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Objective To investigate the effect of miR-200 a mimic on transforming growth factor β1-mediated acti-vation and collagen secretion of rat pancreatic stellate cells .Methods PSCs were isolated and cultured from pan-creatic tissue and identified by desmin , GFAP and α-SMA immunofluorescence staining .PSCs of 2nd generation were divided into control group , TGF-β1 group, TGF-β1+miR-NC group and TGF-β1+miR-200a mimic group.α-SMA and collagen Ⅰ protein were measured by Western blot and immunofluorescence staining .The mRNA ofα-SMA and collagen Ⅰ and the expression of miR-200a were detected by quantitative real-time PCR.Results TGF-β1 stimulates the activation of PSCs and promote collagen synthesis in time-dependment manner ( P<0.05 ) . After transfection of the mimic , treating with the same concentration of TGF-β1, the expressions of protein and mR-NA of both α-SMA and collagen Ⅰ decreases significantly ( P<0.01 ) .Conclusions Over-expression of miR-200 a significantly attenuates α-SMA activity and further affects the collagen synthesis of TGF-β1-dependent activa-tion of PSCs.The mechanisms are potentially related to the biological effects of TGF-β1.
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Objective To explore the expression of urothelial carcinoma-associated 1 ( UCA1 ) in pancreatic cancer cell lines and its influence on the invasion and metastasis of the pancreatic cancer cells .Methods The expression of UCA1 in pancreatic cancer tissues and paired adjacent normal tissues ( 11 cases ) and 5 pancreatic cancer cell lines was analyzed by real-time PCR.The level of UCA1 in BxPC-3 was knocked down by small interfering RNA . The ability of invasion and migration in vitro of transfected BxPC-3 was detected by Transwell invasion assay and wound healing assay .The protein levels of MMP-2 and MMP-9 were measured by Western blot experiment .Results The expression level of UCA1 in pancreatic cancer tissues was higher than that in paired adjacent normal tissues , and UCA1 differentially expressed in 5 pancreatic cancer cell lines .Down-regulation of UCA1 by siRNA suppressed the expression of MMP-2 and MMP-9 in BxPC3, and dramatically impaired the ability of invasion and migration of BxPC-3.Conclusions UCA1 is over-expressed in pancreatic cancer , and down-regulation of UCA1 attenuates the capacity of invasion and metastasis in vitro of BxPC-3 by decreasing MMP-2 and MMP-9.
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<p><b>OBJECTIVE</b>To investigate the reversal effect of targeted modulation of bcl-2 expression by miR-15a and miR-16 on drug resistance of human colon cancer cells.</p><p><b>METHODS</b>Mimics or inhibitors of miR-15a and miR-16 were transfected into HCT8 or HCT8/VCR cells with the help of Lipofectamine 2000. The expressions of miR-15a and miR-16 mRNA were detected by RT-qPCR. The levels of bcl-2 and P-gp proteins were measured by Western blot. The inhibitory effects of VCR on growth of HCT8 and HCT8/VCR cells were detected by CCK8.</p><p><b>RESULTS</b>After transfection of the mimics, the expression of miR-15a in the blank control group, negative control group and miR-15a mimic group was 1.00, 0.87 ± 0.24, and 223.44 ± 59.07, respectively, and miR-15a was increased significantly (P < 0.001). The expression of miR-16 in the blank control group, negative control group and miR-16 mimic group was 1.00, 0.66 ± 0.19, and 107.32 ± 22.58, respectively, and miR-16 expression was increased significantly (P < 0.001). The Western blot assay showed that the relative expressions of bcl-2 protein in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group were 1.00, 0.97 ± 0.02, 0.51 ± 0.06, and 0.65 ± 0.03, respectively, and the expression of bcl-2 protein was decreased significantly (P < 0.05), however, the expressions of P-gp protein showed no significant difference. The CCK8 test showed that at 1, 5, 25 and 125 µg/ml concentration of VCR, the survival rates of HCT8/VCR cells were basically the same in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group, but the survival rate of HCT8/VCR cells was significantly decreased after transfection of mimics (P < 0.05). After transfection of the inhibitors, the expressions of both miR-15a and miR-16 were decreased significantly (P < 0.001). The Western blot showed that the expression of bcl-2 protein was increased (P < 0.05), while the expression of P-gp protein showed no significant difference. The CCK8 test showed that the survival rate of HCT8 cells which were transfected with inhibitors was significantly higher than that of the blank control group (P < 0.05).</p><p><b>CONCLUSIONS</b>miR-15a and miR-16 may reverse the drug resistance in human colon cancer cells. A possible mechanism is regulating the expression of bcl-2.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Cell Line, Tumor , Colonic Neoplasms , Drug Resistance , MicroRNAs , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Genetics , Metabolism , RNA, Messenger , TransfectionABSTRACT
