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Objective@#To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma (HCC) cells.@*Methods@#The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore, the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated by immunohistochemical staining.@*Results@#Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9, conjugated SUMO1, free SUMO1 and AKT1 in HCC cells (P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%, 54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were (59.47±4.66) μm and 89.44±8.36, (56.56±5.37) μm and 93.84±8.79, as well as (34.57±6.61) μm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were (3.78±0.69) g, (3.72±0.72) g and (2.09±0.61) g, respectively. The corresponding apoptotic cell rates were (7.79±2.21)%, (6.45±2.48)% and (33.59±5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group (P<0.05).@*Conclusions@#Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.
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Objective To investigate the inhibitory effect of dexamethasone on residual Lewis lung cancer cells in mice after palliative surgery.Methods The model of residual Lewis lung cancer cells in C57BL mice after palliative surgery were established,then accordance with the random number table,18 mice were randomly divided into 3 groups with 6 animals in each group:the normal saline group,cisplatin group,and dexamethasone group.After operation,the subcutaneous tumor nodules of each mouse were measured on days 4-10 using vernier calipers (accuracy of 0.l mm).The expressions of hypoxia induction factor-1α (HIF-1 α) and mean vascular density (MVD) in the normal saline group,cisplatin group and dexamethasone group were assessed by paraffin immunohistochemical assay.The expressions of HIF-1α,VEGF and PCNA mRNA and protein in the three groups were assessed by real-time quantitative PCR and Western blotting.Results Tumor growth curve showed that the tumor volume in cisplatin group (200.34 ± 20.94) mm3 and in dexamethasone group (436.58 ± 37.94)mm3 were obviously decreased compared with the normal saline group (1 398.81 ± 192.85) mm3,with statistically significant differences (t =-1201.75,P ＜ 0.001;t =-921.52,P ＜ 0.001).As Paraffin immunohistochemical assay showed,in cisplatin group and dexamethasone group,the expressions of HIF-1 α(2.67 ± 0.43,1.67 ± 0.43) and MVD counts (17.01 ± 3.24,9.89 ± 2.25) were decreased significantly compared with the normal saline (4.21 ± 0.35,29.75 ± 5.64),with statistically significant differences (t =-1.55,P＜0.001;t=-1.83,P＜0.001;t=-12.68,P＜0.001;t=-18.35,P＜0.001).The results of real-time quantitative PCR showed that the expressions of HIF-1α (0.56 ±0.11),VEGF (0.61 ±0.18) and PCNA mRNA (0.38 ± 0.07) in dexamethasone group were obviously reduced compared with the normal saline group (1.21 ±0.13,1.13 ± 0.26,1.06 ± 0.08),with statistically significant differences (t =-0.55,P ＜ 0.001;t=-0.62,P＜0.001;t=-0.69,P＜0.001).The expressions of HIF-1α (0.31 ±0.12),VEGF (0.30 ± 0.13) and PCNA mRNA (0.18 ± 0.06) in cisplatin group were also obviously reduced compared with the normal saline group,with statistically significant differences (t =-0.73,P ＜ 0.001;t =-0.76,P ＜ 0.001;t =-0.81,P ＜ 0.001).The results of Western blotting showed that the expressions of HIF-1α (85.98 ± 20.86),VEGF (173.28 ± 30.98) and PCNA protein (228.96 ± 22.97) in dexamethasone group were decreased significantly compared with the normal saline group (198.98 ± 29.89,378.98 ± 28.98,357.98 ± 35.98),with statistically significant differences (t =98.78,P ＜ 0.001;t =85.68,P ＜ 0.001;t =120.86,P ＜ 0.001).The expressions of HIF-1 α (65.78 ± 18.62),VEGF (109.43 ± 19.86) and PCNA protein (176.86 ± 22.76) in cisplatin group were decreased significantly compared with the normal saline group,with statistically significant differences (t =132.86,P ＜ 0.001;t =108.68,P ＜ 0.001;t =154.74,P ＜ 0.001).Conclusion Dexamethasone can effectively inhibit the growth and angiogenesis of the residual Lewis lung carcinoma after palliative surgery in mice,and may also provide a new method of postoperative adjuvant therapy for patients,especially who received palliative surgery.
