Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells / 中华肿瘤杂志

Objective: To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6 (TRAF6) on the biological characteristics of colorectal cancer cells and its mechanism. Methods: lentivirus wild type (pCDH-3×FLAG-TRAF6) and mutation (pCDH-3×FLAG-TRAF6-331mut) of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116, respectively. The infection was observed by fluorescence microscope, and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot. Cell counting kit-8 (CCK-8) and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group, cell scratch test to detect cell migration, Transwell chamber test to detect cell migration and invasion, immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63. Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinase mitogen-activated protein kinase (MAPK)/activating protein-1(AP-1) signal pathway. Results: The successful infection of colorectal cancer cells was observed under fluorescence microscope. Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells. The results of CCK-8 assay showed that on the fourth day, the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively, which were lower than those in TRAF6 group (2.09±0.12 and 2.17±0.45, P=0.036 and P=0.011, respectively). The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively, which was lower than those in TRAF6 group (190±21 and 125±13, P=0.001 and P=0.002, respectively). The results of cell scratch test showed that after 48 hours, the percentage of wound healing distance of HCT116 and SW480 cells in TRAF6-331mut group was (31±12)% and (33±14)%, respectively, which was lower than those in TRAF6 group [(43±13)% and (43±7)%, P=0.005 and 0.009, respectively]. The results of Transwell migration assay showed that the migration numbers of HCT116 and SW480 cells in TRAF6-331mut group were significantly lower than those in TRAF6 group (P<0.001 and P<0.002, respectively). The results of Transwell invasion assay showed that the number of membrane penetration of HCT116 and SW480 cells in TRAF6-331mut group was significantly lower than those in TRAF6 group (P=0.008 and P=0.009, respectively). The results of immunoprecipitation detection showed that the ubiquitin protein of K48 chain pulled by TRAF6-331mut was lower than that of wild type TRAF6 in 293T cells co-transfected with K48 (0.57±0.19), and the ubiquitin protein of K63 chain pulled down by TRAF6-331mut in 293T cells co-transfected with K63 was lower than that of wild type TRAF6 (0.89±0.08, P<0.001). Western blot assay showed that the protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-HCT116 cells were 0.63±0.08, 0.42±0.08 and 0.60±0.07 respectively, which were lower than those in TRAF6-HCT116 cells (P=0.002, P<0.001 and P<0.001, respectively). The expression level of AP-1 protein in TRAF6-HCT116 cells was 0.89±0.06, compared with that in TRAF6-HCT116 cells. The difference was not statistically significant (P>0.05). The protein expression levels of NF-κB, p-NF-κB and p-AP-1 in TRAF6-331mut-SW480 cells were 0.50±0.06, 0.51±0.04, 0.48±0.02, respectively, which were lower than those in TRAF6-SW480 cells (all P<0.001). There was no significant difference in AP-1 protein expression between TRAF6-331mut-SW480 cells and TRAF6-SW480 cells. Conclusion: The ubiquitin site mutation of TRAF6 gene at 331 may prevent the binding of TRAF6 and ubiquitin lysine sites K48 and K63, and then affect the expressions of proteins related to downstream NF-κB and MAPK/AP-1 signal pathways, and inhibit the proliferation, migration and invasion of colorectal cancer cells.

Influencing factors related to lymphatic metastasis of T2 rectal carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the risk factors associated with lymphatic metastasis of T2 rectal carcinoma.</p><p><b>METHODS</b>A consecutive series of 122 patients with T2 rectal cancer who underwent radical surgery in the First Affiliated Hospital of Fujian Medical University from 2006 to 2011 were included for retrospective analysis. Risk factors associated with lymphatic metastasis were investigated.</p><p><b>RESULTS</b>The rate of lymph node metastasis was 21.3% (26/122). Distance to anal verge(P<0.05), morphological type(P<0.05), histological type(P<0.05), tumor differentiation(P<0.05), and depth of invasion(P<0.05) were risk factors for lymph node metastasis in T2 rectal cancer by univariate analysis. The depth of invasion remained statistically significant by multivariate analysis. The rate of lymph node metastasis was 13%(7/54) in patients with shallow muscularis propria involvement, and 28%(19/68) in those with deep muscularis involvement.</p><p><b>CONCLUSION</b>For T2 rectal cancer with shallow muscularis involvement, the risk of lymph node metastasis is low and transanal excision should be considered.</p>

Identification of proteins that interact with murine cytomegalovirus early protein M112-113 in brain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Murine cytomegalovirus (MCMV) early protein M112-113 is involved in viral DNA replication and believed to play a crucial role in the viral pathogenesis. To investigate the biological function of M112-113 protein in the pathogenesis of the brain disorders caused by cytomegalovirus (CMV), a screening for proteins interacting with M112-113 was performed by a yeast two-hybrid system.</p><p><b>METHODS</b>Bait plasmid pGBKT7-M112-113 was constructed and transformed into AH109 yeast. After confirmation of the expression of MCMV M112-113 in yeast, the bait yeast was mated with a prey yeast containing mouse brain cDNA library plasmid to screen the proteins interacting with M112-113. Interactions between M112-113 and the obtained proteins were verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.</p><p><b>RESULTS</b>Two proteins interacting with M112-113 were identified, including metastasis-associated 1 (MTA1) and zinc finger, CCHC domain containing 18 (ZCCHC18). M112-113 protein could interact with MTA1 or ZCCHC18 in yeast and mammalian cells.</p><p><b>CONCLUSION</b>The interactions of M112-113 with MTA1 or ZCCHC18 may be related to the pathogenesis of MCMV-associated disease in central nervous system.</p>

