KLF4 targets RAB26 and decreases 5-FU resistance through inhibiting autophagy in colon cancer.

BACKGROUND: Accumulating studies demonstrated that resistance of colon cancer (CC) to 5-fluorouracil (5-FU) contributes to adverse prognosis. We investigated how Kruppel-like factor 4 (KLF4) affected 5-FU resistance and autophagy in CC cells. METHODS: KLF4 expression and its downstream target gene RAB26 in CC tissues was analyzed by bioinformatics analysis, and the effect of abnormal KLF4 expression on prognoses of CC patients was predicted. Luciferase reporter assay detected the targeted relationship between KLF4 and RAB26. The viability and apoptosis of CC cells were analyzed by CCK-8 and flow cytometry. The formation of intracellular autophagosomes was detected by confocal laser scanning microscopy and immunofluorescence staining. The mRNA and protein levels were assayed by qRT-PCR and western blot. A xenograft animal model was constructed to verify the function of KLF4. Rescue assay was employed to verify whether KLF4/RAB26 could affect 5-FU resistance in CC cells through autophagy. RESULTS: KLF4 and RAB26 were lowly expressed in CC. KLF4 correlated with patients' survival. KLF4 was down-regulated in 5-FU resistant CC cells. KLF4 overexpression suppressed the proliferation and 5-FU resistance of CC cells, and inhibited LC3 II/I expression and autophagosome formation. Autophagy activator Rapamycin or sh-RAB26 treatment reversed the impact of KLF4 overexpression on 5-FU resistance. In vivo assay verified that KLF4 inhibited 5-FU resistance in CC cells. Rescue experiments revealed that KLF4 targeted RAB26 to inhibit CC cell autophagy, resulting in decreasing the resistance to 5-FU. CONCLUSION: KLF4 strengthened the sensitivity of CC cells to 5-FU by targeting RAB26 to restrain autophagy pathway.

Upregulated lncRNA Cyclin-dependent kinase inhibitor 2B antisense RNA 1 induces the proliferation and migration of colorectal cancer by miR-378b/CAPRIN2 axis.

LncRNA Cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) plays a role in the progression of multiple cancers like cholangiocarcinoma, osteosarcoma and several gastrointestinal tumors. Few studies have linked its function and mechanism to the development of colorectal cancer (CRC). The expression of CDKN2B-AS1, microRNA (miR)-378b, and cytoplasmic activation/proliferation-associated protein 2 (CAPRIN2) was analyzed in CRC patients and cell lines. The proliferation and migration of CRC cells were evaluated after gain and loss-of function mutations. Interactions between CDKN2B-AS1 and miR-378b, miR-378b and CAPRIN2 were validated by luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. The role of CDKN2B-AS1 was further confirmed in a xenograft mouse model. We found that the expression of CDKN2B-AS1 and CAPRIN2 was upregulated in CRC and they were linked to the poor differentiation and distant metastasis in CRC patients. CDKN2B-AS1 knockdown attenuated while CDKN2B-AS1 overexpression promoted CRC cell proliferation and migration. Notably, the results of Starbase 2.0 database analysis and in vitro experiments demonstrated that CDKN2B-AS1 could interact with miR-378b and regulate its expression. Furthermore, CAPRIN2 acted as a downstream target of CDKN2B-AS1/miR-378b that involved in modulating ß-catenin expression in CRC cells. Upregulation of CDKN2B-AS1 contributed to CRC progression via regulating CAPRIN2 expression by binding to miR-378b. Downregulation of CDKN2B-AS1 suppressed tumor growth and Ki-67 staining in vivo that was related to the miR-378b/CAPRIN2 pathway. This study indicated that lncRNA CDKN2B-AS1 promoted the development of CRC through the miR-378b/CAPRIN2/ß-catenin axis. CDKN2B-AS1 might serve as a potential and useful target in CRC diagnosis and treatment.

Advanced oxidation protein products trigger apoptosis and block epithelial-to-mesenchymal transition in crypt epithelial cells.