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Objective To evaluate the expression levels of peptidylarginine deiminase 4 (PADI4)and B-cells pecific Moloney leukemia virus insert site-1 (BMI-1 )in esophageal squamous cell carcinoma (ESCC) tissues and pericarcinous tissues.To explore the function and clinical significance in the development of ESCC and their association.Methods The expression levels of PADI4 and BMI-1 were measured by immunohisto-chemistry,Western blotting and quantitative real time PCR in ESCC tissues and pericarcinous tissues from 86 patients.The relationships between the expressions of PADI4 and BMI-1 and the clinicopathologic characte-ristics were analyzed.Results The immunohistochemistry showed that the expressions of PADI4 and BMI-1 in ESCC tissues (68.6% and 73.3%)were significantly higher than those in pericarcinous tissues (37.2% and 30.2%,χ2 =1 7.01 1 ,P =0.000;χ2 =31 .876,P =0.000).Western blotting indicated that the levels of PADI4 and BMI-1 were higher than those in pericarcinous tissues (0.91 9 ±0.098 vs.0.71 8 ±0.1 03,t =2.462,P =0.021 ;0.975 ±0.074 vs.0.71 7 ±0.071 ,t =2.640,P =0.01 4).The expressions of BMI-1 and PADI4 mRNA in ESCC tissues were higher than those in pericarcinous tissues,but the differences were not sta-tistically significant (0.091 ±0.005 vs.0.038 ±0.002,t =1 .701 ,P =0.1 01 ;0.1 1 4 ±0.075 vs.0.048 ± 0.003,t =1 .499,P =0.1 46)by the quantitative real time PCR.The expression of PADI4 was correlated with lymph node metastasis (χ2 =5.771 ,P =0.01 6),depth of invasion (χ2 =6.672,P =0.01 0)and clinical stage (χ2 =5.771 ,P =0.01 6).The BMI-1 gene expression had a correlation with lymph node metastasis (χ2 =7.1 76,P =0.007),the differentiation degree (χ2 =1 3.787,P =0.001 )and clinical stage (χ2 =7.1 76,P =0.007).In addition,there was a positive correlation between PADI4 and BMI-1 expression in ESCC by immunohistochemistry and quantitative real time PCR (r =0.21 4,P =0.047;r =0.534,P =0.005).Conclusion The expression levels of PADI4 and BMI-1 are significantly higher in ESCC compared to pericarcinous tissues.PADI4 and BMI-1 are positively correlated and may contribute to the diagnosis and prog-nosis of the ESCC.
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Objective To explore the regulation mechanism for miR - 125a - 5P in epidermal growth factor receptor(EGFR)signaling pathway in medulloblastoma. Methods The potential targets of miR - 125a - 5P in the EGFR signaling pathway were predicted by TargetScan and Sanger software,there were 3 groups:control group,non -sense group and miR - 125a - 5P group. Their relationship,between miR - 125a - 5P and cyclin - dependent kinase in-hibitor 2B( CDKN2B),E2F transcription factor 3( E2F3),mitogen - activated protein kinase 14( MAPK14)and growth factor receptor - bound protein 10(GRB10),were tested by luciferase experiments. After miR - 125a - 5P oligo-nucleotide was transfected to D341 cells,miR - 125a - 5P level was detected by reverse transcription polymerase chain reaction. Then the thiazolyl blue tetrazolium bromide assay was used to draw the cell growth curves,and Transwell assay was used to detect cell migration ability. The expression levels of GRB10,EGFR,phosphatidylinositol 3 - kinase(PI3K) and Ras were tested by Western blot method. Results The results of luciferase experimental results showed that GRB10 was the only target gene of miR - 125a - 5P. After miR - 125a - 5P being transfected,the D341 cell prolifera-tion obviously declined markedly. Compared with control group[(38. 16 ± 7. 47)% ]and the non - sense group [(36. 79 ± 8. 94)% ],cell migration rate in the miR - 125a - 5P group was lowest[(13. 59 ± 4. 41)% ],and there was a significant difference among 3 groups(χ2 = 11. 495,P < 0. 05);in the miR - 125a - 5P group,the expression level of EGFR increased 1. 67 times,GRB10,PI3K and Ras levels were reduced to 23% ,61% and 42% . Conclusion miR - 125a - 5P can inhibit tumor growth by silenced GRB10 expression targeting EGFR downstream signaling pathways in medulloblastoma.