Objective To investigate the effect of sodium valproate in suppression of cell proliferation and arrest of cell cycle of in human hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988.Methods Hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988 were inoculated on the culture plate,cultured in the DMEM medium,they were intervened with sodium valproate in concentration of 0.2 mmol/L (0.2 mmol/L group),1.0 mmol/L (1.0 mmol/L group),5.0 mmol/L (5.0 mmol/L group) and dimethyl sulfoxide (control group) for 48 h respectively.Absorbance was measured by enzymelinked immunosorbentassay equipment,and inhibition ratio was calculated.Cell cycle was detected by flow cytometry.Results The absorbance of hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988 in 5.0 mmol/L group were significantly lower than those in control group and 0.2 mmol/L group (0.569 ±0.059 vs.0.706 ±0.033 and 0.760 ±0.020,2.068 ±0.178 vs.2.793 ±0.144 and 2.663 ± 0.078,P ＜ 0.05),the absorbance of pancreatic cancer cell line PaTu8988 in 1.0 mmol/L group (2.432 ± 0.084) was significantly lower than that in control group and 0.2 mmol/L group (P ＜ 0.05).With the sodium valproate concentration increased,inhibition rate of tumor cell increased gradually,the inhibition rate of hepatoma cell line SMMOL/LC-7721 and pancreatic cancer cell line PaTu8988 in 5.0 mmol/L group was 23.5％ and 25.9％ respectively.Compared with control group,with the sodium valproate concentration increased in 0.2,1.0,5.0 mmol/L group,the proportion of G1 phase cell increased gradually in hepatoma cell line SMMOL/LC-7721 [(49.25 ± 1.63)％,(65.26 ± 2.34)％,(83.13 ± 1.78)％ vs.(49.22 ± 4.35)％],the proportion of S phase cell decreased gradually [(26.84 ± 2.30)％,(17.76 ± 3.90)％,(3.38 ± 0.65)％ vs.(29.21 ± 2.35)％],cell cycle showed G1 phase arrest,there was significant difference (P ＜ 0.05).Compared with control group and 0.2 mmol/L group,the proportion of G2 phase cell increased in pancreatic cancer cell line PaTu8988 in 1.0 and 5.0 mmol/L group [(26.80 ± 1.50)％,(36.58 ± 3.78)％ vs.(12.00 ± 4.62)％,(16.54 ± 2.26)％],cell cycleshowed G2 phase arrest,there was significant difference (P ＜ 0.05).Conclusion Sodium valproate mightsignificantly suppress the cell proliferation in hepatoma cell line SMMOL/LC-7721 and pancreatic cancercell line PaTu8988 and induce cell cycle arrest,it is clinically promising antitumor drug.
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ObjectiveTo assess the effects of DNA methyhransferase 1 ( DNMT1 ) gene silencing on DNMTs activity and methylated CpG sites of hMLH-1 in pancreatic cancer cell line PaTu8988.Methods DNMT1 siRNA and negative control siRNA was constructed by Ambion Company of United States.Then they were transfected into pancreatic cancer cell line PaTu8988 at the concentrations of 15,30 nmol/L,and the cells without transfection was used as the control group.Real-time PCR and Western blotting were applied to detect the DNMT1 mRNA and protein expression,and DNMTs activity was detected by using DNMTs activity assay kit.Change of methylation of CpG island of hMLH-1 was detected by bisulfite sequencing PCR (BSP).The expression of hMLH-1 mRNA was detected by Real-time PCR.ResultsAt 48 h after transfection,Realtime RT-PCR analysis showed that the levels of DNMT1 mRNA in DNMT1 siRNA group ( 15 nmol/L) and DNMT 1 siRNA group (30 nmol/L) were 0.573 ± 0.026 and 0.143 ± 0.044,which were significantly lower than those in control group 1.020 ±0.217 and negative siRNA 15 nmol/L group 0.900 ±0.475,and negative siRNA 30 nmol/L group 0.938 ± 0.327 (P ＜0.05 ).Western blotting analysis showed that the level of DNMT1 protein of DNMT1 siRNA group was also lower than those of negative siRNA and control groups.DNMT activity in DNMT1 siRNA15,30 nmol/L groups was 0.364 ± 0.124and 0.250 ± 0.072,which were significantly lower than those in control group 0.931 ± 0.065and negative siRNA group 0.665 ± 0.055 and 0.472 ± 0.040.DNMT activity was positively correlated with DNMT1 mRNA expression ( r =0.69,P ＜ 0.01 ).DNMT1 RNA interference decreased 8 methylated CpG sites of hMLH-1 to 1 site.Concluslons DNMT1siRNA can specifically inhibit the expression of DNMT1 gene of PaTu8988 and DNMT activity,and can decrease methylated CpG sites of hMLH-1 gene.
