Objective:To investigate the effect of ribonucleotide reductase regulatory subunit M2 (RRM2) on the malignant biological behavior and aerobic glycolysis of gastric cancer cells by regulating cyclin-dependent kinase (CDK) 1.Methods:Human gastric cancer MKN-45 cells were divided into si-NC group (transfected with blank fragment) , CoCl 2+si-NC group (hypoxia control transfected with blank fragment) , CoCl 2+si-RRM2 group (hypoxia with RRM2 silencing) , CoCl 2+si-RRM2+pcDNA3.1 NC group (hypoxia with RRM2 silencing and blank vector) and CoCl 2+si-RRM2+pcDNA3.1 CDK1 group (hypoxia with RRM2 silencing and CDK1 overexpression) . The mRNA relative expression levels of RRM2 and CDK1 were analyzed by real time fluorescent quantitative reverse transcription PCR. Co-immunoprecipitation (CoIP) was used to analyze the interaction between RRM2 and CDK1 protein. MTT assay was used to analyze the proliferation activity of cells. The cell migration distance was detected by cell scratch assay. Cell apoptosis was detected by flow cytometry. Adenosine triphosphate (ATP) and glucose kit were used to detect ATP production and glucose consumption. The protein expressions of ENO1, RRM2, HK2, PKM2, GLUT1 and p-CDK1/CDK1 were detected by Western blotting. Results:Real time fluorescent quantitative reverse transcription PCR results showed that the relative expression levels of CDK1 mRNA in si-NC group, CoCl 2+si-NC group and CoCl 2+si-RRM2 group were 1.01±0.15, 1.30±0.06 and 0.51±0.18, and the relative expression levels of RRM2 mRNA were 1.03±0.32, 1.59±0.28 and 0.44±0.17, respectively, and there were statistically significant differences ( F=25.52, P=0.001; F=14.47, P=0.005) . The mRNA expressions of RRM2 and CDK1 in CoCl 2+si-NC group were higher than those in si-NC group. Compared with the si-NC group and the CoCl 2+si-NC group, the mRNA expressions of RRM2 and CDK1 were lower in the CoCl 2+si-RRM2 group (all P<0.05) . CoIP results showed that there was interaction between RRM2 and CDK1. MTT assay, cell scratch assay and flow cytometry showed that the cell proliferation activity of si-NC group, CoCl 2+si-NC group, CoCl 2+si-RRM2 group, CoCl 2+si-RRM2+pcDNA3.1 NC group and CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were 1.04±0.01, 1.18±0.04, 0.84±0.03, 0.81±0.03 and 0.93±0.05, respectively. The cell migration distances were (301.83±2.75) , (369.67±0.76) , (176.50±6.38) , (175.83±3.69) , (254.17±1.61) μm, respectively. The apoptosis rates were 8.05%±0.21%, 5.75%± 0.20%, 28.28%±0.04%, 30.18%±1.51% and 17.79%±0.22%, respectively, all with statistically significant differences ( F=73.82, P<0.001; F=1 600.01, P<0.001; F=787.15, P<0.001) . Compared with the si-NC group and CoCl 2+si-NC group, the proliferation and migration ability of cells in the CoCl 2+si-RRM2 group, CoCl 2+si-RRM2+pcDNA3.1 NC group and CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were weaker, and the apoptosis rates were higher (all P<0.05) . Compared with the CoCl 2+si-RRM2+pcDNA3.1 NC group, the proliferation and migration ability of cells in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were stronger, and the apoptosis rate was lower (all P<0.05) . The results of ATP and glucose detection showed that there were statistically significant differences in the amount of ATP production and glucose consumption among the above five groups ( F=12.53, P<0.001; F=19.21, P<0.001) . Compared with the si-NC group, the glucose consumption of cells was lower in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group ( P<0.05) . Compared with the CoCl 2+si-NC group, the ATP production and glucose consumption of cells in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were lower (both P<0.05) . Compared with the CoCl 2+si-RRM2+pcDNA3.1 NC group, the ATP production and glucose consumption of the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were higher (both P<0.05) . Western blotting showed that there were statistically significant differences in the protein expressions of ENO1, RRM2, HK2, PKM2, GLUT1, and p-CDK1/CDK1 among the above five groups (all P<0.001) . Compared with the si-NC group and the CoCl 2+si-NC group, the protein expressions of ENO1, RRM2, HK2, PKM2, GLUT1 and p-CDK1/CDK1 in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were lower (all P<0.05) . Compared with the CoCl 2+si-RRM2+pcDNA3.1 NC group, the protein expressions of ENO1, RRM2, PKM2, GLUT1 and p-CDK1/CDK1 in the CoCl 2+si-RRM2+pcDNA3.1 CDK1 group were higher (all P<0.05) . Conclusions:Silencing RRM2 can inhibit the malignant biological behavior of gastric cancer cells and the occurrence of aerobic glycolysis by regulating CDK1.

