Objective To study the effect of endoscopic treatment for acute appendicitis (AA)without perforation or gangrene.Methods A total of 94 patients with AA were randomly divided into operation group (n =45 ) to receive appendectomy,control group (n =15 ) to accept conventional medicine of metronidazole and Cefoxitin and colonoscopy group (n =34) to undergo conventional medicine plus endoscopic treatment.The time for alleviation of abdominal pain,duration and mean cost of the hospitalization,and the recurrence rate in one year were compared.Results Compared to operation group,colonoscopy group was superior in the duration [ (2.77 ± 0.27) d vs.(6.65 ± 1.68) d ] and mean cost of hospitalization [ ( 1011.35 ± 22.12) yuan vs.(4023.37 ± 32.02 ) yuan ] ( P ＜ 0.05 and P ＜ 0.01,respectively).There were no significant differences in the time for alleviation of abdominal pain or the recurrence rate in one year between 2 groups.Colonoscopy group was superior to control group in all the indices (P ＜ 0.05 ).Conclusion Endoscopic treatment for AA without perforation and gangrene is effective and safe,which can be considered as the first-line treatment.

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.

Objective To explore the molecular mechanisms of resistance to phosphatidyl inositol 3?kinase ( PI3K) inhibitors in triple?negative breast cancer ( TNBC) cells. Methods HCC70 cells ( TNBC) were transfected with siFZD7, siWANT5B or siGSK3 using lipofectamine 2000 transfection reagent. The expression levels of key proteins of WNT/β?catenin and PI3K/AKT/mTOR pathways were determined by Western blot analysis. After HCC70, MCF?7 ( ER?positive ) and SK?BR3 ( HER2?positive ) cells were treated with PI3K/AKT/mTOR inhibitors, the inhibition rates of cell proliferation were measured by MTT assay, and half maximal inhibitory concentrations ( IC50 ) were calculated. The altered activities of WNT/β?catenin and PI3K/AKT/mTOR proteins were detected by Western blot and luciferase report gene assay, respectively. The nuclear translocation of β?catenin protein was examined by immunofluorescence assay. Xenograft nude mouse model was used to evaluate the tumorigenicity of breast cancer cells treated with BKM120 in vivo. The expression levels of p?LRP6, p?4EBP1 and β?catenin proteins in the tumor tissues were determined by immunohistochemical staining. Results The expression levels of FZD7, WANT5B and GSK3 proteins were significantly reduced in the HCC70 cells transfected with the target siRNAs. Meanwhile, the activity of WNT/β?catenin was enhanced and PI3K/AKT/mTOR pathway was inhibited. PI3K/AKT/mTOR inhibitors suppressed MCF?7 and SK?BR3 cell proliferation. The IC50 of GDC?094, BKM120, XL147, perifosine, everolimus, and BEZ235 in MCF?7 cells were 0. 46 mmol/L, 1. 44 mmol/L, 4. 34 mmol/L, 11.35 μmol/L, 53. 71 μmol/L and 12. 87 μmol/L respectively, and 0. 63 mmol/L, 0. 58 mmol/L, 3. 74 mmol/L, 13.22μmol/L, 60.00μmol/L and 11.38μmol/L in the SK?BR3 cells, respectively. The results of luciferase report gene assay showed that the luciferase activities in HCC70, MCF?7 and SK?BR3 cells treated with BKM120 were 1.75±0.05, 1.13±0.02 and 0.43±0.01, respectively. The luciferase activities in HCC70 and SK?BR3 cells were significantly different from that of the control cells (1.00±0.02, P<0.05). The immunohistochemical analysis showed that BKM120 inhibited mTOR activity, and the enhanced WNT/β?catenin activity reversed the phenotype of inhibitory mTOR induced by BKM120. BKM120 suppressed the tumorigenic ability of MCF?7 and SK?BR3 cells in vivo, but had no effect on cultured HCC70 cells. The immunohistochemical analysis showed nuclear translocation of β?catenin protein and increased expression level of p?LRP?6 protein in transplanted tumor tissues from HCC70 cells treated with BKM120, increased the level of p?LRP?6 protein, and no changes of p?4EBP1 protein expression. However, no nuclear translocation of β?catenin protein and no decrease of p?LRP6 and p?4EBP1 protein levels in the transplanted tumor tissue of MCF?7 cells after treatment with BKM120. Conclusions The triple?negative breast cancer HCC70 cells have drugs?resistance to PI3K inhibitors. The WNT/β?catenin signaling pathway may regulate the PI3K/AKT/mTOR pathway, therefore, inducing the drug?resistance of TNBC cells to PI3K inhibitors.

