Objective To explore the effects of siRNA targeting survivin combined with radiotherapy on human carcinoma cells SW480.Methods The human colorectal carcinoma cell line SW480 was treated with siRNA targeting survivin.The expression level of survivin in transfected SW480 cells was detected by RT-PCR and immunocytochemistry.The effects of siRNA on cell cycle were analyzed by FCM with PI staining.Radiotherapy was combined with siRNAs targeting survivin to study the sensitivity of SW480 induced by siRNA targeting survivin to radiotherapy.The effects of the survivin siRNA combined with radiotherapy on the growth of SW480 were studied by MTT assay.The apoptosis rate of SW480 was analyzed by FCM with annexinV/PI staining.Results RT-PCR demonstrated that the expression of survivin was knocked down significantly by the siRNA.Arrest in G2/M phase of the cell cycle was detected by FCM in the SW480 cells transfected with siRNA targeting survivin.The MTT assay showed that siRNA targeting survivin combined with radiotherapy inhibited the growth of SW480 cells more significantly compared with single treatment.More apoptosis cells(almost 35.72%) were detected by FCM in the SW480 cells treated with siRNA combined with radiotherapy.Conclusions The small interfering RNA can inhibit survivin of the human colorectal carcinoma cell line SW480.The siRNA targeting survivin combined with radiotherapy can inhibit the proliferation of SW480 and increase the percentage of apoptotic cells markedly.The siRNA targeting survivin can enhance the sensitivity of colorectal carcinoma cell line SW480 to radiotherapy in vitro.

ObjectiveTo evaluate the intraoperative neuromonitoring of the external branch of the superior laryngeal nerve (EBSLN) during Micooli's endoscopic thyroidectomy in order to avoid nerve injury.MethodsIn this study,36 patients with 56 nerves at risk were enrolled from February 2011 to September 2011.A positive signal is determined by observing contractions of the cricothyroid muscle to locate the EBSLN.The relationship between EBSLN and the upper pole of the thyroid or the inferior constrictor muscle was studied.The VHI-10 table was used for evaluation pre- and postoperatively. ResultsAll 56 nerves were located successfully,26 nerves(46.4％ ) crossed the superior thyroid artery more than 1 cm apart from the upper pole of the thyroid gland,while the other 30 nerves(53.6％ ) did less than 1 cm.In cases where the diameter was longer than 5 cm,the nerves crossed the artery at less than 1.0 cm from the upper pole in 73％ cases(P =0.006).There was no significant difference between VHI-10 results before and after surgery (P ＞ 0.05). ConclusionsIntraoperative neuromonitoring is useful and helpful in avoiding nerve injury by locating EBSLN.

Objective To evaluate the clinical value of breast localization needle placed via mammary ductoscopy in surgical treatment of patients with intraductal neoplasms. Methods In this study,76 patients with intraductal neoplasms diagnosized by mammary ductoscopy from January 2009 to March 2010 in the Second Affiliated Hospital of Soochow University were randomly divided into two groups.For methylene blue group,ducts with the lesion were marked by methylene blue injection before operation.For localization needle group,ducts were marked by localization needle placed via ductoscopy.The operative time,specimen weight,incision length and diagnostic coincidence rate were compared among the two groups. Results Compared to the methylene blue group,the localization needle group had a significantly shorter operative time (31 ± 8 min vs.42 ± 9 min),lighter specimen weight (1.51 ± 1.36 g vs.2.95 ± 2.07 g),and shorter incision (23.2 ± 7.8 mm vs.34.4 ± 7.1 mm).All the breast cancer cases dianosised by mammary ductoscopy were confirmed by postoperative pathology,but the localization needle group had a higher diagnostic coincidence rate than the methylene blue group (94.7% vs. 76.3%). Conclusion Localization needle under mammary ductoscopy is a reliable technique for localizing intraductal neolasms.The surgical excision guided by localization needle is accurate and less traumatic,and should be a routine method marking the tumor involved duct before operation.

Objective To evaluate the mechanism of external branch of the superior laryngeal nerve (EBSLN) injury during thyroid surgery as showed by intraoperative neuromonitoring.Methods 70 patients with 109 nerves at risk were enrolled in this study from March 2011 to October 2011.A positive signal is determined by observing contractions of the cricothyroid muscle.The relationship between EBSLN and the upper pole of the thyroid or the inferior constrictor muscle was studied.Results 108 nerves (99.1％) were located successfully,42 of which were visualized (38.9％).55 nerves (50.9％) crossed the superior thyroid artery more than 1 cm apart,while the other 53 nerves (49.1％) went less than 1 cm including 24 nerves(more than 0.5 cm,less than 1 cm) and 29 nerves (less than 0.5 cm) or coursed below the superior pole of the thyroid.The rate of the latter type was significantly elevated in patients with the top to botton diameter of the gland more than 5 cm.One patient suffered from impairing in the production of high tones postoperatively.Conclusions Intraoperative neuromonitoring is useful and helpful in providing instructive information for operations by locating EBSLN.