Advanced oxidation protein products (AOPPs) are uremic toxins. The present study aimed to investigate the effects of AOPPs on the epithelial mesenchymal transition (EMT) and apoptosis of rat crypt epithelial cells, and to assess the signaling pathways involved. The oxidized rat serum albumin was obtained by sodium hypochlorite modification as AOPPs, and the rat serum albumin (RSA) without sodium hypochlorite modification was set as the control. Different concentrations of AOPPs or RSA were incubated with rat crypt epithelial cells (IEC-6 cells). After culturing for 48 and 72 h, apoptosis was detected by flow cytometry. IEC-6 cells were divided into three groups: A normal group, an AOPPs group and an RSA group. Three groups of cells were collected following treatment for 2 h, and the phosphorylation levels of Akt and p65 NF-κB were detected by western blotting. After 72 h of treatment, the cells were collected and the apoptotic rate was detected by flow cytometry. The expression of EMT-related proteins was detected by reverse transcription-quantitative polymerase chain reaction and western blotting. The apoptotic rate of IEC-6 cells increased with the concentration of AOPPs, and the apoptotic rate of the AOPPs group was higher than that of the RSA group. The expression of fibronectin, snail, slug and collagen I in the AOPPs group was lower than that in the RSA group, while the expression of E-cadherin was not significantly different between the two groups. In addition, the expression of fibronectin, snail, slug and collagen I genes in the AOPPs-treated group was equal to or lower than that in the normal group. Compared with the normal group, the Akt phosphorylation level was decreased and the p65 phosphorylation level was increased in the AOPPs- or RSA-treated groups. Compared with the AOPPs-treated group, Akt and p65 phosphorylation levels in RSA-treated group were slightly higher. In conclusion, AOPPs trigger apoptosis and inhibit the EMT of rat crypt epithelial cells, which may be associated with the inhibition of Akt phosphorylation and the promotion of p65 phosphorylation.

Extracellular vesicles derived from cancer-associated fibroblast carries miR-224-5p targeting SLC4A4 to promote the proliferation, invasion and migration of colorectal cancer cells.

More and more studies indicated that extracellular vesicles (EVs) carrying miRNAs have been potential biomarkers of various cancers including colorectal cancer (CRC). This study aims to explore the function of miR-224-5p carried by EVs derived from cancer-associated fibroblasts (CAFs) in CRC. Here, we found that miR-224-5p was highly expressed while SLC4A4 was lowly expressed in CRC cells. Moreover, dual-luciferase reporter gene assay testified that miR-224-5p targeted SLC4A4. The expression of miR-224-5p in CAFs-derived EVs was found to be elevated. It was also testified that CAFs-derived EVs could transfer miR-224-5p into CRC cells. miR-224-5p in CAFs-derived EVs facilitated the proliferation, migration, invasion and anti-apoptosis of CRC cells. Overexpressing miR-224-5p increased the proliferative, migratory and invasive abilities of CRC cells and inhibit CRC cell apoptosis, while overexpressing SLC4A4 caused the opposite result. Research in vitro and in vivo further indicated that miR-224-5p promoted CRC cell progression via binding to its downstream target gene SLC4A4. Rescue assay also demonstrated that overexpressing miR-224-5p reversed the inhibitory effect of overexpressed SLC4A4 on cancer cell growth. In addition, in vivo assay identified that high level of miR-224-5p promoted the growth of cancer cells in mice in vivo. In conclusion, we explored the effect of miR-224-5p in CRC, which helps for further exploration of new methods for CRC targeted therapy.

Risk factors for gastric cancer and related serological levels in Fujian, China: hospital-based case-control study.

