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Objective:To investigate the molecular mechanism for regulation of trophoblast invasion by piR-3127964, which is differentially expressed in placental tissues of preeclamptic and healthy pregnant women.Methods:Placenta samples of healthy (control group, n=12) and preeclamptic pregnant (PE group, n=10) women who delivered by caesarean section and chorionic villi specimens of patients undergoing artificial abortion were collected in the Department of Obstetrics of the First Affiliated Hospital of Chongqing Medical University during November 2020 to August 2021. Total RNA was extracted from placenta samples and sequenced and the expression of piR-3127964 in different tissues was determined by real-time quantitative-polymerase chain reaction (qRT-PCR). The expressions of PIWI proteins including PIWIL-1, PIWIL-2 and PIWIL-3 in different tissues were detected by Western blot. The expressions of two candidate targets, guanine nucleotide-binding protein-like 3-like (GNL3L) mRNA and sialophorin (SPN) mRNA were evaluated by qRT-PCR after exogenous treating HTR-8/SVneo cells with mimics, inhibitor or negative control of piR-3127964, respectively. qRT-PCR was also used to detect the relative expression of GNL3L and SPN at mRNA level in placentas of all women. The interactions between GNL3L/SPN and piR-3127964 were analyzed by double luciferase reporter gene detection. The localization of piR-3127964 and SPN in chorionic villi was detected by fluorescence in situ hybridization and immunofluorescence. Transwell assay was performed to analyze the influence of piR-3127964 on the invasion of HTR-8/SVneo cells and the possible mechanism. Independent sample t-test, analysis of variance, and LSD post test were used for analysis Results:(1) Enrichment pathways of candidate targets predicted by differentially expressed piR-3127964 were associated with cell motility. There were statistically significant differences in piR-3127964 expression in villi, healthy and preeclamptic placentas (2.950±0.853 vs 1.036±0.303 vs 0.254±0.155, F=27.35, P<0.05), and piR-3127964 was predominantly expressed in extravillous cytotrophoblasts (EVTs). (2) The expression of PIWIL-3 protein in placentas of preeclamptic patients was significantly lower than that in healthy placentas and villi (0.810±0.400 vs 3.175±0.429 and 6.843±1.379, F=49.36, P<0.05). (3) Compared with the control group, exogenous piR-3127964 mimics (piR-mimics) and inhibitors (piR-inhibitor) significantly affected the expression of SPN mRNA (0.971±0.045 vs 0.732±0.010, F=6.50; 1.076±0.073 vs 1.293±0.092, F=7.58; both P<0.05), while the expression of GNL3L mRNA had no significant correlation with piR-3127964 level. (4) The luciferase activity of wild-type SPN (SPN-WT) plasmids was significantly affected by piR-mimics (1.010±0.049 vs 0.645±0.047, t=9.34, P<0.05) and piR-inhibitor (1.035±0.058 vs 1.397±0.015, t=－10.60, P<0.05). (5) SPN mRNA was significantly upregulated in placentas of preeclamptic patients than in healthy placentas (2.097±0.239 vs 1.305±0.290, t=－4.22, P<0.05), but no significant difference in the expression of GNL3L mRNA was observed. Immunofluorescence experiment showed that SPN was expressed in EVTs. (6) The invasive potential of HTR8/SVneo cells treated with piR-inhibitor was significantly inhibited, but this effect could be reversed by SPN knockdown (160.714±53.860 vs 371.667±103.061 and 344.333±120.267, F=9.76, both P<0.05). Conclusions:piR-3127964 expression is abnormally downregulated in placentas of preeclamptic patients, resulting in inhibition of trophoblasts invasion through upregulation of SPN expression, which may be related to the pathogenesis of preeclampsia.
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Objective:To investigate the correlation between Piwi-interacting RNA (piRNA) and diabetic nephropathy (DN).Methods:The differential expression profiles of piRNAs in renal tissues of patients with DN (experimental group) and renal tissues adjacent to tumors of patients with renal tumors (control group) were detected by high-throughput sequencing. The biological function of differentially expressed piRNAs was described by gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis. Real-time fluorescence quantitative PCR was used to detect the serum expression level of target piRNAs in patients with DN. Spearman correlation analysis was used to analyze the correlation between serum target piRNAs and clinical indexes of patients with DN.Results:The results of high throughput sequencing showed that there were 127 differentially expressed piRNAs between DN group and control group, with screening condition of |log 2(fold changes)|≥2 and P<0.05. Among them, there were 99 up-regulated piRNAs and 28 down-regulated piRNAs. The top 5 up-regulated piRNAs were piRNA-hsa-161686, piRNA-hsa-349255, piRNA-hsa-355720, piRNA-hsa-151229 and piRNA-hsa-154959, respectively. The top 5 down-regulated piRNAs were piRNA-hsa-1929960, piRNA-hsa-174194, piRNA-hsa- 148658, piRNA-hsa-172594 and piRNA-hsa-172421, respectively. The PCR verification results of 3 up-regulated genes and 3 down-regulated genes with low P values and high expression levels showed that serum expression level of piRNA-hsa-77976 was significantly down-regulated in patients with DN ( P=0.028), which was consistent with that of sequencing, while the expression levels of other genes were inconsistent with the sequencing results or had no statistical significance. Bioinformatics analysis results predicted that significantly differentially expressed piRNAs might participate in the regulation of DN through Rap1, Ras, PI3K-Akt and axon guiding pathways. The results of correlation analysis showed that the expression level of piRNA-hsa-77976 was negatively correlated with blood urea nitrogen ( r=-0.584, P=0.028), serum creatinine ( r=-0.637, P=0.014), cystatin C ( r=-0.738, P=0.003) and β2 microglobulin ( r=-0.822, P<0.001), and positively correlated with estimated glomerular filtration rate ( r=0.661, P=0.010). Conclusion:The differential expression of piRNA is closely related to DN, and may be used as a new biomarker for the diagnosis and prognosis of DN.