Objective To construct recombinant adenovirns containing small interfering RNA (siRNA) targeting human VEGF-C mRNA,then to study the inhibitory effects of adenovirus-mediated VEGF-C siRNA on growth and lymphangiogenesis of human colon cancer in BABL/c nude mice model.Methods In vitro:the specific siRNA sequence targeting human VEGF-C mRNA was selected.The homologous double-strand DNA was designed and synthesized.After such DNA was inserted into pDC316-EGFP-U6 by BamHI and HindⅢ,pDC316-VEGF-C siRNA-EGFP-U6 was obtained,then it was co-transfected into 293 cells with the hone plasmid pBHGF35.After generated by homologous recombination,Ad5F35-VEGF-C siRNA-EGFP-U6 was obtained,and it was packaged and amplified in 293 cells.The human colon cancer model was established in nude mice by hypo-injection LoVo cells.They were divided randomly into four groups (n=6):adenovirus,virus without target gene,single siRNA and PBS control group.Injecting intervention intra-tumorly in each groups,calculating the tumors volume and drawing the growth curve,calculating micro-vascular density (MVD) and micro-lymphatic density (LVD) by staining respectively of the lymphatic and vascular with anti-LYVE-1 monoclonal antibody and anti-CD<,34> monoclonal antibody were completed.Results Adenovirus group tumor sizes were smaller than other groups.The LVD were 8.47±2.1 and 17.35±4.7 (P<0.05) in adenovirns group and the PBS control group.The MVD were 22.65±6.04 and 23.19±7.63 (P>0.05) in adenovims group and the PBS control group respectively.Conclusion The adenovirus-mediated VEGF-C siRNA can significantly inhibit the growth and lymphangiogenesis of human colon cancer in nude mice model,hut have no effect on microangium vessels growth.

ObjectiveTo rapidly detect the mutation frequency of DBC2 gene 7776C ＞ T in breast cancer and breast fibroadenoma by applying the mutation-sensitive molecular switch ( comprised of high-fidelity DNA polymerase and phosphorothioate-modified allele-specific primers) and agarose gel electrophoresis.Methods Allelic specific primers targeting mutation type and wild type were designed with the primers'3'terminal phosphorothioate modification.When the primers matched with the tissue DNA,the primers could be extended with highfidelity polymerase; when they mismatched with the tissue DNA,the primers could not be extended.DNA samples from 85 cases of breast cancer and 10 cases of breast fibroadenoma tissues were chosen and analyzed by PCR amplifications mediated by high-fidelity DNA polymerase.Gel imaging system was employed to make analysis of PCT products.ResultsThe mutation-sensitive molecular switch system showed that the mutation rate of 7776C ＞ T was 2.4％ ( 2/85 ) in the 85 cases of breast cancer,and no mutation was found in the 10 cases of breast fibroadenoma.ConclusionsThe mutation-sensitive molecular switch combined with agarose gel electrophoresis can rapidly detect the mutation of breast cancer DBC2 gene 7776C ＞ T.It is applicable in single nucleotide polymorphisms assay and has enormous application value in detecting gene mutation.

OBJECTIVETo explore the effect and mechanism of autophagy specific gene Beclin-1 in gastric cancer cell SGC7901 on vasculogenic mimicry (VM) forming ability.

METHODSPlasmid vectors with and without integrated shRNA were transfected respectively into SGC7901 cell line (Beclin1-inhibited group and negative control group). Simple SGC7901 cell line was used as blank group. RT-PCR and Western blot were performed to examine the expression of Beclin-1 in 3 groups. Culture was used to construct the VM model in vitro. Different VM forming ability was measured and genes (beclin-1, notch-1) expression of each group was detected before and after VM formation.

RESULTSBeclin-1 and notch-1 expression increased significantly in the process of VM forming. When beclin-1 was inhibited, the formation of VM was limited and VM formative genes expression decreased. As compared to cells of negative control group, those of Beclin1-inhibited group had less number of VM forming cellular tube-like construction (15.4±1.1 vs. 37.8±1.9, P<0.05), shorter length of such construction [(316.8±24.6) mm vs. (385.1±14.2) mm, P<0.05], and less crossing point (11.6±1.1 vs. 27.2±1.1, P<0.05).

CONCLUSIONSBeclin-1 can promote VM formation through maintaining stable expression of gastric cancer cell VM regulating genes. Beclin-1 inhibition may be a new target for gastric cancer gene therapy.

Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Membrane Proteins ; metabolism ; RNA, Small Interfering ; Stomach Neoplasms ; blood supply ; metabolism ; Transfection

Purpose: The incidence of adenocarcinoma of the esophagogastric junction (AEG) has increased in recent years, and the optimal surgical strategy for AEG remains highly controversial. We aimed to evaluate the safety and efficacy of proximal gastrectomy with double-tract reconstruction (PG-DT) for the treatment of patients with AEG. Materials and Methods: We retrospectively analyzed patients with Siewert type II/III AEG between January 2013 and July 2018. Clinicopathological characteristics, survival, surgical outcomes, quality of life (QOL), and nutritional status were compared between the PG-DT and total gastrectomy with Roux-en-Y anastomosis (TG-RY) groups. Results: After propensity score matching, 33 patients in each group were analyzed. There were no statistical differences between the 2 groups in terms of disease-free survival and overall survival. The surgical option was not an independent prognostic factor based on the multivariate analysis. In addition, no differences were found in terms of surgical complications. There were no significant differences in QOL assessed by the Visick grade, Gastrointestinal Symptom Rating Scale, or endoscopic findings. Furthermore, the long-term nutritional advantage of the PG-DT group was significantly greater than that of the TG-RY group. Conclusions PG-DT is a safe and effective procedure for patients with local Siewert type II/III AEG, regardless of the TNM stage.

Objective:To investigate the effects of rapamycin on the autophagy activation of M2 macrophages and the radiosensitivity in colorectal cancer xenograft.Methods:THP-1 cells were induced into Type-Ⅱ macrophages with PMA and/or IL-4. Rapamycin and Bafilomycin A1 were uesd to activate and suppress autophagy of M2 macrophage, respectively. Colorectal cancer LoVo cells were inoculated on BALB/c-nu/nu nude mice. After the xenograft tumor size approached to 10 mm in diameter, the nude mice were divided into the following groups randomly: M2 macrophage autophagy inactive group and active group, autophagy downregulation of the activated group, and nontreatment control group. The tumors in mice were irradiated with 8 Gy X-rays in two fractions, and the radiosensitivity of colorectal cancer xenograft in each group was analyzed.Results:The expression levels of M2 macrophage markers Arg-1 and CCL-22 were significantly higher than those in M0 macrophage. The tumor weight, volume [(1.93±0.05)g, (2.14±0.06)cm 3] and micro-vessel density (36.37±1.04) in M2 autophagy inactive group were higher than those in control group [(1.35±0.05)g, (1.77±0.02)cm 3, 25.69±1.34] ( t=20.07, 14.56, 10.92, P < 0.05). After activation of M2 autophagy, the tumor weight, volume and micro-vessel density were significantly decreased to (0.89±0.03)g, (1.24±0.01)cm 3, and 13.60±1.52 ( t=44.37, 40.32, 21.43, P < 0.05). After down-regulation of M2 autophagy with bafilomycin A1, the tumor weight, volume and micro-vessel density were increased to (1.02±0.07)g, (1.37±0.02)cm 3, and 21.06±1.41 ( t=4.67, 13.79, 6.23, P < 0.05). Autophagy inaction suppressed the expression of Livin and Survivin in tumor ( t=2.64, 7.90, P < 0.05), and the activation of M2 autophagy further down-regulated the expression of Livin, Survivin ( t=5.43, 9.39, P < 0.05). The expression levels of Livin and Survivin were increased after the treatment with bafilomycin A1 ( t=2.80, 3.17, P<0.05). Conclusions:M2 macrophagy promoted the growth of colorectal cancer xenograft by inducing the formation of micro-vessels in the tumor, which is one of the mechanisms of tumor-associated macrophages participating in the radiotherapy resistance of colorectal cancer. Activation of M2 autophagy by rapamycin inhibited the ability of M2 macrophagy in promoting tumor growth, and induced apoptosis of colorectal cancer cells after radiotherapy by down-regulating the expression of anti-apoptotic genes Livin and Survivin, thus increased the radiosensitivity of colorectal cancer.

Background/Aims: This study aimed to investigate the biological function and regulatory mechanism of TCN1 in colorectal cancer (CRC). Methods: We studied the biological function of TCN1 by performing gain-of-function and loss-offunction analyses in HCT116 cell lines; examined the effects of TCN1 on the proliferation, apoptosis, and invasion of CRC cells; and determined potential molecular mechanisms using HCT116 and SW480 CRC lines and mouse xenotransplantation models. Tumor xenograft and colonization assays were performed to detect the tumorigenicity and metastatic foci of cells in vivo. Results: TCN1 knockdown attenuated CRC cell proliferation and invasion and promoted cell apoptosis. Overexpression of TCN1 yielded the opposite effects. In addition, TCN1-knockdown HCT116 cells failed to form metastatic foci in the peritoneum after intravenous injection. Molecular mechanism analyses showed that TCN1 interacted with integrin subunit β4 (ITGB4) to positively regulate the expression of ITGB4. TCN1 knockdown promoted the degradation of ITGB4 and increased the instability of ITGB4 and filamin A. Downregulation of ITGB4 at the protein level resulted in the disassociation of the ITGB4/plectin complex, leading to cytoskeletal damage. Conclusions TCN1 might play an oncogenic role in CRC by regulating the ITGB4 signaling pathway.