OBJECTIVE: To explore the relationships between gastric cancer and serum pepsinogen I (PG I), PG II, PG I/II ratio, gastrin 17 (G-17) and Helicobacter pylori infection, and to investigate dietary and lifestyle risk factors for gastric cancer in Fujian Province, China. DESIGN: A hospital-based, 1:1 matched case-control study. SETTING: Patients with newly diagnosed gastric cancer were recruited from the Fujian Provincial Hospital and the No. 900 Hospital of the Joint Support Force of the Chinese People's Liberation Army between July 2014 and December 2016. PARTICIPANTS: A total of 180 pairs of patients with gastric cancer and control subjects were recruited in the study, including 134 (74.4%) male pairs and 46 (25.6%) female pairs. INVESTIGATION AND ANALYSIS MEASURES: Serological indicators were tested with ELISA kits. Dietary, lifestyle and psychological factors were investigated through face-to-face questionnaire. Relationships between gastric cancer and these influencing factors were examined by Χ2 test and conditional logistic regression. RESULTS: Serum PG II and G-17 levels and H. pylori infection rate were higher in patients with gastric cancer than in control subjects (p<0.05), while PG I/II ratio was lower in patients with gastric cancer (p<0.05). Serum G-17 levels were higher in patients with corpus gastric cancer than in patients with antral gastric cancer (p<0.05). Serum PG II levels were higher in patients with advanced gastric cancer than in patients with early-stage cancer (p<0.05), however, PG I/II ratio was lower in patients with advanced-stage gastric cancer than in patients with early-stage cancer (p<0.05). Eating hot food (OR=2.32), eating pickled vegetables (OR=4.05) and often feel troubled (OR=2.21) were found to significantly increase the risk of gastric cancer (all p<0.05), while consuming onion or garlic (OR=0.35), drinking tea (OR=0.26), eating fresh fruits (OR=0.55), and high serum PG I (OR=0.99) or PG I/II ratio (OR=0.73) were found to be protective against gastric cancer. CONCLUSION: Study results showed that serum PG, G-17 and H. pylori antibodies could be useful indicators for early diagnosis of gastric cancer. Increase in serum G-17 level might indicate the location of gastric cancer. Increase in serum PG II level and decrease in PG I/II ratio might imply the clinical stage. Eating hot food, eating pickled vegetables and often feel troubled may be risk factors for gastric cancer, while eating fresh fruits, eating onion or garlic, and drinking tea may be protective factors against the disease.

[Histological study on the safety of the controllable ileostomy with pipe].

OBJECTIVE: To investigate the safety of the controllable ileostomy with pipe in view of histology. METHODS: Twenty-eight Beagle dogs undergoing controllable ileostomy with pipe were studied. The special fistula tube with balloon was placed into the hole locating at the cecal root opposing the mesenteric side, and fixed by double knot compression method. RESULTS: The fistula tube was removed 14 days after surgery, then the safety of the procedure was preliminarily evaluated by gastrointestinal radiography and anatomical observation. The small intestine tissue at the compression suture was used as the experimental segment, and the small intestine tissue at the proximal non-compression suture was used as the control segment. The histological staining and the immunohistochemical staining of S-100 protein, c-kit protein and α-smooth muscle actin(α-SMA) protein between two segment were compared, while quantitative comparison of myenteric plexus, intestinal Cajal cell(ICC) and smooth muscle cells in intestinal wall was carried out. After removal of fistula tube at 14 days postoperative, the dogs were normal in feeding and defecation. The digestive tract radiography showed that the intestine was patent without obvious stenosis and obstruction. The dogs were dissected 21 days after operation. The abdominal sinus ostium was well healed and the internal sinus was well formed. Under gross inspection, blood supply, morphology and motor function of experimental intestine segment were similar from the proximal and distal segments of control intestine. S-100 immunohistochemical staining showed that the morphology and distribution of S-100 protein positive cells and "blank area" cells in the experimental and control segments were consistent. Myenteric plexus counting showed that the experimental segment was 3.62±1.82/field and the control segment was 3.27±1.62/field, whose difference was not statistically significant(t=1.30, P=0.20). Immunohistochemical staining of c-kit showed that the distribution of c-kit positive cells in both segments was consistent. Counting of the number of ICCs in myenteric plexus revealed that experimental segment was 2.96±2.57/plexus, and control segment was 2.49±1.80/plexus without significant difference(t=1.81, P=0.07). Immunohistochemical staining of α-SMA showed that the morphology and distribution of smooth muscle cells in whole intestinal wall(muscle layer, longitudinal muscle, ring muscle) in experimental and control segments were consistent. The average absorbance(A) value of α-SMA staining in ring muscle layer was detected and quantified. The experimental segment was 0.15±0.03 and control segment was 0.14±0.04 without significant difference(t=1.16, P=0.25). CONCLUSION: The technique of controllable ileostomy with pipe is safe in view of histology, which may replace the traditional protective ileostomy.