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The manuscript introduced the overview, training objectives, policy advantages, training process,curriculum, examination of the Australian College in Rural and Remote Medicine and further contrasted that with China's domestics.The authors held that Australia's training is better targeted due to its colleges tailored to this end;the training duration for general practitioners of rural and remote areas is longer,and the training schedule is reasonable;the curriculum design and training content are more targeted;and the homogeneous training is better achieved as its examination is run by the college in a standardized manner.The authors therefore hold that China should develop detailed regulations for general practitioners from rural and remote areas and explore the feasibility of setting up second-level disciplines institutes for internal medicine, surgery, gynecology and obstetrics, pediatrics and general at national and provincial level.
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<p><b>OBJECTIVE</b>To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).</p><p><b>METHODS</b>Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T. The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry. The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot. The cell growth curve was drawn by MTT test. The Ki-67-positive rate was calculated using immunofluorescence staining. The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.</p><p><b>RESULTS</b>Western blot results showed that BTG2 relative expression levels were 0.83 ± 0.12, 0.18 ± 0.04, 0.20 ± 0.05 and 0.36 ± 0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively. The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5% (33/40), 77.5%(31/40) and 17.5% (7/40), respectively, with a significant difference between two groups (P < 0.05). Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2 ± 8.0)%, (81.4 ± 9.7)% and (50.1 ± 7.1)%, respectively, showing a significant difference between two groups (P < 0.05). The scratch test results showed that in the control group, empty vector group and BTG2 transfection group, the distance of SW620 cells between two sides was (79.27 ± 11.24) µm, (80.65 ± 12.17) µm and (124.77 ± 19.63) µm, respectively, with a significant difference between two groups (P < 0.05). Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5 ± 13.1)%, (73.2 ± 12.9)% and (47.4 ± 9.1)%, respectively, showing a significant difference between two groups (P < 0.05). The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.</p><p><b>CONCLUSIONS</b>BTG2 gene is involved in colon cancer cell proliferation and metastasis, and effectively restores the function of BTG2 protein. Therefore, it may be expected to become a new option in gene therapy for colon cancer.</p>
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Humanos , Linfocitos B , Fisiología , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Genética , Neoplasias del Colon , Vectores Genéticos , Proteínas Inmediatas-Precoces , Genética , Inmunohistoquímica , Plásmidos , Transfección , Proteínas Supresoras de Tumor , GenéticaRESUMEN
Objective To study the effect of carbonated drinks on primary and permanent teeth replacement in Chil-dren. Method Dog tooth enamel samples were soaked in coca-cola, sprite and pure soda, and the calcium, phosphorus lev-el were analysed. Dental papilla stem cells were separated and cultured in the conditioned medium by adding three drinks. PCR and western blot were used to detect mRNA and protein levels of activator of nuclear factor-k B receptor ligand (RANKL), osteoprotegerin (OPG) and vitamin D receptor (VDR) , then the possible role of each gene and interactions rela-tionship were analyzed. Results Compared with saline, coca-cola and sprite showed their significantly decalcification and dephosphorization role, while plain soda water showed calcium and phosphorus protective effect. These three drinks had no effect on mRNA and protein levels of RANKL gene (P>0.05). Coca-cola and sprite can reduce OPG mRNA and protein lev-els, and at the same time increase transcription and expression of the VDR gene. Plain soda water has no effect on the OPG gene manifestation, but can significantly reduce the transcription and translation level of the VDR gene. Conclusion Car-bonated drinks may affect the dental health of the children's primary and permanent teeth replacement by regulating bone re-lated gene expression and vitamin D receptor family.