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Objective:To explore whether thyroxine (T 4) could promote differentiated thyroid cancer (DTC) progression by binding to integrin α vβ 3in vitro and its downstream mechanism. Methods:Papillary thyroid cancer cell lines TPC-1, K1 and follicular thyroid cancer (FTC) cell line FTC133 were cultured in vitro, and the expressions of integrin α vβ 3 in those 3 DTC cell lines were determined with immunofluorescence and flow cytometry analysis. After the treatment of T 4, tetraiodo thyroacetic acid (Tetrac) and Arg-Gly-Asp (RGD) peptide alone or in combination, the proliferation and metastatic potential of DTC cell lines were detected by cell counting kit-8 (CCK-8), Transwell migration and invasion assays. The small interfering RNA (siRNA) transfection was used to verify whether integrin α v or β 3 subunit knockdown could reverse the effect of T 4 on DTC cells. The expression levels of downstream signaling proteins phosphorylated extracellular signal-regulated kinase (p-ERK)1/2 and total extracellular signal-regulated kinase (ERK)1/2 were detected by Western blot. The effects of mitogen-activated protein kinase kinase (MEK)1/2 inhibitor (GSK1120212) on the proliferation, migration and invasion of T 4-treated cells were detected. One-way analysis of variance and Tukey test were used for data analysis. Results:The integrin α vβ 3 expressions in TPC-1, K1 and FTC133 cells were all positive, with the relative mean fluorescence intensity (MFI) of 61.93±18.61, 16.89±2.43 and 32.36±0.83, and the percentages of positive cells of (94.38±1.30)%, (74.11±3.87)% and (50.67±1.78)%, respectively ( F values: 13.36 and 217.30, P=0.006 and P<0.001). Compared with control group, the proliferation, migration and invasion in the three DTC cell lines treated with T 4 were significantly enhanced (96 h, F values: 62.67-297.50, q values: 13.15-20.73, all P<0.001). T 4-induced cell proliferation, migration and invasion were markedly reversed by Tetrac or RGD (96 h, q values: 8.61-17.54, all P<0.001). T 4-induced cell proliferation, migration and invasion were also significantly inhibited by the knockdown of integrin α v or β 3 subunit (72 h, F values: 7.75-70.98, q values: 4.77-15.21, all P<0.05). Western blot results showed that the phosphorylation levels of ERK1/2 in DTC cells were significantly increased by T 4 treatment, and the T 4-induced activation of ERK1/2 signaling pathway could be blocked by Tetrac, RGD, integrin α v or β 3 subunit knockdown. T 4-induced cell proliferation, migration and invasion were significantly reversed by GSK1120212 (96 h, F values: 47.53-151.40, q values: 10.32-16.65, all P<0.001). Conclusion:T 4 can promote cell proliferation and metastasis of DTC cells by binding to integrin α vβ 3 and activating the ERK1/2 pathway.
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Objective:To explore new methods of treating Graves′ disease (GD) by targeting thyroid stimulating hormone receptor (TSHR) and intercellular adhesion molecule-1 (ICAM-1).Methods:The small interfering RNA (siRNA) targeting TSHR and the ICAM-1 monoclonal antibody (mAb) were designed and synthesized. Thirty GD model mice were randomly divided into siRNA treatment group, ICAM-1 mAb treatment group, and untreated GD group (10 mice in each group), and 10 normal mice were taken as blank control. Serum thyroxine (T 4), thyroid stimulating hormone (TSH), TSH receptor-stimulating antibody (TSAb) and TSH-stimulation blocking antibody (TSBAb) were measured before and after treatment. At the end of the treatment, body mass and heart rate of mice in each group were measured, and thyroid uptake of 99Tc mO 4-, thyroid size and pathological changes were evaluated. Independent-sample t test, paired t test and one-way analysis of variance were used to analyze data. Results:After three treatments, the body mass of mice in siRNA group and ICAM-1 mAb group were significantly lower than that of normal mice ( F=3.50, P=0.025); the heart rates of the mice in two groups were significantly lower than that of untreated GD mice ( F=24.73, P<0.001). Heart rate of mice treated with siRNA decreased significantly, close to that of normal mice. After treatment, the serum T 4((27.58±1.94) vs (65.71±6.89) μg/L, (27.24±3.50) vs (70.84±8.46) μg/L), TSAb ((331.44±43.38) vs (457.33±45.85) mU/L, (275.16±45.80) vs (443.91±42.32) mU/L) and TSBAb ((13.94±1.11) vs (15.83±5.92) mU/L, (14.59±1.02) vs (17.05±6.16) mU/L) levels of mice in both siRNA group and ICAM-1 mAb group significantly decreased ( t values: 4.45-10.87, all P<0.05), while the serum TSH levels of mice in two groups significantly increased ((0.13±0.05) vs (0.04±0.05) mU/L, (1.46±0.34) vs (0.06±0.03) mU/L; t values: -2.22, -5.87, P values: 0.007, <0.001). The elevated TSH level and decreased TSAb level of mice treated with ICAM-1 mAb were significantly different from those treated with siRNA ( t values: 1.03, -1.63, P values: 0.002, 0.031). After treatment, the uptake of 99Tc mO 4- in part of the thyroid lobes of mice was decreased, and the enlargement degree of the corresponding lobes was reduced. The thyroid pathology of mice in the treated groups showed that the absorption vacuoles of thyroid follicles were reduced, and the phenomenon of thinner colloids was improved. No obvious damage was observed in the heart, liver and kidneys of the mice. Conclusions:Both the siRNA targeting TSHR and ICAM-1 mAb have therapeutic effects on GD model mice. The siRNA is better at controlling heart rate, and ICAM-1 mAb is better at increasing TSH and decreasing TSAb. Each of the above treatment methods is safe and effective, which can provide new ideas for GD targeted therapy.
