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Objective:To investigate the effect of BMAL1 gene on the proliferation, migration and invasion ability of radiation-resistant nasopharyngeal carcinoma cell line (5-8FR) and the molecular mechanism. Methods:A multi-target click model was constructed for radiation-resistant nasopharyngeal carcinoma cell line 5-8FR by low-dose fractionated irradiation, and the results of clone formation assay were used to fit the multi-target click model and calculate the sensitization ratio of radiotherapy. The expression levels of PI3K/Akt/MMP-2/9 signaling pathway-related proteins in 5-8FR and control 5-8F cell lines were detected by Western blot. The overexpression and knockdown vectors of BMAL1 gene were constructed and transfected with 5-8F and 5-8F cell lines, respectively. The BMAL1 gene overexpression (pcDNA-BMAL1) and its control (pcDNA) and interference (BMAL1-shRNA) and control (con-shRNA) cell lines were stably transfected with nasopharyngeal carcinoma cell line 5-8F and radiation-resistant cell line 5-8FR, respectively. Western blot was performed to verify the infection efficiency and detect the changes of PI3K/Akt/MMP-2/9 signaling pathway-related proteins after overexpression or interference of BMAL1 gene in both groups of cells. CCK-8 assay, cell scratch test and Transwell chamber test were conducted to investigate the proliferation, migration and invasion capabilities of 5-8FR cell line after overexpression or interference of BMAL1 gene. Results:BMAL1 gene expression was down-regulated, and those of PI3K/Akt pathway proteins and downstream related molecules of MMP-2 and MMP-9 were up-regulated, and TIMP-2 and TIMP-1 expression was down-regulated in nasopharyngeal carcinoma radiation-resistant cell lines. Overexpression of BMAL1 gene inhibited the expression of PI3K/Akt pathway proteins and downstream related molecules of MMP-2 and MMP-9, promoted the expression of TIMP-2 and TIMP-1, and inhibited the proliferation, migration and invasion capabilities of radiation-resistant nasopharyngeal carcinoma cells, while interference with BMAL1 gene yielded the opposite results. Conclusions:BMAL1 gene can reverse the expression of PI3K/Akt/MMP-2/9 signaling pathway-related proteins in radiation-resistant nasopharyngeal carcinoma cell lines and inhibit the proliferation, migration and invasion capabilities of radiation-resistant nasopharyngeal carcinoma cell lines.
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Objective@#To evaluate the long-term effect and safety of chrono-chemotherapy combined with intensity modulated radiotherapy (IMRT) in locally advanced nasopharyngeal carcinoma (NPC).@*Methods@#160 patients with locally advanced NPC were randomly divided into a chrono group and conventional group according to random number table. In the first stage, all patients underwent two cycles of induced chemotherapy, consisting of docetaxel, cisplatin and 5-Fu every 21 days. Notably, patients received chrono-moduated chemotherapy according to circadian rhythm in the chrono group, and conventional chemotherapy in the conventional group. Then, 21 days after the completion of first stage, three cycles of concurrent cisplatin chemotherapy every 21 days were given to all patients during IMRT. The median follow-up after the completion of radiotherapy was 31 months. Long-term side effects and the survival of patients were observed.@*Results@#Patients in the chrono group had significantly lower rates of hearing loss (22.72%), dysphagia (0) and neck fibrosis (4.54%) compared with those in the conventional group (39.13%、8.69%, 15.94%, respectively, all P<0.05). Meanwhile, the 1- year overall survival rates (97.0% vs 92.8%), 3-year overall survival rates (80.3% vs 81.2%), 1-year progression free survival rates (95.5% vs 87.0%), 3-year progression free survival rates (71.2% vs 73.9%), 1-year locoregional relapse-free survival rates (97.0% vs 95.7%), 1-year locoregional relapse-free survival rates (92.4% vs 92.8%), 1-year distant metastasis-free survival rates (97.0% vs 98.6%) and 3-year distant metastasis-free survival rates (90.9% vs 91.3%) between the chrono group and the conventional group were not statistically significant (all P>0.05).@*Conclusions@#Compared with conventional chemotherapy, chrono-chemotherapy combined with IMRT didn′t affect long-term survival, but reducing the incidence of adverse events in patients with locally advanced NPC.
