate cell proliferation and angiogenesis and migration (VEGF and MMP-9). Results:Effect of siRNA of Smad4 gene in PanIN cells was conifrmed by real-time RT-PCR and western blot. In vitro, silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. Soft agar assay showed that there were more colony cell numbers in PanIN-S cells compared with PanIN cells (P<0.05). Using the transwell assay, we observed that PanIN-S cells migrated faster than PanIN cells and similar results were obtained by Matrigel assay (P<0.05). Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with cell proliferation (PCNA reactivity) and angiogenesis and migration (VEGF and MMP-9), and the expressions of PCNA, VEGF and MMP-9 in PanIN-S group were signiifcantly increased (P<0.05). Conclusion:Silence of Smad4 in PanIN cells enhanced progression to invasive adenocarcinoma of the pancreas by promoting cell growth, migration and invasion. Smad4 might be a new diagnostic marker in pancreatic cancer and prove to be a feasible and novel target for therapeutic intervention.
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To explore how cytohesin-1 (CYTH-1) small interfering RNA (siRNA) influences the insulin-like growth factor receptor (IGFR)-associated signal transduction in prostate cancer, we transfected human prostate cancer PC-3 cell lines with liposome-encapsulatedCYTH-1 siRNA in serum-free medium and exposed the cells to 100 nM IGF-1. The mRNA and protein levels of the signal molecules involved in the IGFR signaling pathways were determined by real-time PCR and detected by Western blotting. The relative mRNA levels of CYTH-1, c-Myc, cyclinD1 and IGF-1R (CYTH-1 siRNA group vs scrambled siRNA group) were 0.26 vs 0.97, 0.34 vs 1.06, 0.10 vs 0.95, and 0.27 vs 0.41 (P < 0.05 for all), respectively. The relative protein levels of CYTH-1, pIGF-1R, pIRS1, pAkt1, pErk1, c-Myc, and cyclinD1 (CYTH-1 siRNA group vsscrambled siRNA group) were 0.10 vs 1.00 (30 min), 0.10 vs 0.98 (30 min), 0.04 vs 0.50 (30 min), 0.10 vs 1.00 (30 min), 0.10 vs 1.00 (30 min), 0.13 vs 0.85 (5 h), and 0.08 vs 0.80 (7 h), respectively. The tyrosine kinase activity of IGF-1R was associated with CYTH-1. The proliferative activity of PC-3 cells transfected with CYTH-1 siRNA was significantly lower than that of cells transfected with scrambled siRNA at 48 h (40.5 vs87.6 percent, P < 0.05) and at 72 h (34.5 vs 93.5 percent, P < 0.05). In conclusion, the interference of siRNA with cytohesin-1 leads to reduced IGFR signaling in prostate cancer; therefore, CYTH-1 might serve as a new molecular target for the treatment of prostate cancer.
Subject(s)
Humans , Male , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Prostatic Neoplasms/metabolism , RNA, Small Interfering/pharmacology , Receptors, Somatomedin/metabolism , Signal Transduction/drug effects , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors/genetics , Insulin-Like Growth Factor I/metabolism , Phosphorylation , Prostatic Neoplasms/pathology , Real-Time Polymerase Chain Reaction , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolismABSTRACT
Objective To explore the relationship of the ADP-ribosylation factor-like 8A (Arl8a)with TLR4-TRIF-GEFH1 -RhoB pathway in dendritic cells(DCs). Methods DCs were prepared from wildtype and TRIF-knockout (TRIFKO) mice. After LPS stimulation, the cells were collected for cDNA amplification. Real-time PCR method was used to detect Arl8a mRNA levels. DCs from wild-type mice were transfected with guanine nucleotide-exchange factors H1 ( GEFH1 ) small interference RNA ( siRNA ), Arl8a mRNA levels were examined with or without LPS stimulation. Then RhoB mRNA expression was analyzed in DCs transfected with the siRNA of GEFH1 and Arl8a gene, respectively. Results LPS induced the up-regulation of Arl8a mRNA in DCs from control mice but not in DCs from TRIFKO, indicating that LPS-mediated up-regulation of Arl8a was suppressed in TRIFKO DCs. In addition, siRNA of GEFH1 significantly suppressed the LPS-mediated up-regulation of Arl8a mRNA, RNAi of Arl8a and GEFH1 significantly decreased RhoB mRNA level in DCs after LPS stimulation ( P ＜ 0. 01 ). Conclusion The expression of Arl8a is involved in the TLR4-TRIF pathway in DCs, and Arl8a is closely associated with GEFH1 and RhoB at transcriptional level.