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Objective To investigate the expression of DNMT3b and its correlation with clinicopathological parameters of pancreatic cancer.Methods The expressions of DNMT3b protein in 12 pancreatic cancer tissues and para-cancerous tissues were detected by Western blot.The expressions of DNMT3b protein in 59 pancreatic cancer tissues were detected by immunohistochemistry.The association of DNMT3b expression and clinicopathological parameters of pancreatic carcinoma were analyzed.Results Western blot results showed the expressions of DNMT3b protein in pancreatic cancer tissues and para-cancerous tissues were 0.69 ±0.13and 0.14 ±0.03,and the protein level of DNMT3b in pancreatic cancer tissues were significantly higher than that in para-cancerous tissues (t =4.464,P ＜0.05 ).Immunohistochemistry examination showed that the positive rates of DNMT3b protein were 59％ in pancreatic cancer tissues and negative in para-cancerous tissues.DNMT3b expression was positively correlated with the TNM stage and lymph node metastasis.Conclusions DNMT3b is highly expressed in pancreatic cancer tissues,and it is related with malignant biological behaviors of pancreatic cancer cells.
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Objective To investigate the correlation between the ratio change of circulating myeloid-derived suppressor cells (MDSCs) and cellular immune function in healthy volunteers,chronic gastritis patients,gastric intraepithelial neoplasia patients and gastric cancer patients.Methods From February 2011 to July 2011,129 peripheral blood samples were collected,including 32 healthy volunteers,48 chronic gastritis patients,27 gastric intraepithelial neoplasia patients and 22 gastric cancer patients.The percentages of peripheral blood MDSC,T lymphocyte subsets and regulatory T cells (Treg) were determined by flow cytometry.The data were analyzed by one way analysis of variance,pearson and spearman correlation.Results The percentages of circulating MDSC,CD8+ T lymphocyte and Treg were highest in gastric cancer patients (9.63％±3.24％,10.03％ ± 1.26％,69.45％±3.42％) and lowest in healthy volunteers (0.92％±0.33％,4.12％ ±0.99％,32.35％ ±4.83％).Those of gastric intraepithelial neoplasia patients (5.13％ ± 1.30％,7.54％ ± 0.79％,53.26％±4.30％) were lower than gastric cancer patients but higher than chronic gastritis patients (2.76％ ±0.64％,6.28％ ±0.61％,42.37％ ±4.02％).The differences among each groups were statistically significant (F=24.85,20.88,37.84,all P＜0.05).However,the percentage of circulating CD4+T lymphocyte was highest in healthy volunteers (65.10％±4.10％),55.15％ ± 4.00％ in chronic gastritis group,42.23％ ± 3.91％ in gastric intraepithelial neoplasia group,and lowest in gastric cancer group (26.84％ ± 3.69％).The differences among each groups were statistically significant (F=46.80,P＜0.05).A significant correlation between circulating MDSC and TNM stages of gastric cancer was also observed (r=0.856,P＜0.01).The percentage of circulating MDSC was positively correlated with Treg percentage (r =0.862,P ＜ 0.01),and negatively correlated with CD4+/CD8+ ratio (r=-0.768,P＜0.01).Conclusion The increase of MDSC percentage in peripheral blood is correlated with human cellular immune function,which might play an important role in the tumor immune evasion during the development of gastric cancer.