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Objective @# To investigate the expression of N6-methyladenosine modification ( m6 A) and related en- zymes FTO,METTL3 and YTHDF2 in gastric epithelial cells infected with Helicobacter pylori (Hp) ,and to analyze its expression and clinical significance in gastric cancer by using online websites.@*Methods @#Gastric epithelial cells GES-1 and AGS were infected with Hp GZ7 strain,and the multiplicity of infection was 30 ∶ 1.Total RNA and total protein were collected after infecting 24 h,and the m6 A modification level of total RNA was detected by RNA dot hybridization.The mRNA and protein expressions of FTO ,METTL3 and YTHDF2 were detected by q-PCR and Western blot.The expression difference of FTO,METTL3 and YTHDF2 in gastric cancer tissues and normal tissues and their expression in Stage classification were analyzed by GEPIA,and the relationship between the expression of FTO,METTL3 and YTHDF2 and prognosis was analyzed by Kaplan-Meier Plotter. @*Results @# Compared with the un- infected group,the m6 A modification level of total RNA in both Hp-infected GES-1 and AGS cells significantly de- creased,the mRNA and protein expression levels of FTO increased,and the mRNA and protein levels of METTL3 and YTHDF2 decreased,with statistical significance (P<0. 05) .The expression level of FTO in gastric cancer tis- sues was higher than that in normal tissues,with statistical significance (P<0. 05) ,YTHDF2 was higher than that in normal tissues,with no statistical significance,and the expression level of METTL3 was lower than that in nor- mal tissues,with no statistical significance.Patients with high expression of FTO and METTL3 had a lower survival rate,while patients with high expression of YTHDF2 had a higher survival rate.The expression of FTO was signifi- cantly correlated with Stage classification (P<0. 05) ,while the expression of METTL3 and YTHDF2 was not sig- nificantly correlated with Stage classification.@*Conclusion @# Hp infection can change the level of m6 A and the ex- pression of related enzymes FTO,YTHDF2 and METTL3,suggesting that m6 A methylation may play a role in the occurrence and development of Hp-induced gastric cancer.
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Objective To evaluate the differences of toxicities,therapeutic efficacy and immune function between induction chemotherapy followed by sinusoidal chrono-modulated infusion and flat intermittent infusion of cisplatin (DDP)with intensity-modulated radiotherapy (IMRT) in patients with locoregionally advanced nasopharyngeal carcinoma (NPC).Methods Seventy patients with biopsydiagnosed stages Ⅲ and Ⅳ B NPC (according to the 2010 UICC staging system) were treated with two-cycle induction chemotherapy before chemoradiotherapy in Guizhou Cancer Hospital.The TPF chemotherapy regimen was administered as follows:The TXT and DDP with the dose of 75 mg/m2 was carried out by bolus infusing for the first day,the 5-FU with 750 mg · m-2 · d-1 was carried out by continuous intravenous pumping for the first day to fifth day(120 h).The induction chemotherapy was 21 days per cycle,for two cycles.After that all patients were randomly treated with 2-3 cycles of sinusoidal chronomodulated infusion or flat intermittent constant rate infusion of DDP with IMRT.Using a multi-channel programmed pump,the patients were given 12 h continuous infusions of DDP (100 mg/m2) for day one,repeated every 3 weeks for 2-3 cycles.DDP was administered from 10:00 am to 10:00 pm.Concurrent radiotherapy regimen was administered as follows:GTVnx 69.96-73.92 Gy/33 f,PTVnx 69.96 Gy/33 f,PTVnd 69.96 Gy/33 f,PTV1 60.06 Gy/33 f,PTV2 50.96 Gy/28 f.Results The main toxicities of chemoradiotherapy in the group of sinusoidal chrono-modulated infusion were bone marrow suppression:leukocytes,and then nausea,oral mucositis and hemoglobin.The main toxicities of chemoradiotherapy in the group of flat intermittent constant rate infusion were bone marrow suppression:hemoglobin,leukocytes,and then nausea,oral mucositis.No significant differences were observed for toxicities(P > 0.05).After concurrent chemoradiotherapy,the complete response rate (CR),partial response rate (PR),stable disease rate(SD),progressive disease rate (PD) and overall response rate (ORR) were 11.4%,85.7%,2.9%,0 and 97.1% in the group of sinusoidal chrono-modulated infusion.The CR,PR,SD,PD,ORR in the group of flat intermittent constant rate infusion were 22.9%,74.2%,2.9%,0,97.1%,respectively.However,there was no significant differences of effect in the two Arms (P > 0.05).For sinusoidal ehrono-modulated infusion and flat intermittent infusion group,the 2-year overall survival(OS) were 82.9% and 94.3% respectively,the 2-year progression-free survival(PFS) were 77.1%,91.4% respectively,and the 2-year distant metastasis free survival (DMFS) were 82.9%,91.4% respectively.The value of CD3 + in the group of sinusoidal chrono-modulated infusion was higher than the group of flat intermittent constant rate infusion after therapy (Z =3.254,P < 0.05).The value of CD4 +,CD8 +,CD16 + CD56 +,CD19 +,and CD4 +/CD8 + had no differences in two Arms (P > 0.05).Conclusions No significance differences on the toxicities,therapeutic efficacy and survival were observed between the two groups,but immune function might be improved in the sinusoidal chrono-modulated infusion group.