Background/Aims: The increased mortality of gastric cancer (GC) is mainly attributed to the development of chemoresistance. Circular RNAs, as the novel type of biomarkers in GC, have attracted wide attention. The purpose of this study was to investigate the functional role of circ_0081143 in GC with doxorubicin (DR) resistance and its potential action mechanism. Methods: The expression of circ_0081143, miR-129-2-3p and YES proto-oncogene 1 (YES1) in GC tissues and cells was measured by quantitative real-time polymerase chain reaction. The half maximal inhibitory concentration value was calculated based on the MTT cell viability assay. Cell proliferation and apoptosis were monitored by MTT and flow cytometry assays. Transwell assays were employed to check cell migration and invasion. The protein levels of YES1 and apoptosis-related proteins were detected by western blotting. The interaction between miR-129-2-3p and circ_0081143 or YES1 was verified by dual-luciferase reporter and pull-down assays. A tumorigenicity assay was conducted to verify the role of circ_0081143 in vivo. Results: Circ_0081143 was highly expressed in DR-resistant GC tumor tissues and cells. Depletion of circ_0081143 reduced DR resistance and inhibited DR-resistant GC cell proliferation, migration and invasion. Circ_0081143 targeted miR-129-2-3p and inhibited the role of miR-129-2-3p. In addition, YES1 was a target of miR-129-2-3p, and its function was suppressed by miR-129-2-3p. Importantly, circ_0081143 positively modulated the expression of YES1 through mediating miR-129-2-3p. Circ_0081143 knockdown weakened the DR-resistant GC tumor growth in vivo. Conclusions Circ_0081143 knockdown weakened DR resistance and blocked the development of DR-resistant GC by regulating the miR-129-2-3p/YES1 axis. Our data suggest that circ_0081143 is a promising target for the treatment of GC with DR resistance.

Objective:To investigate the signaling pathway of inhibiting macrophage phagocytosis of TIR domain-containing protein encoded by Escherichia coli (TcpC) N-terminal ubiquitin ligase active fragments of uropathogenic Escherichia coli. Methods:Bioinformatics software was used to analyze the amino acid sequences and the function of TcpC N-terminal ubiquitin ligase active fragments as well as the functional sites. PCR was performed to amplify tcpc-330, tcpc-450 and tcpc-510 genes and a prokaryotic expression system was constructed to express the target proteins. The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were purified by Ni-NTA affinity chromatography. LPS in the recombinant proteins was removed by Detoxi-gel chromatography. The expression of MyD88 at protein and mRNA levels in macrophages incubated with rTcpC-N110, rTcpC-N150, rTcpC-N170 or rTcpC-TIR was detected by Western blot and qRT-PCR. The activation of NF-κB signal pathway and the levels of proinflammatory factors in macrophages incubated with the above TcpC protein fragments were measured by Western blot and ELISA, respectively. Results:Cys12, Trp104 and Trp106 in the N-terminal fragment of TcpC were crucial amino acids in maintaining its ubiquitin ligase activity. The target recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were successfully expressed and purified. After Detoxi-gel chromatography, rTcpC-N110, rTcpC-N150 and rTcpC-N170 extracts were undetectable for LPS. TcpC ubiquitin ligase fragments inhibited the expression of MyD88 at protein level, but not affect its expression at mRNA level in macrophages. LPS-induced phosphorylation of NF-κB signaling pathway-related proteins p50 and p65 was significantly inhibited in macrophages treated with TcpC ubiquitin ligase fragments. Moreover, LPS-induced production of pro-inflammatory factors was also significantly inhibited.Conclusions:The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 could inhibit the expression of MyD88 at protein level and suppress the activation of NF-κB signaling pathway, suggesting that they were closely related to the inhibition of innate immune activity of macrophages.