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Objective To detect the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in the early secondary infection of acute necrotizing pancreatitis (ANP) and to probe its diagnostic value for early infection. Methods Twenty-four male SD rats were randomly divided into the control (C) group, the ANP group and the secondary infection of ANP (SIANP) group. The constructions of the models were achieved through intraperitoneal injection of L-arginine and E. coli. After 24 hours, the blood and peritoneal fluid samples were collected for bacterial culture, and the serum levels of amylase, CRP, TNF-α and TREM-1 were detected. The pathological changes in the pancreas were observed. The expression of TREM-1 mRNA and TREM-1 protein in pancreatic tissue was detected by Real-time PCR and Western Blot. Results The histological score of pancreas, and serum amylase in ANP group and SIANP group were significantly higher than those in C group; the positive rate of bacterial culture of blood and peritoneal fluid in SIANP group was 100% , which suggested the model was successfully established. CRP and TNF-a levels in SIANP group were (8.7 ±3.1)mg/L and (185.7 ± 10.9) mg/L, which were not significantly different from that in ANP group [( 16.5 ±3.6) , ( 176.0 ± 18.6) mg/L]. The serum level of TREM-1, expression of TREM-1 mRNA and TREM-1 protein in pancreatic tissue was (9.3 ±0.9) ng/ml, 14.84 ± 3.45, 316.2 ± 59.2, which were significantly higher than those in ANP group [ (5.5 ±0.3)ng/ml, 4.51 ±1.44, 188.6 ±42.4, P <0.05]. Conclusions TREM-1 has diagnostic value for early secondary infection of ANP.
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Objective To explore the diagnosis value and the mechanism of triggering receptor expressed on myeloid cells-1 (TREM-1) in early liver damage of severe acute pancreatitis with secondary infection. Methods Twenty-four male SD rats were randomly divided into the control group, the severe acute pancreatitis (SAP) group and the secondary infection of SAP (SISAP) group.The animal model was established by intraperitoneal injection of L- arginine and E. coli. After 24 hours, the serum levels of amylase, glutamate aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor (TNF)-α, C-reactive protein (CRP) and the variation of TREM-1 expression were tested. The blood and peritoneal fluid samples were collected for bacterial culture.Part of the pancreas and liver tissue were taken for histopathological score under microscope. The expression of TREM-1 at mRNA and protein level in liver tissue was detected through Real-time PCR and Western Blot. Results The histological score of pancreas and liver, serum amylase, ALT and AST were significantly higher in the SAP and SISAP groups than those in C group (P<0. 05), and higher in SISAP group than in SAP group (P<0. 05). The CRP and TNF-a expression in SAP and SISAP groups were higher then those in control group, while there was no significant difference between the two groups (P=0. 262 and 0. 359 , respectively). The positive ratio of bacterial culture in blood and peritoneal fluid was 0(0/8), 12. 5% (1/8), and 100% (8/8) in control group, SAP group and SISAP group respectively. The expression of TREM-lmRNA in liver was 2. 10 ± 0. 33 in SAP group and 4. 58+ 1. 00 in SISAP group, which were significantly higher than that in control group (1. 00,P<0. 05) , and the expression of TREM-1 mRNA in SISAP group was higher than that in SAP group (P < 0.05). The expression of TREM-1 at protein level was higher in SISAP group,significantly stronger than that in control and SAP group. Conclusions TREM-1 may play an important role in the early liver damage caused by severe acute pancreatitis.
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Objective To investigate the expressions of DNA methyltransferase 1 (DNMT1) in pancreatic carcinoma and its clinical significance.Methods 30 samples of pancreatic cancer tissues and paired para-cancerous tissues were collected from patients who underwent curative pancreatectomy.The levels of DNMT1 mRNA were detected by real-time RT-PCR.Expressions of DNMT1 protein were detected by streptavidin peroxidase immunohistochemistry.The relationships between expression of DNMT1 and clinicopathological findings were analyzed.Results The value of relative quantification (RQ) of DNMT1 mRNA in human pancreatic cancer tissues was 2.32 (1.17 ～ 5.17 ), which was significant higher than 0.78 (0.07 ～3.14) in para-cancerous tissues(P ＜0.05).The index of expression of DNMT1 protein in human pancreatic cancer tissues was (54.5 ±21.2)% ,which was significant higher than( 10.9 ± 15.0)% in paracancerous tissues (P ＜ 0.01 ).Patients were divided into the high DNMT1 group (n = 19) with above 54.5% of the DNMT1 positively cancer cells and the low DNMT1 group ( n = 11 ) with less than 54.5% of the DNMT1 positively cancer cells.The high expression of DNMT1 was correlated significantly with clinical staging ( x2 =6.897, P = 0.029), lymph node metastasis ( x2 = 4.739, P = 0.029) and neural invasion ( x2 = 5.44, P =0.020).On the other hand, no association between DNMT1 expression and age, gender, tumor location,tumor size, tumor differentiation, the serum CEA and CA19-9 levels could be found.Conclusions DNMT1 mRNA and protein was highly expressed in pancreatic cancer tissues.High expression of DNMT1 might be related to the aggressiveness of pancreatic cancer, lymph node metastasis and neural invasion.