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Objective To investigate the efficacy,adverse reactions and immune function of time-adjusted chemotherapy combined with intensity-modulated radiation therapy (IMRT) and conventional chemotherapy combined with IMRT for locally advanced nasopharyngeal carcinoma.Methods Random number grouping method was used to divide 66 cases of locally advanced nasopharyngeal carcinoma into 2 groups,of which 36 cases in the time-adjusted chemotherapy group and 30 cases in the conventional group.Both of them received docetaxel + cisplatin + fluorouracil regimen to induce chemotherapy for 2 cycles.The time-adjusted chemotherapy group was treated with intravenous injection of electronic automatic injection pump,the conventional group was treated with conventional intravenous infusion,and both groups were treated with synchronous cisplatin combined with IMRT.Calculated survival rate was generated by Kaplan-Meier method and long-term adverse reactions was evaluated according to CTC 3.0 criteria.Results The 3-year overall survival (OS) rate was 86.1% and 93.3% in the time-adjusted chemotherapy group and the regular group,the 3-year progress-free survival (PFS) was 83.3% and 93.3%,the 3-year RFS was 88.5% and 93.3%,and the 3-year recurrence-free survival was 94.1% and 100% respectively with no statistically significant difference (P > 0.05).The dryness and hearing loss of the time-adjusted chemotherapy group had a decreasing trend compared with the conventional group.However,CD3 +,CD3 + CD4 +,CD3 + CD4 + CD8 +,and CD4 +/CD8 + of the time-adjusted chemotherapy group had an increasing trend compared with the conventional group.Conclusions Both time-adjusted chemotherapy and conventional chemotherapy combined with IMRT had comparable mid-term efficacy,but the former had lower adverse reactions,improved quality of life and immune function.Trial registration Chinese clinical trial registry,ChiCTR1800016809
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Objective To investigate the effects of blocking gastrin receptor on the proliferation,apoptosis and expression of key proteins in the related pathway in gastric cancer cell lines.Methods In the experimental group,the gastric cancer cell lines SGC-7901 and AGS cells were treated with 5 mmol/L proglumide,a kind of a gastrin receptor antagonist.And the normal cultured gastric cancer cells SGC-7901 and AGS were used in control group.The growth of each group was detected by MTT assay;the cell growth curve was drawn by flow cytometry;the cell cycle of each group was detected by flow cytometry.Annexin V-FITC/PI double staining was used to detect the cell growth of apoptosis.The relative mRNA expression of β-catenin,nuclear factor-P65,mammalian target of rapamycin and glycogen synthase kinase 3 beta in Wnt,NF-κB and PI3K-AKT-MTOR pathways were detected by RT-qPCR.The expression of β-catenin protein was detected by Western blotting.Results After treatment with proglumide,the growth of the cells in the experimental group was lower than that in the control group;and the proportion of S phase cells in the cell cycle was also lower than that in the control group,but the proportion of cells in G0/G1 phase was higher than that in the control group(P<0.05).The percentage of apoptotic cells was also increased after treatment with proglumide(P<0.05).Furthermore,proglumide treatment significantly reduced the expression of β-catenin at both mRNA and protein levels(P<0.05).Conclusion Blocking gastrin receptor can down-regulate the expression of β-catenin,inhibit the cell proliferation and promote the cell apoptosis in gastric cancer cells.