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Objective To study the functional roles of 1,25(OH)2D3 in osteogenic differentiation of the dental papilla stem cells. Methods The dental papilla stem cells were isolated and cultured in medium supplemented with different con-centrations of 1,25(OH)2D3 (1, 10 and 100 nmol/L). MTT assay was used to detect the cell growth, and flow cytometry was used to detect the cell cycle. Western blot assay was used to detect protein expression levels of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG) and vitamin D receptor (VDR). After siRNA silencing VDR expression, protein levels of RANKL and OPG were detected. Results MTT and flow cytometry results showed that there were no sig-nificant differences in the cell proliferation between different concentrations of 1,25(OH)2D3 (1, 10 and 100 nmol/L) and con-trol groups (P>0.05). Western blot results showed that there were protein expressions of VDR, RANKL and OPG in control group. The protein expressions of VDR, RANKL and OPG were increased after adding 1,25(OH)2D3, in which the upward trend was the most significant in VDR. After VDR expression was silenced by siRNA, the protein expression levels of VDR, RANKL and OPG were decreased. Conclusion 1,25(OH) 2D3 affects the osteoblast differentiation process of the dental pa-pilla stem cells by adjusting the VDR expression.
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Objective To analysis the malignant performance characteristics of tumor stem cell-like side popula-tion cells in patients with cervical cancer. Methods The cervical cancer cells were obtained from surgical resection tumor tissue. The tumor stem cell-like side population cells were isolated by flow cytometry. The cell growth curve was drawn by MTT assay. The invasion ability of tumor cells was compared by transwell assay. The clonogenic capacity was detected by clone formation in soft agar. The expression level of ABCG2 protein, a drug-resistant gene, was detected by immunofluores-cence method. Finally, these cells were transplated into the subcutaneous of de thymus mice. The rate of tumor formation was compared between groups. Results The results from flow cytometry assay showed the percentage of cervical cancer stem cell-like side population cells was 1.39%. Compared with the non-side population cells, the side population cells grow quickly, showed the enhanced invasion ability and colony forming ability. There was more high expression level in ABCG 2 protein of side population cells. The tumor form rate was 100%(10/10) in the side population cells and the non-side popula-tion cells was 20%(2/10). Conclusion The cervical cancer stem cell-like side population cells have more malignant perfor-mance characteristics than that of non-side population cells, which maybe a core target for cancer gene therapy in the future.
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Objective To study the function of the nano-molecular polyamide-amine (PAMAM) as microRNA(miR) carrier targeting gastric adenocarcinoma, and the foundation of developing an efficient delivery of small molecule drugs tar-geting gastric cancer thereof. Methods The folic acid (FA)/PAMAM comoles compound was prepared by dialysis method. After transfection of miR-7 or liposomes into SGC-7901 cell line, fluorescence microscope was used to detect the gene trans-fect efficiency. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the miR-7 level. The immu-nocytochemistry assay was used to test the protein expressions of epidermal growth factor receptor (EGFR), protein kinase B (PKB) and proliferating cell nuclearantigen (PCNA). The transwell system was utilized to explore the migration ability of tu-mor cells. Results Compared with liposme, FA/PAMAN complex compound can significantly improve the level of miR-7 in SGC-7901 cells,reduce the protein levels of EGFR, PKB and PCNA in SGC-7901 cells, and also reduce the percentage of cancer cell migration (P<0.05). Conclusion PAMAM can effectively transfect miR into gastric cancer cells, which is expected to become an efficient delivery of small molecule drugs.