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Objective:To explore the effect of knockdown of the homeobox gene paired-box 6 ( Pax6) on the biological behavior and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs). Methods:The SRA01/04 human LECs were divided into small interfering RNA-Pax6 (siRNA-Pax6) group transfected with siRNA-Pax6 and siRNA negative control (siRNA-NC) group transfected with disordered siRNA.Cell survival rate was detected by cell counting kit-8 method at 24, 48 and 72 hours after transfection.Cell cycle distribution and apoptosis were analyzed by flow cytometry at 48 hours after transfection.Migratory capability of cells was examined by cell scratch test at 24 hours after transfection.The mRNA relative expression levels of Pax6, α-crystallin A (CRYAA), α-crystallin B (CRYAB), Sox2, α-smooth muscle actin (α-SMA) and E-cadherin were detected by quantitative real-time PCR at 48 hours after transfection.The relative expression of Pax6 protein was detected by Western blot at 48 hours after transfection.Results:There was a significant difference in cell survival rates at different time points between the two groups ( Fgroup=4.776, P<0.05; Ftime=13.535, P<0.05). The cell survival rate of siRNA-Pax6 group was obviously lower than that of siRNA-NC group at 48 and 72 hours after transfection, and the differences were statistically significant (both at P<0.05). Compared with siRNA-NC group, the proportion of cells in G 0/G 1 phase was significantly increased and the proportion of cells in S phase was significantly reduced in siRNA-Pax6 group ( t=9.971, -5.063; both at P<0.05). The cell migration rate of siRNA-Pax6 group was (19.73±6.07)%, which was lower than (70.56±2.97)% of siRNA-NC group, showing a statistically significant difference ( t=-7.245, P<0.05). The relative expressions of Sox2 mRNA and α-SMA mRNA were lower, and the relative expression of E-cadherin mRNA was higher in siRNA-Pax6 group than siRNA-NC group, with statistically significant differences between them ( t=-23.254, -5.294, 6.062; all at P<0.01). The relative expression of CRYAA mRNA and CRYAB mRNA was significantly higher in siRNA-Pax6 group than siRNA-NC group, and the differences were statistically significant ( t=5.521, 8.270; both at P<0.01). The relative expressions of Pax6 mRNA and protein in siRNA-Pax6 group were 0.27±0.01 and 0.24±0.05, respectively, which were both lower than 1.00±0.05 and 1.14±0.10 in siRNA-NC group, showing statistically significant differences ( t=-14.456, -4.458; both at P<0.001). Conclusions:Silence of Pax6 can suppress the proliferation and EMT of human LECs and enhance the expression of crystallin.
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Objective:Apt-A10-3.2 (aptamer of prostate specific membrane antigen (PSMA)) can be used as a specific ligand for early diagnosis and targeted treatment of prostate cancer. Mouse double minute 2 homolog (MDM2) is closely related to the malignancy of prostate cancer, and MDM2 small interfering RNA (siRNA) can silence MDM2 gene through RNA interference. To design a novel chimera of PSMA Apt-MDM2 siRNA and combine it with docetaxel (DTX) to explore a new diagnosis and treatment model combining targeted therapy of PSMA-positive prostate cancer with 99Tc m-chimera imaging monitoring. Methods:Apt-siRNA were obtained by covalent connection of PSMA Apt-A10-3.2 and MDM2 siRNA, which was combined with DTX to treat PSMA-positive prostate cancer cell lines (22RV1 and LNCaP). Cell lines were treated with Apt-siRNA alone or in combination with DTX. The levels of MDM2 and apoptosis-related proteins (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), poly ADP-ribose polymerase (PARP), caspase-3) were detected by Western blot, which were used to evaluate the therapeutic effect. Fifteen BALB/c mice bearing 22RV1 xenografts were treated with PBS, DTX+ Apt-siRNA (200 pmol) and DTX+ Apt-siRNA (400 pmol), respectively. Tumor volume and MDM2 level were observed, and 99Tc m-Apt-siRNA SPECT imaging was performed to obtain the tumor/muscle (T/M) ratio. One-way analysis of variance, Tukey′s test and linear regression analysis were used for data analysis. Results:The levels of MDM2 protein were significantly decreased by Apt-siRNA (0.25±0.02, F=183.40, P<0.001; 0.56±0.03, F=37.15, P<0.001) in 22RV1 and LNCaP cells. After the treatment of Apt-siRNA+ DTX, the levels of Bcl-2 were significantly decreased, and the levels of Bax, PARP and caspase-3 were significantly increased. MDM2 protein level (400 pmol: 0.59±0.12; F=49.99, P=0.023) and tumor volume (400 pmol: (0.22±0.07) cm 3;F=71.30, P=0.039) were significantly inhibited by Apt-siRNA+ DTX in mice bearing 22RV1 xenografts. As for 99Tc m-Apt-siRNA SPECT imaging in vivo, T/M ratio of treatment group was significantly decreased (400 pmol: 2.07±0.22; F=34.99, P=0.022), and there was a linear regression relationship between T/M ratio and the expression level of MDM2 ( R2=0.875, P<0.001). Conclusion:Apt-siRNA combined with DTX can effectively inhibit the progression of prostate cancer, and realize visual targeted diagnosis and treatment of PSMA-positive prostate cancer by coupling radionuclide technetium.