Clinical analysis of 8 cases with acute invasive pulmonary aspergillosis in younger children / 中华儿科杂志

<p><b>OBJECTIVES</b>To analyze the clinical features of acute invasive pulmonary aspergillosis in younger children, in order to improve the levels of early recognition, diagnosis and management of this disease.</p><p><b>METHOD</b>Clinical data of 8 patients aged below 15 months who were diagnosed as acute invasive pulmonary aspergillosis from August 2010 to February 2011 in general pediatric wards in our hospital were retrospectively analyzed for the high-risk factors of the hosts, clinical manifestations, laboratory findings and lung CT imaging, the processes of diagnosis and treatment, and the outcomes.</p><p><b>RESULT</b>Five cases were tested for serum GM test absorbent index (GMI) ranged from 1.92 to 3.27; in 2 cases sputum culture was positive for Aspergillus fumigatus for twice, and 1 infant was serum GMI 2.85 and a sputum culture was positive for Aspergillus fumigatus positive, all these findings were accordant with the clinical diagnosis. Seven cases had a history of receiving intravenously broad-spectrum antibiotics or plus corticosteroids (6 hospitalized, 1 out-patient), and one was only 1 month old, whose parents had severe tinea pedis. 4 patients of high-fever type had sustained high temperature, severe changes of lungs without obvious respiratory symptoms and signs in early phase, and significant increase of the rod granulocyte rate (0.25 - 0.68), which was apparently discordant with the normal WBC count and high sensitivity C-reactive protein (hs-CRP) value. Another 4 cases of non-high-fever type were present with normal WBC count, hs-CRP value and the percentage of rod granulocyte. Among them, 3 infants had low-grade fever, with serious respiratory symptoms and signs and changes of lungs CT. Another 1-month-old case only showed lower vigor and response. Lung CT imaging often showed multiple irregular large nodules, patches and streaks of density (6 cases) and unilateral lobar consolidation (1 case), with some involving the pleura; one appeared severe peri-main bronchus lesions with stenoses of bilateral main bronchi. The first case died of multiple organ failure because of severe sepsis complication. Another 7 cases were treated with voriconazole promptly after clinical or suspected diagnosis, and the state of patients relieved rapidly within 1 - 3 d.</p><p><b>CONCLUSION</b>The abuse of broad-spectrum antibiotics and corticosteroids may increase the risk of invasive pulmonary aspergillosis in younger children. There may be the risk of nosocomial infection and spread of aspergillus in general pediatric wards. Cases of high-fever type in early period of disease had two inconsistency: few symptoms and signs, while severe changes of lungs CT; apparent increase of peripheral rod granulocyte, while normal WBC count and hs-CRP value. Preemptive voriconazole therapy could obtain significant effect and reduce the mortality rate.</p>

Effects of allergen and intranasal glucocorticoid on Th17 and RORgamma t in peripheral blood in patients with allergic rhinitis / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the effect of specific immunotherapy and intranasal glucocorticoid on T help 17 (Th17) cells and RORgammat in peripheral blood in patients with allergic rhinitis (AR).</p><p><b>METHODS</b>Forty patients with allergic rhinitis (group A) were divided randomly into two subgroups (group A1 and A2), and each subgroup had 20 patients. The patients in group A1 were treated with intranasal glucocorticoid (INGS) for one-year. The patients in group A2 were treated with special immunotherapy (SIT) for 4 weeks. Blood samples were respectively taken from 10 healthy individuals (group B), 20 AR patients (group A1) before and after SIT with specific standardized allergen and 20 AR patients (group A2) before and after INGS. The ratio of Th17 cells in peripheral blood monouclear cells (PBMC) were analysed by flow cytometry. The expression of RORgammat mRNA were detected by real-time polymerase chain reaction and the interleukin-23(IL-23), IL-17, IL-6 were detected by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The ratio of Th17 cells in PBMC and the expression of RORgammat mRNA in group A [(18.97 +/- 1.05)% and (0.604 +/- 0.027)] were respectively higher than those in group B [(15.12 +/- 1.09)% and (0.447 +/- 0.024)] and the difference reached statistical significance (t were respectively -10.056 and -17.986, each P < 0.01). The level of IL-6, IL-17 and IL-23 in group A were respectively higher than those in group B and the difference reached statistical significance (t were respectively -41.149, -17.618 and -26.824, all P < 0.01). The ratio of Th17 cells in PBMC, the expression of RORgammat mRNA, the level of IL-6, IL-17 and IL-23 before INGS did not show significant difference from those of after INGS in group A1 (t were respectively 0.298, 0.240, -1.136, 0.283 and -1.670, all P > 0.05). The ratio of Th17 cells in PBMC and the expression of RORgammat mRNA were respectively (18.99 +/- 1.14)% and (0.603 +/- 0.027) before SIT and were respectively (16.30 +/- 1.63)% and (0.429 +/- 0.023) after SIT in group A2, and the difference reached statistical significance (t were respectively 6.035 and 22.015, all P < 0.01). The level of IL-6, IL-17 and IL-23 before SIT were lower respectively than those of after SIT in group A2 and the difference reached statistical significance (t were respectively 9.235, 11.289, 7.267, all P < 0.01).</p><p><b>CONCLUSIONS</b>The ratio of Th17 cells in PBMC, the expression of RORgammat mRNA, the level of IL-6, IL-17 and IL-23 were up-regulated in patients with AR. The treatment of SIT could get the 5 items down and the treatment of INGS couldn't.</p>