Objective:To investigate whether the hypervirulent Klebsiella pneumoniae (hvKP) induces liver abscess through activating NLRP3 inflammasome. Methods:K1-hvKP and K35-non-hvKP bacterial suspensions were intraperitoneally injected into C57BL/6 mice to establish the models of liver abscess. Human peripheral blood neutrophils were sorted by immunomagnetic beads with CD45 + and Gr-1 + , and the purity was detected by flow cytometry. The concentrations of capsular polysaccharide of K1-hvKP and K35-non-hvKP were detected by total carbohydrate assay kit. The expression of IL-18 and IL-33 by neutrophils at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and ELISA, respectively. The activation of NLRP3 inflammasome in neutrophils was detected by Western blot. Neutrophil extracellular trap formation (NETosis) was observed under confocal laser scanning microscope. Results:The C57BL/6 mice with K1-hvKP infection had significantly serious liver abscess as compared with the K35-non-hvKP-infected mice. The purity of human neutrophils was more than 95%. The concentration of capsular polysaccharide in K1-hvKP was significantly higher than that in K35-non-hvKP. Compared with K35-non-hvKP, K1-hvKP significantly promoted the neutrophils to express IL-18 and IL-33 at both mRNA and protein levels, enhanced the activation of NLRP3 and induced NETosis.Conclusions:This study suggested that hvKP could promote NETosis by activating NLRP3 inflammasome to cause liver abscess.

Objective:To investigate the role of TcpC in uropathogenic Escherichia coli (UPEC)-induced cystitis in mice and to preliminarily analyze the pathogenic mechanism. Methods:C57BL/6 mice were injected with 10 9 CFU wild-type UPEC CFT073 (CFT073 wt) or tcpc gene-deleted mutant (CFT073 Δ tcpc) from urethra into bladder to construct the mouse model of cystitis. The mice were sacrificed 3 d after infection and the bladders were taken to observe the gross pathological changes. Histopathological changes in bladder tissues were observed after HE staining. Immunohistochemistry was used to detect TcpC in bladder tissues. Bacterial loads in urine samples of UPEC-infected mice were counted by tenfold dilution method, and the presence of tcpc gene in the genomic DNA of bacteria from the bladder and urine samples of CFT073 wt-infected mice was measured by PCR. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to detect the expression of TcpC at mRNA and protein levels in macrophages after CFT073 wt infection. The influence of UPEC strains on the activation of NF-κB signaling pathway in macrophages were determined by Western blot. The levels of proinflammatory factors and the bacterial and cell activity after infecting macrophages with UPEC strains were detected by ELISA, laser confocal microscope and fluorescence microscope, respectively. Results:Compared with the mice with CFT073 Δ tcpc infection, CFT073 wt-infected mice had significantly enlarged bladder and severe neutrophil infiltration and abundant TcpC in bladder tissues. The number of bacteria in the urine of CFT073 wt-infected mice was significantly greater than that of the CFT073 Δ tcpc group. PCR results showed that the bacteria in bladder or urine were CFT073 wt. The expression of TcpC at both mRNA and protein levels in macrophages increased significantly after CFT073 wt infection. Moreover, in CFT073 wt-infected macrophages, the expression of IκBα was promoted and the phosphorylation of p65 and the production of proinflammatory factors were suppressed. TcpC was instrumental in the survival and invasion of CFT073 wt in macrophages. Conclusions:TcpC expression increased significantly in mice with CFT073 wt-induced cystitis. TcpC inhibited the activation of NF-κB signaling pathway and the production of proinflammatory factors in macrophages to improve the survival rate of CFT073 wt, which was closely related to the pathogenesis and immune evasion of UPEC.