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ObjectiveTo investigate the expression of the neutral cholesterol ester hydrolase 1 (NCEH1) gene in liver cancer tissue and human hepatoma cell lines and the effect of NCEH1 gene knockdown on the proliferation, apoptosis, invasion, and metastasis abilities of human hepatoma SMMC-7721 cells. MethodsLiver cancer tissue samples and adjacent tissue samples were collected from 32 patients with liver cancer who underwent surgical treatment in Guangzhou Red Cross Hospital Affiliated to Jinan University from January 2013 to June 2019, and quantitative real-time PCR was used to measure the relative expression level of the NCEH1 gene. Gene expression data of liver cancer samples up to September 2020 were downloaded from the ICGC database, and R software was used to analyze the data and obtain the expression level of the NCEH1 gene in each sample. The paired Wilcoxon signed-rank test and the Wilcoxon rank-sum test were used to investigate the differences between liver cancer tissue and adjacent tissue. Quantitative real-time PCR was used to measure the expression level of the NCEH1 gene in human hepatoma SMMC-7721, Bel-7402, HepG2, and Hep3B cells and normal human HL7702 liver cells. The lentivirus-mediated small interfering RNA (siRNA) technique was used to establish a human hepatoma SMMC-7721 cell line with NCEH1 gene knockdown, and the cells were divided into NCEH1 knockdown group (KD group) and negative control group (NC group); quantitative real-time PCR was used to measure the knockdown efficiency of the NCEH1 gene, and then MTT assay, flow cytometry with Annexin V-APC single staining, wound healing assay, Transwell assay, and Transwell chamber invasion assay were used to measure the proliferation, apoptosis, metastasis, and invasion abilities of SMMC-7721 cells in both groups. The t-test was used for statistical analysis of data between the two groups. ResultsThe mean expression level of the NCEH1 gene in liver cancer tissue was significantly higher than that in adjacent tissue (specimens from our hospital: Z=2.263, P=0.024; ICGC database: U=18 768, P＜0.001). SMMC-7721 cell line with moderate potential of invasion and metastasis had the highest expression level of the NCEH1 gene, followed by BEL-7402 and HepG2 cell lines with low potential of invasion and metastasis, and Hep3B cell line without the potential of invasion and metastasis had the lowest expression level. The KD group had a significantly lower expression level of the NCEH1 gene than the NC group (t=11.578, P=0000 3), and the knockdown efficiency of the NCEH1 gene was as high as 74.0%. Compared with the NC group, the KD group had a significant reduction in cell growth rate, a significant increase in apoptosis rate, and significant reductions in migration rate and the number of metastatic and invasive cells (t=32.100, 27.303, 9.51, 38.123, and 22.331, all P＜0.001). Conclusion There is a significant increase in the expression of the NCEH1 gene in liver cancer tissue and cell lines, and the NCEH1 gene can promote the growth, proliferation, invasion, and metastasis of hepatoma cells and inhibit their apoptosis, suggesting that it may be a potential therapeutic target for liver cancer.
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Background: Colon cancer is one of the common malignant tumors with high morbidity and mortality. Cullin7 is located at chromosome 6p21.1, which is closely related to the occurrence and development of malignant tumors. Aims: To investigate the effect of down-regulating Cullin7 on proliferation and apoptosis of colon cancer cells. Methods: qRT-PCR and Western blotting were used to detect the expressions of Cullin7 mRNA and protein in colon cancer tissue and adjacent tissue, respectively. The Cullin7 gene was silenced by siRNA, and the silencing effect was detected by qRT-PCR and Western blotting. Cell proliferation was detected by CCK-8 assay, and apoptosis was detected by flow cytometry. The protein expressions of cleaved caspase-3, β-catenin and C-myc were detected by Western blotting. Colon cancer cells were treated with siRNA Cullin7 plus Wnt signaling pathway inhibitor FH-535, cell apoptosis was detected by flow cytometry, the protein expressions of cleaved caspase-3, β-catenin and C-myc were detected by Western blotting. Results: The expressions of Cullin7 mRNA and protein in colon cancer tissue were significantly higher than those in adjacent tissue (P<0.05). Compared with the control group, the expressions of Cullin7 mRNA and protein in siRNA Cullin7 1 group, siRNA Cullin7 2 group and siRNA Cullin7 3 group were significantly decreased (P<0.05), especially in the siRNA Cullin7 2 group. Compared with the control group, after silencing the expression of Cullin7, the proliferation activity of colon cancer cells was significantly decreased, apoptosis rate was significantly increased, expression of cleaved caspase-3 protein was significantly up-regulated, and expressions of β-catenin and C-myc protein were significantly down-regulated (P<0.05). Compared with siRNA Cullin7 2 group, apoptosis rate was significantly increased, expression of cleaved caspase-3 protein was significantly up-regulated, and expressions of β-catenin and C-myc protein were significantly down-regulated in siRNA Cullin7 2 plus Wnt signaling pathway inhibitor FH-535 group (P<0.05). Conclusions: Cullin7 gene is involved in the proliferation and apoptosis of colon cancer HCT116 cells. Silencing Cullin7 can inhibit cell proliferation and induce cell apoptosis through Wnt/β-catenin signaling pathway.
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Objective@#To investigate the function and mechanism of hsa_circ_0014130 in lung adenocarcinoma cell line and to find potential molecular inhibitors.@*Methods@#The hsa_circ_0014130 expression level detection and overexpression and subtraction experiments were performed using common cell lines of lung cancer (PC9, H1299, A549, HCC827, and BEAS-2B). qPCR was used to verify the proliferation and invasion of lung cancer cells by MTS and invasion assay, and then the targeted microRNA was searched through the database. Western blot was used to detect the downstream signaling pathways, and finally the effect of small molecule inhibitors was investigated on proliferation and invasion of non-small cell lung cancer.@*Results@#The expression level of hsa_circ_0014130 was up-regulated in the three cell lines, and both the overexpression plasmid and the subtractive siRNA were effectively transfected into the cells. Overexpression of hsa_circ_0014130 was able to promote the proliferation and invasion of tumor cells, and knockdown of hsa_circ_0014130 inhibited the proliferation and invasion of tumor cells. hsa_circ_0014130 was capable to target hsa-miR-566 to reduce its expression level and to inhibit epithelial-to-mesenchymal transition. The use of the small molecule inhibitor SB-431542 and simultaneous reduction of hsa_circ_0014130 significantly inhibited the proliferation and invasion of tumor cells.@*Conclusions@#The hsa_circ_0014130 promotes the invasion and proliferation of lung cancer cells by targeting hsa-miR-566 to enhance the expression of TWIST1, and its expression level can be significantly inhibited by the small molecule inhibitor SB-431542, which significantly inhibits the proliferation and invasion of lung cancer cells. Therefore,hsa_circ_0014130 is a potential lung cancer treatment target.