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Objective To investigate etiologies,treatment approaches and prophylaxis for delayed thoracostomach emptying in esophageal or gastric cardiac cancer patients treated with esophagogastrostomy.Methods We performed a retrospective review of the clinical data of 24 patients suffering delayed thoracostomach emptying among 1985 post-surgical patients with esophageal or gastric cardiac cancer from January 2000 to June 2011.Results Eighteen patients in the 24 patients were cured by conservative managements including endoscopic dilatation procedures.The remaining 6 patients were treated with surgery.Conclusion The main etiology of delayed thoracostomach emptying is gastroparesis,which can be treated with nonsurgical conservative approaches; whereas mechanical emptying disturbance requires surgery.Endoscopic examination appears to be the most important diagnostic approach in identifying and differentiating the etiologies of delayed thoracostomach emptying in post-surgical patients.Endoscopic dilatation procedure is proved to be effective for the treatment of delayed thoracostomach emptying in post-surgical patients in this study.
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Objective: To investigate the relationship of loss of P16 expression with biologlical behaviors and prognosis of gastrointestinal stromal tumors(GISTs). Methods: The expression of P16 protein and mRNA was detected in GISTs tissues by immunohistochemistry,Western blot and RT-PCR.The prognosis was evaluated through follow up. Results: The expression rates of P16 protein and mRNA in GISTs were 53.8%(21/39，Frozen tissue)，51.3%(20/39，Frozen tissues)and 51.4%(37/72，Paraffin-embedded tissues)，respectively.The expression of P16 was significantly different among GISTs of different aggressive risk(P<0.05).With the incease of aggressive risk，the expression of P16 was deceased.The expression of P16 was negtively correlated with Ki-67 and patient survival(P<0.05). Conclusion: The loss of P16 expression has a positive correlation with the infiltration and progression of GIST.Detection of P16 protein and mRNA is helpful for the evalutaion of biological behaviors and prognosis of gastrointestinal stromal tumors.
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Objective To evaluate the efficacy of sufentanil combined with propofol for video-assisted endoscopic transthoracic sympathectomy.Methods Twenty ASA I or II patients of both sexes aged 17-40 yr weighing 52-75 kg undergoing video-assisted endoscopic transthoracic sympathectomy were enrolled in this study.Anesthesia was induced with propofol 2.0-2.5 mg/kg and sufentanil 0.5 μg/kg.Tracheal intubation was facilitated with atracurium 0.6 mg/kg.The patients were mechanically ventilated (VT=8-10 ml/kg,RR=10-12 bpm,I:E =1:2,FiO2=80%).Anesthesia was maintained with infusion of propofol 2-4 mg·kg-1·h-1 and sufentsnil 0.2-0.3/.μg·kg-1 h-1 and intermittent iv boluses of atracurium.At the 30 rain before the end of operation propofol infusion was reduced to 1-2 mg.kg-1·h-1 and sufentanil infusion to 0.1 μg·kg-1 h-1 .BP (SP,DP) and HR were recorded and venous blood samples were taken before induction of anesthesia (baseline),at tracheal intubation at the moment of CO2 insnfflation 10 min and 30 min after CO2 insufflation,5 min after deflation and at extubation for determination of plasma corticesteroid,aldosterone and glucose levels.The duration from termination of infusion of the anesthetics to recovery of spontaneous breathing,eye opening at command and tracheal extubation were recorded.Results SP,DP and HR were within the normal range.Plasma levels of comcesteroid,aldosterone and blood glucose were significantly increased during operation as compared with the baseline values.The duration from termination of infusion of the anesthetics to recovery of spontaneous breathing,eye opening at command and tracheal extubation were4.5±1.9,6.4±2.7 and (12.6±1.5)min respectively.Conclusion Sufentanil 0.1-0.3 μ·kg-1·h-1 combined with propofol 1-4 mg·kg-1.h-1 can inhibit stress response during video-assisted endoscopic transthoracic sympathectomy with stable hemodynamics.