Objective To investigate the therapeutic efficacy of laparoscopic surgery for treatment of the patients with advanced gastric cancer.Methods A total of 120 patients with advanced gastric cancer treated by D2 radical resection of distal gastric cancer from January 2015 to January 2017 in Affiliated Hospital of Guangdong Medical University were retrospectively analyzed.According to the method of operation,the patients were divided into laparoscopic group (60 cases) and laparotomy group (60 cases).The operation-related indexes,operation curative effect indexes and postoperative recovery were compared between the two groups by using x 2 test and t test.Results The operation time of the laparoscopic group was longer than that of the laparotomy group [(197±21) min vs.(178±23) min,t =4.759,P ＜ 0.001].Some parameters in the laparoscopic group were lower than those in the laparotomy group (all P ＜ 0.05),including intraoperative blood loss [(111±30) ml vs.(221±52) ml,t =14.103],the length of the surgical incision[(6.1±1.3) cm vs.(17.3±3.2) cm,t =25.117],postoperative anal evacuation time [(90±14) h vs.(110±18) h,t =6.983],the postoperative bed-out time [(2.9±0.8) d vs.(4.8±1.4) d,t =9.127],the postoperative liquid dietary intake time [(4.3±0.9) d vs.(5.7±1.2) d,t =7.230],and hospital stay time [(13.2±2.5) d vs.(15.5±3.2) d t =4.387].There were no statistical differences in the number of removed lymph nodes,the number of first-site removed lymph nodes,the number of second-site removed lymph nodes,the distance from the distal margin to the tumor,and the distance from the proximal margin to the tumor (t values 1.351,0.912,1.240,0.876,and 0.840 respectively,all P ＞ 0.05);The incidence of operative complications in the laparoscopic group was lower than that in the laparotomy group [6.67 ％ (4/60) vs.20.00 ％ (12/60),X 2 =4.615,P =0.032].Conclusion Laparoscopic surgery in the treatment of advanced gastric cancer patients has a favorable effect,with reliable results,less trauma,postoperative recovery and fewer complications.

OBJECTIVETo evaluate the association of early diarrhea and fecal volume with anastomotic leakage after low anterior resection (LAR) of rectal cancer.

METHODSClinical data of 541 patients with rectal cancer undergoing LAR at The Affiliated Hospital of Guangdong Medical College between January 2007 and January 2017 were analyzed retrospectively. Early postoperative diarrhea was defined as at least one occurrence of more than 50 ml watery stool or at least four times defecation per day within 7 days after surgery. The volume of fecal discharge from the transanal drain was measured at daily intervals for 3 days after surgery. Association of early diarrhea and anastomotic leakage was analyzed using logistic regression model. The accuracy of fecal volume in predicting anastomotic leakage was evaluated using receiver operating characteristics (ROC) curve.

RESULTSThere were 319 males and 222 females with mean age of 59.3 years. Early postoperative diarrhea occurred in 99(18.3%) patients, and 41(7.6%) patients developed anastomotic leakage. The incidence of anastomodc leakage in patients with early diarrhea was significantly higher as compared to those without early diarrhea (15.2% vs. 5.9%, P=0.000). Multivariate analysis revealed that early diarrhea (OR=33.940, 95%CI: 8.423 to 89.240) and the distance between the tumor and the anal verge less than 7 cm (OR=13.085, 95%CI: 2.117 to 44.556) were independent risk factors for anastomotic leakage, while the presence of a transanal tube was an independent protective factor (OR=0.474, 95%CI: 0.122 to 0.881). The total fecal volume for 3 days after surgery was calculated in 162 patients with a transanal tube. The median fecal volume was 210 (100 to 4360) ml and 60 (0 to 480) ml in patients with and without anastomotic leakage respectively(P=0.000). ROC curve showed that the cut-off value of fecal volume for anastomotic leakage was 110 ml and the area under the curve was 0.824 with a high sensitivity of 85.7% and specificity of 81.3%.

CONCLUSIONSEarly postoperative diarrhea after LAR procedure of rectal cancer may be an early predictor of anastomotic leakage, and fecal volume for 3 days after surgery ≥110 ml can accurately predict anastomotic leakage. Active prevention and management of early postoperative diarrhea may reduce the risk of anastomotic leakage.