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Objective@#To investigate the inhibitory effect of small interference RNA (siRNA) targeted against transforming growth factor β1 (TGFβ1) in mice with hepatic fibrosis infected with Schistosoma japonicum.@*Methods@#Three short hairpin RNAs (shRNA) targeting different positions of TGFβ1 and one unrelated control sequence (HK) were designed and cloned to a plasmid pGenesil-1 respectively to obtain four recombinant expression vectors. Thirty male BALB/c mice were randomly divided into six groups, including normal group, model group, control group (pGenesil-HK) and three treatment groups (pGenesil-TGFβ1-m1, pGenesil-TGFβ1-m2 and pGenesil-TGFβ1-m3) and each group had five mice. The hepatic fibrosis animal models infected with Schistosoma japonicum were constructed. The levels of hydroxyproline (HYP) in liver tissue were examined by biochemistry. Liver histopathology was examined by hematoxylin-eosin and Masson staining. The mRNA expression and protein expression levels of TGFβ1, mothers against decapentaplegic homolog (Smad) 3, Smad 7 and α-smooth muscle actin (α-SMA) in the livers were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot. Two independent samples t test was used to compare the measurement data between groups.@*Results@#The liver fibrogenesis was obviously improved in all treatment groups compared with model group.The levels of HYP of liver tissue in all treatment groups were significantly lower than that in model group (t=14.870, 7.097 and 10.741, respectively, all P<0.01). The mRNA expression levels of TGFβ1, Smad 3 and α-SMA(model group vs pGenesil-TGFβ1-m1 group, t=3.235, 5.141 and 10.026, respectively; model group vs pGenesil-TGFβ1-m2 group, t=3.396, 5.145 and 4.951, respectively; model group vs pGenesil-TGFβ1-m3 group, t=3.511, 5.429 and 6.485, respectively)and protein (model group vs pGenesil-TGFβ1-m1 group, t=8.847, 8.044 and 10.746, respectively; model group vs pGenesil-TGFβ1-m2 group, t=9.709, 7.484 and 10.847, respectively; model group vs pGenesil-TGFβ1-m3 group, t=9.672, 8.766 and 11.508, respectively) were significantly decreased in all treatment groups compared with model group (all P< 0.01), while the levels of Smad 7 mRNA and protein were significantly increased in all treatment groups compared with model group(t=11.742 and 11.211, respectively in pGenesil-TGFβ1-m1 group; t=14.446 and 13.736, respectively in pGenesil-TGFβ1-m2 group; t=10.892 and 10.908, respectively in pGenesil-TGFβ1-m3 group, all P< 0.01).@*Conclusions@#Specific siRNA targeting TGFβ1 could significantly inhibit the liver fibrogenesis in mice infected with Schistosoma japonicum. The anti-fibrosis mechanisms of the siRNA maybe associated with the down-regulation of TGFβ1, Smad 3 and α-SMA expressions and up-regulation of Smad 7 expression in liver tissue, which results in suppressing the activation of hepatic stellate cells.
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Objective@#To explore the mechanism of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.@*Methods@#Twist siRNA interference expression vector was constructed and NCI-H1299 cells were divided into three groups: blank control group, negative control group and experimental group. The blank control group was the untreated cell group, while the negative control group was the lentivirus transfected cell group by the blank vector. The experimental group was the lentivirus transfected cell group constructed by the lentivirus interference vector of shRNA Twist. The siRNA interference expression vector of Twist was constructed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot to detect the expression of Twist. Transwell kit was used to detect cell invasion. Cell counting kit-8 (CCK-8) kit was used to detect cell proliferation.@*Results@#⑴ The titer of lentivirus was detected. The transfection titer of lentivirus vector: shRNA-Twist vector was 3×108 TU/ml. ⑵ The results of qRT-PCR test showed that compared with the negative control group, the mRNA expression of Twist in the experimental group was decreased (q=3.177, P=0.0234). ⑶ The results of Western blot showed that compared with the negative control group, the protein expression of Twist in the experimental group was decreased (q=4.071, P=0.0304). ⑷ The results of Transwell test showed that there was significant statistical difference among the three groups (F=19.472, P=0.000). Compared with the negative control group, the number of cell imigration in the experimental group was decreased (q=3.567, P=0.0318). ⑸ The results of CCK-8 showed that there was significant statistical difference among the three groups (F=20.983, P=0.000). Compared with the negative control group, the proliferation rate in the experimental group was decreased (q=5.272, P=0.0157).@*Conclusions@#ShRNA Twist gene can significantly inhibit the proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.