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Objective: To design and prepare siRNAs targeting ski gene and to observe its influence on the biological functions of HepG2 cells, such as proliferation, cell cycle, apoptosis, etc.. Methods: Three specific siRNAs of ski were designed and synthesized, and were transiently transfected into HepG2 via cathodolyte liposome transfection method. Real-time PCR and Western blotting were used to measure ski expression at mRNA and protein levels. The cell proliferation was assessed by MTT assay and the changes in cell cycle and apoptosis were evaluated by flow cytometry. Results: All the 3 specific ski-siRNA(A, B, C) effectively inhibited the expression of ski gene, with ski-siRNA-B having the highest inhibition rate(70%). Furthermore, the ski expression had a decreasing tendency with the transfection time. The proliferation of HepG2 cells was markedly inhibited by ski-siRNAs(P<0.05); the number of cells at S stage was obviously decreased, being 2 folds that of the negative control group. Conclusion: The siRNA of ski gene can effectively induce growth inhibition of HepG2 cells and reduce cells of S stage, which is possibly through down-regulation of ski gene.
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Glucose-6-phosphate dehydrogenase (G6PD) derives from the expression of the house-keeping gene G6PD. Recent studies have indicated that G6PD is related to tumor genesis, growth, clinical phenotype, therapy, and prognosis. To elucidate the relationship between G6PD and cancer, three siRNA sequences and one negative control sequence were designed based on the 3' noncoding region of the human G6PD gene. Two complementary single-strand DNA (sense and antisense) were designed and synthesized based on siRNA sequences. The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-U6.2/Lenti. One siRNA with higher interference efficiency than the other two was found after siRNA plasmid transfecting human skin A375 melanoma cells. After lentivirus particle packaging and virus production, the A375 cells were infected, and the single cell clone was acquired and cultured to establish the stable cell strain. Western blotting showed that the endogenous G6PD in the stable A375 cell strain was 0.257 ? 0.074, which was 11.17% of G6PD expression (2.301 ? 0.286) in wild type A375 cells. The final siRNA interference efficiency in this stable cell strain was 88.83%. The G6PD activity of A375-G6PD?驻 was 21.53% of A375-WT. Further study showed that A375-G6PD△ doubling generation time prolonged, and its proliferation was greatly inhibited and the cloning efficiency lowered 25%(P
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Objective To detect the effect of osteopontin (OPN) siRNA on the growth,invasion and apoptosis of human gastric cancer cell line MKN28. Methods The eukaryotic expression vector of siRNA specific for OPN was constructed by liposome transfection,and the silencing effect was identified by fluorescence quantitative PCR and immunofluorescence staining. According to the inhibitory effect of the siRNA vectors,the experiment was divided into control group,non-specific OPN siRNA group,24-hour specific OPN siRNA group,36-hour specific OPN siRNA group,and 48-hour specific OPN siRNA group. The cell proliferation,apoptosis,migration and adhesion was finally detected among control group and OPN siRNA groups at different time points. Results For 48-hour specific OPN siRNA group,the expression of OPN in MKN28 cells was the lowest. Accordingly,the cell viability was decreased and the cell proliferation inhibitory rate was reduced to 30.20%; the ability of migration and adhesion was decreased,the number of cells attached on stromal cell membrane was much lesser (21.8?6.9 vs 42.0?9.4 in control group),and the ratio of cell adhesion on basement membrane matrix and fibronectin was lower [basement membrane matrix: (41.5?8.4)% vs (20.5?4.5)%; fibronectin: (25.3?4.5)% vs (14.6?2.5)% in control group],as well as the cell apoptotic rate was increased compared with the control group. Conclusion OPN gene silencing can inhibit the growth and invasion,as well as accelerate apoptosis in human gastric cancer MKN28 cells.