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Objective:To study the effects of ascorbic acid on BMP-2 mRNA expression of osteoblast cell sheets.Methods:Rat os-teoblasts were primaryly cultured and identified;osteoblast cell sheets were built by physical scraping method in vitro;the osteoblast cell sheets were cultured with 1 5,50 and 85 mg/L ascorbic acid for 1 and 2 weeks respectively,and the expression of BMP-2 mRNA of the cell sheets was detected by RT-qPCR.Results:The obtained cells were conformed to be osteoblasts.The osteoblast cell sheets could be rolled into tube in vitro.The expression of BMP-2 mRNA of osteoblast cell sheets in experiment group,whether in week one or week two was higher than that in control group,50 mg/L group showed the highest expression(first week P 0.05);the expression of any group in week two was higher than that in week one(P <0.05).Conclusion:Ascorbic acid may pro-mote the expression of BMP-2 mRNA in osteoblast cell sheets.
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<p><b>OBJECTIVE</b>To establish an animal model of gastric cancer by long-term infection of Helicobacter pylori (H.pylori) and to elucidate the pathogenesis by proteomics analysis.</p><p><b>METHODS</b>Fifty male Mongolian gerbils (4-5 week-old and weighted 60-100 g) were infected with H.pylori and the gastric tissues were obtained after the infection at 3, 6, 12 and 24 months. Histological changes were evaluated by H-E staining of the gastric tissue sections. Detection of H.pylori was performed by in-vitro culture of fresh gastric tissue samples, PCR amplification of H.pylori 16s rRNA and localization by silver staining. In addition, proteins extracted from gastric tissue samples were subjected to two-dimensional electrophoresis (2-DE) at various infection time points. Protein spots with increased quantity over the course of H.pylori infection were selected and analyzed by LC-MS/MS. Finally, differentially expressed proteins between human gastric cancer tissue samples and lymph nodes were analyzed by real-time RT-PCR.</p><p><b>RESULTS</b>Colonization of H.pylori was observed in gastric tissue of gerbils as early as 3 months after H.pylori infection, and persisted till 24 months. Pathological examination of infected animals showed various histological changes including acute gastritis, atrophic gastritis, intestinal metaplasia and gastric carcinoma. Seventy-eight differentially expressed proteins were identified by proteomics analysis, among which 36 proteins were up-regulated and 42 were down-regulated. Analyzed by LC-MS/MS, ten proteins were identified, including lactate dehydrogenase, ATP synthase, fatty acid-binding protein, COX5B, peroxiredoxin-4, peroxide reductase, transgelin, succinyl-CoA ligase, keratin and protein disulfide-isomerase A2, among which transgelin, ATP synthase and lactate dehydrogenase were highly expressed in human gastric carcinoma and lymph nodes.</p><p><b>CONCLUSIONS</b>H.pylori infection induces the expression of transgelin, ATP synthase and lactate dehydrogenase, implying possible roles in the pathogenesis of gastric diseases including cancer.</p>
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Animales , Humanos , Masculino , Modelos Animales de Enfermedad , Gastritis , Microbiología , Patología , Gerbillinae , Infecciones por Helicobacter , Metabolismo , Helicobacter pylori , Genética , L-Lactato Deshidrogenasa , Metabolismo , Metaplasia , Proteínas de Microfilamentos , Metabolismo , Proteínas Musculares , Metabolismo , Proteómica , ATPasas de Translocación de Protón , Metabolismo , ARN Ribosómico 16S , Neoplasias Gástricas , Metabolismo , Microbiología , Espectrometría de Masas en TándemRESUMEN
Objective To study the effect of gastrin on the proliferation and angiogenesis of human umbilical vascular endothelial (HUVE) cell in vitro.Methods The immunocytochemistry assay,realtime-PCR,and Western blot were used to detect the gastrin receptor (CCK-BR) expression in HUVE cells.After HUVE cells were treated with 10 and 100 nmol/L gastrin for 72 h,MTT and soft agar colony formation assay were used to test the cell proliferation rate and colony formation rate,and the vascular-like structures were observed by three-dimensional culture of HUVE cells.The half-ring and ring vascular numbers were counted with five random visions.The vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1a (HIF-1a) mRNA and protein levels in HUVE cells were detected by realtime PCR and ELISA assay respectively.Results HUVE cells expressed CCK-BR.After the treatment with 10 and 100 nmol/L gastrin,the cell proliferation rate was increased by 48.48% and 82.82 % compared to the control group,and colony formation rate was increased to 31.33% and 45.67 % as compared with 15.33% in the control group(P<0.01).Relative expression quantities of VEGF and HIF-1a genes were 2.3 and 4.6 folds (VEGF) and 20.76 and 26.77 folds (HIF-1 a) than those in the control group.The concentration of VEGF protein in culture medium was 221 and 392 μg/mg protein higher than that in control group.The numbers of half-ring and ring vascular structures were (14.00 ± 3.00,39.33 ± 7.57 and 34.33 ± 4.50)/vision and (8.33 ± 2.51,41.33 ± 5.85 and 37.67 ± 3.51)/vision in control,10 and 100 nmol/L gastrin-treated groups,respectively (P<0.01).Conclusion Gastrin up-regulates the expression of VEGF and HIF-1a genes in HUVE cells and promotes cell proliferation and vascular-like structure formation of HUVE cells in vitro by being combined to CCK-BR,which may be involved in the development and metastasis of gastric cancer.