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Objective To investigate the inhibitory effect of small interference RNA (siRNA) targeted against transforming growth factor β1 (TGFβ1) in mice with hepatic fibrosis infected with Schistosoma japonicum.Methods Three short hairpin RNAs (shRNA) targeting different positions of TGFβ1 and one unrelated control sequence (HK) were designed and cloned to a plasmid pGenesil-1 respectively to obtain four recombinant expression vectors.Thirty male BALB/c mice were randomly divided into six groups,including normal group,model group,control group (pGenesil-HK) and three treatment groups (pGenesil-TGFβ1-m1,pGenesil-TGFβ1-m2 and pGenesil-TGFβ1-m3) and each group had five mice.The hepatic fibrosis animal models infected with Schistosoma japonicum were constructed.The levels of hydroxyproline (HYP) in liver tissue were examined by biochemistry.Liver histopathology was examined by hematoxylin-eosin and Masson staining.The mRNA expression and protein expression levels of TGFβ1,mothers against decapentaplegic homolog (Smad) 3,Smad 7 and α-smooth muscle actin (α-SMA) in the livers were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot.Two independent samples t test was used to compare the measurement data between groups.Results The liver fibrogenesis was obviously improved in all treatment groups compared with model group.The levels of HYP of liver tissue in all treatment groups were significantly lower than that in model group (t =14.870,7.097 and 10.741,respectively,all P ＜ 0.01).The mRNA expression levels of TGFβ1,Smad 3 and α-SMA(model group vs pGenesil-TGFβ1-m1 group,t =3.235,5.141 and 10.026,respectively;model group vs pGenesil-TGFβ1-m2 group,t =3.396,5.145 and 4.951,respectively;model group vs pGenesil-TGFβ1-m3 group,t =3.511,5.429 and 6.485,respectively) and protein (model group vs pGenesil-TGFβ1-m1 group,t =8.847,8.044 and 10.746,respectively;model group vs pGenesil-TGFβ1-m2 group,t =9.709,7.484 and 10.847,respectively;model group vs pGenesil-TGFβ1-m3 group,t =9.672,8.766 and 11.508,respectively) were significantly decreased in all treatment groups compared with model group (all P ＜ 0.01),while the levels of Smad 7 mRNA and protein were significantly increased in all treatment groups compared with model group(t=11.742 and 11.211,respectively in pGenesil-TGFβ1-m1 group;t =14.446 and 13.736,respectively in pGenesil-TGFβ1-m2 group;t =10.892 and 10.908,respectively in pGenesil-TGFβ1-m3 group,all P ＜ 0.01).Conclusions Specific siRNA targeting TGFβ1 could significantly inhibit the liver fibrogenesis in mice infected with Schistosoma japonicum.The anti-fibrosis mechanisms of the siRNA maybe associated with the down-regulation of TGFβ1,Smad 3 and α-SMA expressions and up-regulation of Smad 7 expression in liver tissue,which results in suppressing the activation of hepatic stellate cells.
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Objective To explore the mechanism of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.Methods Twist siRNA interference expression vector was constructed and NCI-H1299 cells were divided into three groups:blank control group,negative control group and experimental group.The blank control group was the untreated cell group,while the negative control group was the lentivirus transfected cell group by the blank vector.The experimental group was the lentivirus transfected cell group constructed by the lentivirus interference vector of shRNA Twist.The siRNA interference expression vector of Twist was constructed by quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot to detect the expression of Twist.Transwell kit was used to detect cell invasion.Cell counting kit-8 (CCK-8) kit was used to detect cell proliferation.Results (1) The titer of lentivirus was detected.The transfection titer of lentivirus vector:shRNA-Twist vector was 3 × 108 TU/ml.(2)The results of qRT-PCR test showed that compared with the negative control group,the mRNA expression of Twist in the experimental group was decreased (q =3.177,P =0.0234).(3) The results of Western blot showed that compared with the negative control group,the protein expression of Twist in the experimental group was decreased (q =4.071,P =0.0304).(4) The results of Transwell test showed that there was significant statistical difference among the three groups (F =19.472,P =0.000).Compared with the negative control group,the number of cell imigration in the experimental group was decreased (q =3.567,P =0.0318).(5) The results of CCK-8 showed that there was significant statistical difference among the three groups (F =20.983,P =0.000).Compared with the negative control group,the proliferation rate in the experimental group was decreased (q =5.272,P =0.0157).Conclusions ShRNA Twist gene can significantly inhibit the proliferation and invasion of lung adenocarcinoma NCI-H1299 cells.
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Objective To study the effect of small interfering ribonucleic acid (siRNA) silencing apoptosis signal-regulating kinase 1 (ASK1) on inflammatory response of lipopolysaccharide-induced alveolar epithelial A549 cells and its mechanism.Method Cell inflammation model of A549 cells was induced by lipopolysaccharide.The expression of ASK 1 in A549 cells was silenced by liposome transfection of siRNA.The mRNA and expression levels of ASK1,interleukin 6 (IL-6),interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in A549 cells were detected by immunoblotting,real-time fluorescence quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.Result The expression of IL-6,IL-8 and TNF-α in the experimental group was significantly higher than that in the control group (P＜0.001),which indicated that the inflammatory model of A549 cells was successfully constructed.The mRNA level and expression of ASK1 in the interference group was significantly lower than that in the negative control group and the blank control group (P＜0.01),indicating that silencing ASK1 was also successful.The expressions of IL-6,IL-8 and TNF-α in the interference group (0.37±0.04,0.32±0.04,0.48 ±0.13) were significantly lower than those in the negative control group (1.04±0.11,1.22±0.19,0.93±0.14) and the blank control group (1.01±0.14,1.01 ±0.23,1.02±0.25).The expression of IL-6,IL-8 and TNF-α protein in the interference group (pg/ml) (122.6± 11.0,537.2±42.4,159.2± 19.6) were also significantly lower than those in the negative control group (267.4±20.4,1 289.8±55.3,327.0±26.3) and blank control group (246.6±18.7,1 300.3±35.6,325.2± 18.3),with significant difference (P＜0.05).There was no significant difference in each value between negative control group and blank control group (P＞0.05).Conclusion Silencing ASK1 by siRNA can down-regulate the expression of IL-6,IL-8 and TNF-α in A549 cells,suggesting that ASK 1 may be involved in the regulation of lipopolysaccharide-induced inflammation in A549 cells.
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Piwi-interacting RNA (piRNA) is a new class of small non-coding RNAs that function by specifically binding to the Piwi subfamily members of the Argonaute protein family. piRNA regulates the expression of target genes at epigenetic and post-transcriptional levels, and participates in the development and progression of cerebral ischemia by mediating pathophysiological processes such as inflammation, angiogenesis, and neuronal synaptic plasticity.