Objective To explore the molecular mechanisms of resistance to phosphatidyl inositol 3?kinase ( PI3K) inhibitors in triple?negative breast cancer ( TNBC) cells. Methods HCC70 cells ( TNBC) were transfected with siFZD7, siWANT5B or siGSK3 using lipofectamine 2000 transfection reagent. The expression levels of key proteins of WNT/β?catenin and PI3K/AKT/mTOR pathways were determined by Western blot analysis. After HCC70, MCF?7 ( ER?positive ) and SK?BR3 ( HER2?positive ) cells were treated with PI3K/AKT/mTOR inhibitors, the inhibition rates of cell proliferation were measured by MTT assay, and half maximal inhibitory concentrations ( IC50 ) were calculated. The altered activities of WNT/β?catenin and PI3K/AKT/mTOR proteins were detected by Western blot and luciferase report gene assay, respectively. The nuclear translocation of β?catenin protein was examined by immunofluorescence assay. Xenograft nude mouse model was used to evaluate the tumorigenicity of breast cancer cells treated with BKM120 in vivo. The expression levels of p?LRP6, p?4EBP1 and β?catenin proteins in the tumor tissues were determined by immunohistochemical staining. Results The expression levels of FZD7, WANT5B and GSK3 proteins were significantly reduced in the HCC70 cells transfected with the target siRNAs. Meanwhile, the activity of WNT/β?catenin was enhanced and PI3K/AKT/mTOR pathway was inhibited. PI3K/AKT/mTOR inhibitors suppressed MCF?7 and SK?BR3 cell proliferation. The IC50 of GDC?094, BKM120, XL147, perifosine, everolimus, and BEZ235 in MCF?7 cells were 0. 46 mmol/L, 1. 44 mmol/L, 4. 34 mmol/L, 11.35 μmol/L, 53. 71 μmol/L and 12. 87 μmol/L respectively, and 0. 63 mmol/L, 0. 58 mmol/L, 3. 74 mmol/L, 13.22μmol/L, 60.00μmol/L and 11.38μmol/L in the SK?BR3 cells, respectively. The results of luciferase report gene assay showed that the luciferase activities in HCC70, MCF?7 and SK?BR3 cells treated with BKM120 were 1.75±0.05, 1.13±0.02 and 0.43±0.01, respectively. The luciferase activities in HCC70 and SK?BR3 cells were significantly different from that of the control cells (1.00±0.02, P<0.05). The immunohistochemical analysis showed that BKM120 inhibited mTOR activity, and the enhanced WNT/β?catenin activity reversed the phenotype of inhibitory mTOR induced by BKM120. BKM120 suppressed the tumorigenic ability of MCF?7 and SK?BR3 cells in vivo, but had no effect on cultured HCC70 cells. The immunohistochemical analysis showed nuclear translocation of β?catenin protein and increased expression level of p?LRP?6 protein in transplanted tumor tissues from HCC70 cells treated with BKM120, increased the level of p?LRP?6 protein, and no changes of p?4EBP1 protein expression. However, no nuclear translocation of β?catenin protein and no decrease of p?LRP6 and p?4EBP1 protein levels in the transplanted tumor tissue of MCF?7 cells after treatment with BKM120. Conclusions The triple?negative breast cancer HCC70 cells have drugs?resistance to PI3K inhibitors. The WNT/β?catenin signaling pathway may regulate the PI3K/AKT/mTOR pathway, therefore, inducing the drug?resistance of TNBC cells to PI3K inhibitors.

OBJECTIVETo investigate the expression of cyclin D1 in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma and its relationship with human papillomavirus 16 (HPV16) E7 gene expression.

METHODSBoth SiHa and Hcc94 cell lines were obtained from cervical epithelial cells of squamous cell carcinoma. E6/E7 gene was silent in Hcc94 cell line.Expression levels of cyclin D1 mRNA and protein in CIN and squamous cell carcinoma were detected by QT-PCR and immunohistochemistry (IHC) respectively. SiRNA was constructed for targeting the promoter of HPV16 E7 and then transfected into SiHa cells to establish cm-16 line with stable silencing of E7. Control cell line B3 was obtained by blank plasmid transfection into SiHa cells. RT-PCR and Western blot were used to detect cyclin D1 mRNA and protein expression in the SiHa, B3, and cm-16 cells, respectively.

RESULTSCyclin D1 was expressed in the basal cells of normal cervical squamous epithelia and the expression gradually decreased in the progression from CIN1 to CIN3. Squamous cell carcinoma showed negative or scattered expression of cyclin D1 (P<0.05). Both mRNA and protein of cyclin D1 in E7(+) SiHa cells were lower than those in cm-16 and Hcc94 cells.

CONCLUSIONSquamous cell carcinoma with high HPV E7 expression shows low level of cyclin D1, suggesting that HPV16 E7 gene inhibits the expression of cyclin D1.

Carcinoma, Squamous Cell ; metabolism ; virology ; Cell Line, Tumor ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Cyclin D1 ; genetics ; metabolism ; Female ; Human papillomavirus 16 ; Humans ; Immunohistochemistry ; Papillomavirus E7 Proteins ; genetics ; Promoter Regions, Genetic ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Uterine Cervical Neoplasms ; metabolism ; virology