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<p><b>OBJECTIVE</b>To test expression of carbonic anhydrase II (Ca II) mRNA in osteoclasts which were applied with fluid shear stress.</p><p><b>METHODS</b>The bone marrow cells of Sprague-Dawley rats were cultured with the presence of 1,25-(OH)2D3 and dexamethasone. The osteoclast-like cells were identified by tartrate-resistant acid phosphatase(TRAP) staining and scanning electron microscope (SEM) observation, then purified with trypsin/ethylenediamine tetraacetic acid (EDTA). Different values and lasting time of steady fluid shear stress were exerted on the osteoclasts with parallel plate flow system. The Ca II expression of osteoclasts were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and nested polymerase chain reaction(PCR).</p><p><b>RESULTS</b>The levels of Ca II mRNA were down-regulated correspondingly with the increase of stress and time (P < 0.05).</p><p><b>CONCLUSION</b>It's indicated that steady fluid shear stress within a certain range may down-regulate the expression of Ca II in osteoclasts.</p>
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Animales , Ratas , Células de la Médula Ósea , Anhidrasa Carbónica II , Células Cultivadas , Osteoclastos , Reacción en Cadena de la Polimerasa , ARN Mensajero , Ratas Sprague-Dawley , Estrés MecánicoRESUMEN
Objective To study whether Helicobacter pylori CagA protein can control gastrin gene expression and the detailed mechanism. Methods First, pcDNA3. 1ZEO (-)/cagA7 was transfected into gastric cancer cell lines AGS and SGC-7901 cells. At the same time, culturing the Helicobacter pylori NCTC11637 and infecting AGS and SGC-7901 cells with it. Next, in the infected and transfecled AGS and SGC-7901 cells, respectively adding the JAK2 signaling pathway inhibitor AG490 and the ERK signaling pathway inhibitor U0126 to inhibit the two signaling pathway. Untreated gastric cancer cells and empty vector transfected cells as the control. Using real-time fluorescence quantitative PCR to detect the levels of gastrin mRNA in transfected and infected cells. Results After AGS and SGC-7901 cells were transfected with pcDNA3. lZE0(-)/cagA7 and infected with NCTC11637, the results showed that the expression of gastrin mRNA increased significantly (P < 0. 05) in transfected and infected cells as compared with the control group, but after adding the inhibitor AG490 and U0126 respectively, the expression of gastrin mRNA decreased significantly(P<0.05). Conclution These results suggest that CagA may up-regulate the expression of the gastrin gene, and CagA is one of the important proteins in regulating gastrin gene expression. The ERK/MAPK and JAK/STAT signaling pathways may be involved in controlling of gastrin gene expression by CagA.
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This study was conducted to explore whether cholecystokinin-B/gastrin receptor (CCKBRwt) gene and its alternative splicing variant (CCKBRi4sv) are expressed in human gastric carcinomas cell line and tissues, and to find out their relationship with the development and progression of human gastric carcinoma. The mRNA expression levels of CCKBRwt and CCKBRi4sv were detected in 30 human gastric carcinomas and normal tissues adjacent to cancer, 10 gastritis specimens, 2 autopsied normal stomach specimens as well as in a gastric carcinoma cell line SGC-7901 cells. The results revealed that the transcripts of CCKBRwt and CCKBRi4sv were observed in all of the human gastric specimens tested, but only CCKBRwt was expressed in gastric cancer cell line SGC-7901 cells. The expression levels of the two receptors were not correlated with the differentiation and metastases of gastric cancers. From the results, we infer that human gastric tissues simultaneously express CCKBRwt and CCKBRi4sv, and CCKBRi4sv may have unknown physiological functions in gastric epithelial cells.