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Objective To study the effects and mechanism of evodiamine on human colon cancer cell. Methods After human colon cancer SW620 cell were treated with different doses of evodiamine, the growth of anchorage independence of cancer cell was studied by colony formation in soft agar, and invasion ability was determined by Boyden chamber,and the level of mRNA and protein of midkine gene was detected by real time RT-PCR and Western blot assay, respectively. Results Ecodiamine could significantly inhibit both invasion ability and anchorage independence growth in dose-dependent manners. The level of mRNA and pro-tein of midkine of cancer cells treated with evodiamine reduced in time-and dose-dependent manners Conclusion Evodiamine could inhibit invasion of colon carcinoma cell through down-regulating of midkine expression.
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ObjectiveTo study the effects and mechanism of survivin gene on invasion of prostate cancer cell.MethodsAfter PC-3 prostate cancer cell lines were transfected by survivin small interfering RNA (siRNA), the mRNA and protein of survivin and matrix metalloproteinase-2 and-9 were determined by real time RT-PCR and western blot assay, respectively. The anchorage-independent growth was examined by clon formation in soft agar, and invasion ability was evaluated by boyden chamber model.The invasion ability of cancer cells in vivo was determined by nude mice model. ResultsThe results from clon formation in soft agar showed that the colonies numbers of group of 3.125,6.250 and 12.500 nmol/L of siRNA were 17.8±1.6,13.6±1.5 and 8.8±1.4, and the control group was 22.6±1.8(P<0.05). The results from boyden chamber model exhibited that the cells numbers crossing filter membrane in group of 3.125,6.25 and 12.5 nmol/L of siRNA were 33.6±2.1,19.5±1.9,8.1±1.83, and the control group was 49.4±2.3(all P<0.05). The results in vivo showed that cancer cells of control groups invaded into striped muscle and blood vessel, and there were no these phenomenons in transferred group with survivin siRNA. Survivin siRNA could reduce expression level of MMP-2 and MMP-9 in prostate cancer cells(P<0. 01).Conclusions Survivin-directed RNA interference can inhibit invasion of human prostate cancer cell through down-regualting MMP-2,-9 genes.
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OBJECTIVE: To establish a HPLC method for the determination of the content of Demethylbellidifolin in different parts of Swertia davidi Franch. METHODS: The analysis was carried out on Hypersil C18 column (150mm?4.6mm,5 ?m) at room temperature with mobile phase consisted of CH3OH-0.5%H3PO4(56∶44) at a flow-rate of 1.0mL?min-1.The detection wavelength was set at 254 nm. RESULTS: The linear range of Demethylbellidifolin was 0.52～2.60?g (r=0.999 4) and the average recovery was 99.77%(RSD=0.95%).CONCLUSION: The method is simple, rapid, reproducible, and suitable for the determination of the content of Demethylbellidifolin in Swertia davidi Franch..
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AIM: To study the effects of Cripto gene on vascular endothelial growth factor(VEGF) of colon carcinoma cells.METHODS: Cripto siRNA was designed and constructed.Colon cancer LS-174T cells were divided into 4 groups: control group and different dose (3.125,6.25 and 12.5 nmol/L) of siRNA groups.After transfected for 24,48 and 72 h,colon cancer cells were harvested to carry on the next tests.Expression of Cripto mRNA was determined with real-time PCR,and immunofluorescence isothiocyanate(FITC) labeling assay and Northern blotting were performed to examine the expression of protein and mRNA of VEGF,respectively.The cells in control group and cells transfected with 12.5 nmol/L siRNA were inoculated into nude mice respectively.30 days after inoculated,the mice of two groups were executed,and immunohistochemical(ICH) assay was used to evaluate the VEGF protein of mice tumor.RESULTS: siRNA down-regulated the Cripto mRNA in a dose and time dependent manner.Protein and mRNA of VEGF in transfected cells reduced in a dose and time dependent manner.Compared to control,the expression of VEGF protein from ICH assay was lowered significantly(P