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Objective To explore the effects of graphene oxide (GO)-polyethylene glycol (PEG)-folic acid (FA)-pyrenemethylamine hydrochloride (PyNH2)-mediated RNA interference (RNAi) of hypoxia-inducible factor-1α (HIF-1α) on the biological behaviors of human pancreatic cancer Patu8988 cells.Methods GO-PEG-FA-PyNH2 and RNAi targeting HIF-1α gene (GO-PEG-FA-PyNH2-HIF-1α-RNAi)was constructed.The expressions of HIF-1α and glucose transporter 1 (Glut-l) in Patu8988 cells were determined after knockdown of HIF-1α by RNAi.The invasive ability,the proliferation and the cell cycle of Patu8988 cells were investigated.The effect of HIF-1α knockdown on the uptake of 18F-fluorodeoxyglucose (FDG) in Patu8988 cells was also detected.Comparison of data was conducted by one-way analysis of variance and least significant difference t test.Results The GO-PEG-FA-PyNH2 was successfully constructed,and no cytotoxicity was found.Under the hypoxia or normoxia state,the mRNA and protein levels of HIF-1α and mRNA level of Glut-1 in cells transfected with GO-PEG-FA-PyNH2-HIF-1α-RNAi (study group) were lower than those in cells transfected with GO-PEG-FA-PyNH2 (negative group) and cells transfected with Opti-minimal essential medium (Opti-MEM,control group;F=30.25-32.58,t=3.66-5.81,all P＜0.05);the numbers of migrated cells in the study group were much lower than those in the negative group and the control group (F=38.63 and 41.35,t=20.51-35.25,all P＜0.01);the cell proliferation in the study group was significantly lower than that in the negative group and the control group (F=35.19 and 38.11,t =15.11-22.19,all P＜0.05).The proportions of G0/G1 cells in the study group were higher than those in the negative group and the control group (F=34.60 and 34.83,t=11.55-34.56,all P＜0.05);the 18 F-FDG uptake in the study group was lower than that in the negative group and control group (F=29.85 and 31.69,t =3.35-4.35,all P＜0.05).Conclusion GO-PEG-FA-PyNH2-mediated HIF-1α RNAi inhibits the expression of HIF-1α in pancreatic cancer cells,leading to changes in related biological behaviors.
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Objective To investigate the effects of human leukocyte-associated antigen-G (HLA-G) expression in silencing trophoblast cell line JEG-3 under normal and hypoxic conditions on invasion and proliferation of JEG-3 cells. Methods Inhibition of HLA-G expression in JEG-3 cells by transfection of small interfering RNA (siRNA),the transfected JEG-3 cells were divided into 4 groups: normoxia control group, hypoxia control group, normoxia inhibition group and hypoxia inhibition group. The levels of HLA-G mRNA and protein in 4 groups of cells were detected by real-time quantitive PCR and western blot. The proliferation activity and invasion ability of 4 groups of cells were determined by methylthiazolyl tetrazolium (MTT) assay and invasion assay.Results (1) Real-time quantitive PCR technology showed: the level of HLA-G mRNA in the hypoxic inhibition group (0.220±0.050) was significantly different (P<0.05), when compared with that in the hypoxic control group (0.630±0.030) and normoxic inhibition group (0.400± 0.020). (2) Western blot analysis showed: the expression level of HLA-G protein in the hypoxic inhibition group was 0.260±0.010, statistically different from that in the hypoxic control group (0.850±0.100) and the normoxic inhibition group (0.560±0.020; P<0.05).(3) MTT showed: proliferative activity of JEG-3 cells in the normoxic inhibition group was 0.490 ± 0.070, the ability of cell proliferation was reduced. When compared with that in the normoxic control group (0.850±0.050), the differences was statistically significant (P<0.05). The proliferative activity of JEG-3 cells in the hypoxic inhibition group (0.330±0.070) was lower than that in the normoxic inhibition group (0.490±0.070), and there was a significant difference (P<0.05). (4) Invasion assay showed: compared with the normoxic control group (98±7), the invasive ability of JEG-3 cells in the normoxic inhibition group (73 ± 7) was weakened, and the difference was statistically significant (P<0.05). The number of transmembrane cells (52±11) of JEG-3 cells in the hypoxic inhibition group was lower than that in the hypoxic control group (72±7), and the difference was statistically significant (P<0.05). Compared with the normoxic inhibition group, the invasion ability of JEG-3 cells in the hypoxic inhibition group decreased, and the difference was statistically significant (P<0.05). Conclusion Under hypoxia, using siRNA technology to down-regulate the expression of HLA-G may affect the proliferation and invasion ability of trophoblast cells, which may be involved in the occurrence of hypertensive disorder of pregnancy.