Objective To observe and compare the safety and short-term outcomes of laparoscopic surgery (LS)versus conventional open surgery(OS)for stage Ⅱor Ⅲ rectal cancer(RC). Methods One hundred and six patients with stage ⅡorⅢRC were divided into LS group(n=53)and OS group(n=53)according to the random double blind method. The related outcomes of two groups were compared ,including surgical duration , intraoperative blood loss , length of incision , distal margin length , proximal margin length , the number of lymph node dissection,residual cancer rate,exhaust time,first ambulation time,and postoperative hospital stay. Results Intraoperative blood loss,length of incision,postoperative hospital stay,exhaust time and the first ambu-lation time in LS group were significantly different from those in OS group (P < 0.05 for all comparisons). And there were no significant differences between the two groups in surgical duration ,the number of lymph node dissec-tion,distal margin length,proximal margin length and residual cancer rate(P > 0.05). Conclusions Laparo-scopic technology is safe and feasible in treating rectal carcinoma,with less trauma,quicker recovery,and shorter hospital stay.

Objective To explore the etiologies of fever of unknown origin(FUO)and methods for confirming di-agnosis in patients at a hospital,and provide reference for clinical diagnosis and treatment of FUO.Methods Pa-tients with FUO admitted to a hospital between January 2008 and July 2014 were performed clinical diagnosis with methods of serology,bacteriology,molecular biology,bone marrow aspiration,tissue biopsy,and diagnostic thera-py,the etiologies and final diagnosis of 224 patients were analyzed retrospectively.Results Of 224 FUO cases,189 (84.38%)eventually got confirmed diagnosis,35 (15.62%)were not confirmed.The percentage of infectious dis-eases,connective tissue diseases,malignant tumor,and other diseases were 50.45%,18.75%,9.82%,and 5.36%respectively.Among infectious diseases,the major pathogens were bacteria,followed by virus.The major connec-tive tissue diseases were systemic lupus erythematosus and polyarteritis nodosa;the main malignant tumor was he-matological tumor,lymphoma was the main form.Among 189 patients with confirmed diagnosis,30.16% and 24.34% were performed pathogenic and pathologic detection respectively,and 20.11% were performed the other (compre-hensive)methods.Conclusion Infectious diseases,connective tissue diseases,and tumor are major etiologies of FUO.

PURPOSE: The aim of this study is to further understand the status of BRCA1 and BRCA2 mutation among Chinese high-risk breast cancer patients in multiple-ethnic regions of China. METHODS: A total of 79 blood samples of high-risk breast cancer patients from Xinjiang Uyghur autonomous region were analyzed by PCR-DHPLC sequencing analysis. RESULTS: Analysis with full length of the two genes identified a total of 6 deleterious mutations (2073delA, 2394C-T [Q759X] and IVS16+1G>A in BRCA1; 1627A-T [K467X], 6873delCTCC and 9481delA in BRCA2) in this cohort. The prevalence of BRCA1/2 germline mutation was about 7.6% (6/79) in the Xinjiang multiple ethnic region of China. Among them, 3 novel deleterious mutations, 2073delA in BRCA1 (Han ethnic Chinese) and BRCA2 variants 6873delCTCC and 9481delA (both are Kazakh ethnic Chinese), were identified and they had never been reported in breast cancer information core (BIC) database before. 2394C-T (Q759X) and IVS16+1G>A, in BRCA1 and BRCA2 variants 1627A-T were previously reported in other populations but not Chinese. Among 6 of the BRCA-related tumors, three BRCA1- and one BRCA2-associated tumors were in triple negative (estrogen receptor, progesterone receptor, and HER2 negative expressed) status and exhibited a high tumor grade. So far none of these 6 deleterious mutations were reported in ethnic Han Chinese. CONCLUSION: BRCA germline mutation in Chinese multiple ethnicity region may exhibit different genotypes compared to ethnic Han Chinese in other regions. These differences may arise from interaction of genetic background and environmental factors.
Asian Continental Ancestry Group ; Breast ; Breast Neoplasms ; China ; Cohort Studies ; Ethnic Groups ; Female ; Genetic Variation ; Genotype ; Germ-Line Mutation ; Humans ; Prevalence ; Receptors, Progesterone