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Objective: To investigate the effect of silencing rapamycin insensitive companion of mammalian target of rapamycin (RICTOR) gene expression on the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and to explore its possible molecular mechanism. Methods: The expression level of RICTOR protein in esophageal squamous cell carcinoma TE1, ECa109, EC9706, KYSE450 and KYSE790 cells were detected by Western blotting. RICTOR-shRNA or the Control-shRNA was transfected into ECa109 cells by LipofectAMINE, and the ECa109 cells stably expressing RICTOR-shRNA or the Control-shRNA were screened and named as ECa 109-RICTOR-shRNA or ECa109-control-shRNA cells. The effect of everolimus on the proliferation of ECa109-RICTOR-shRNA cells was detected by CCK-8 assay. The expression levels of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt)/mammalian target of rapamycin (mTOR) signal pathwayrelated RICTOR, Akt, phospho-Akt (p-Akt) (Ser473), ribosome protein subunit 6 kinase of 70 kDa (p70S6K), phospho-p70S6K (p-p70S6K), proline-rich Akt substrate of 40 kDa (PRAS40) and phospho-PRAS40 (p-PRAS40) (Thr246) proteins in everolimus-treated ECa109-control-shRNA and ECa109-RICTOR-shRNA cells were detected by Western blotting. The nude mouse xenograft tumor models of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells were established and treated with everolimus, then the effect of everolimus on tumor growth in nude mice was evaluated. Results: RICTOR protein was expressed in five esophageal squamous cell carcinoma cell lines, especially in ECa1 09 cells. Compared with the Control-shRNA, RICTOR-shRNA inhibited the proliferation of ECa1 09 cells. Everolimus inhibited the proliferation of ECa1 09-RICTOR-shRNA and ECa109-control-shRNA cells, especially to the former; the values of half maximal inhibitory concentration (IC50) were (17.68± 1.25) μmol/L and (36.84±1.57) μmol/L, respectively. The RICTOR-shRNA decreased the expression levels of p-Akt (Ser473) and p-PRAS40 (Thr246) (both P 0.05), which indicated that RICTOR-shRNA inhibited the phosphorylated activation of Akt and PRAS40 induced by everolimus. Both RICTOR-shRNA and everolimus inhibited the growth of ECa109 cell xenografts in nude mice (all P < 0.05), while the inhibitory effect was strongest in RICTOR-shRNA+everolimus group (P < 0.001). Conclusion: Silencing RICTOR gene can improve the sensitivity of esophageal squamous cell carcinoma cells to everolimus, and the molecular mechanism may be associated with the down-regulation of RICTOR expression to inhibit the phosphorylated activation of Akt and PRAS40 induced by everolimus.
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Objective To analyze differentially expressed proteins in A375 melanoma cells before and after short hairpin RNA (shRNA)-mediated Cbl-b gene silencing.Methods The label-free quantitative proteomics approach was performed to identify differentially expressed proteins between A375 cells transfected with lentiviral vectors containing Cbl-b shRNA (Cbl-b shRNA group) and those with control lentiviral vectors (control group).Then,the properties of differentially expressed proteins were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis.Western blot analysis was conducted to determine the expression of differential proteins (EphA2 and GSK3β) and phosphorylated protein kinase (p-AKT) after shRNA-mediated Cbl-b gene silencing.Statistical analysis was carried out by t test of two independent-samples for comparison of protein expression abundance between the two groups with SPSS 23.0 software.Additionally,the results of GO and KEGG enrichment analysis were analyzed by Fisher's exact test.Results A total of 3 449 proteins were identified and quantified,and 74 of them were differentially expressed between the Cbl-b shRNA group and control group.Compared with the control group,52 proteins were up-regulated and 22 were down-regulated in the Cbl-b shRNA group.GO enrichment analysis of differential proteins revealed that the top five significantly enriched biological processes were integrin-mediated cell adhesion,single-organism metabolic process,regulation of integrin-mediated cell adhesion,regulation of protein-targeting mitochondria and nucleic acid metabolic process.The top five significantly enriched molecular functions included DNA binding,2-iron,2-sulfur cluster binding,signaling receptor activity,cadherin binding and cell adhesion molecule binding.The top five significantly enriched cell components included nucleosome,DNA packaging complex,photoreceptor connecting cilium,DNA-protein complex and extracellular region part.KEGG enrichment analysis demonstrated that the top five significantly enriched melanoma-related signaling pathways were folate biosynthesis,axon guidance,extracellular matrix-receptor interaction,adherens junction and Wnt signaling pathways.As Western blot analysis revealed,the Cbl-b shRNA group showed lower protein expression of EphA2 (0.369),but higher protein expression of GSK3β (3.524) compared with the control group (1),which were consistent with the results of proteomics analysis.Additionally,the protein expression of p-AKT was down-regulated in Cbl-b shRNA group (0.453) compared with the control group (1).Conclusion Cbl-b may be involved in the occurrence of melanoma through a variety of biological pathways,and the EphA2/PI3K/AKT signaling pathway may be one important pathway.
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Objective To evaluate the effect of small interfering RNA (siRNA)targeting survivin gene on the expression of survivin and proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro.Methods A survivin-specific siRNA was designed and synthesized.Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA (survivin siRNA group),50.0 nmol/L liposome complexes containing unrelated siRNA (negative control group) and 50.0 nmol/L prepared vesicles (blank control group).Real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of survivin in A431 cells,respectively.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate cellular proliferative activity,flow cytometry using annexin-V/propidium iodide (PI) staining to detect cell apoptosis,Transwell assay to estimate migratory and invasive activities of A431 cells,and flow cytometry to detect cell cycle changes.Results At 48 hours after transfection,the mRNA and protein expression of survivin both significantly differed among the survivin siRNA group,negative control group and blank control group (mRNA:0.56 ± 0.15,0.88 ± 0.37,0.90 ± 0.43,F =276.67,P ＜ 0.001;protein:0.59 ± 0.04,0.86 ± 0.05,0.91 ± 0.07,F =243.61,P ＜ 0.001),the survivin siRNA group showed significantly lower mRNA and protein expression of survivin compared with the negative control group and blank control group (all P ＜ 0.05),and there were no significant differences between the negative control group and blank control group (both P ＞ 0.05).Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells (F =13.19,P =0.004),the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group (both P ＜ 0.05),and no significant difference was observed between the negative control group and blank control group (P ＞ 0.05).At 24 hours after transfection,the apoptosis rate significantly differed among the 3 groups (F =83.97,P =0.002).The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group (both P ＜ 0.05),and there was no significant difference between the negative control group and blank control group (P ＞ 0.05).At 48 hours after transfection,the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase,but lower number of migratory cells and invasive cells compared with the negative control group and blank control group (all P ＜ 0.05).Conclusion Survivin-specific siRNA can inhibit the expression of survivin gene and the proliferation of A431 cells,promote cell apoptosis,and suppress cell migration and invasion,indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.