Functions of the AP-2α gene in activating apoptosis and inhibiting proliferation of gastric cancer cells both in vitro and in vivo.

INTRODUCTION: This study was designed to investigate the potential function of the activating protein 2α (AP-2α) gene in controlling the proliferation and apoptosis of gastric cancer. MATERIAL AND METHODS: Gastric cancer cell line MCG-803 cells and normal cell line GES-1 cells were selected to transfect pcDNA3.1(+)-AP-2α and pcDNA3.1(+) plasmids, respectively. Both mRNA and protein levels of AP-2α in each group transfected with the pcDNA3.1(+)-AP-2α plasmids were up-regulated after 48 h by real-time PCR and Western blotting analysis, leading to marked proliferation inhibition and significant cell cycle arrest. RESULTS: pcDNA3.1(+)-AP-2α reduced tumor tissue growth in a subcutaneous tumor gastric carcinoma nude mouse model. Protein over-expression of AP-2α in the nude mouse model was accompanied by down-regulation of Blc-2 and ErbB2, resulting in the up-regulation of caspase-3, -8, and -9, ERα and p21WAF1/CIP1. CONCLUSIONS: The reintroduction of the AP-2α gene by pcDNA3.1 could inhibit gastric tumor growth in vitro and in vivo, which may be an alternative future therapeutic molecular target for human gastric cancer.

[Application research of presacral space drainage tube combined with subcutaneous vacuum pressure suction in the laparoscopic-assisted abdominoperineal resection].

OBJECTIVE: To study the management for the perineal incision after laparoscopic-assisted abdominoperineal resection for rectal cancer. METHODS: Clinical data of 87 patients undergoing laparoscopic Miles operation for lower rectal cancer from June 2009 to February 2014 were collected and studied. Presacral space drainage group: presacral space drainage tube was applied in 42 patients. Combined drainage group: presacral space drainage tube combined with subcutaneous vacuum pressure suction was applied in 45 cases. In combined drainage group, except the presacral drainage tube, another drainage tube was placed subcutaneously and connected to a negative pressure ball, which was fixed on the lateral anterior of perineal wound by the further incision and drainage. After subcutaneous tube was placed for 2 weeks, as drainage fluid was limpid and <15 ml/d for 3 days, meanwhile no obvious pelvic fluid was detected by ultrasound, and the wound healed quite well without redness and edema, then the subcutaneous tube with the negative pressure ball could be removed. RESULTS: There were 51 males and 36 females with the mean age of 26-78(56.9±10.8) years old. The laparoscopic Miles operation was successfully completed in all the cases without death and complications. The drainage tube was placed for 4-13(8.0±2.5) days in presacral space drainage group, and for 4-14(6.7±2.4) days in combined drainage group. The subcutaneous tube was placed for 14-24(15.8±3.0) days. The primary healing rate of perineal wound in presacral space drainage group and combined drainage group was 66.7%(28/42) and 91.1%(41/45) respectively, while the perineal wound infection rate was 21.4%(9/42) and 4.4%(2/45) respectively, whose differences between two groups were both significant (χ2=7.911, P=0.005 and χ2=5.674, P=0.017). CONCLUSION: Presacral space drainage tube combined with subcutaneous vacuum pressure suction in laparoscopic-assisted abdominoperineal resection for rectal cancer has better efficacy and lower infection rate for perineal incision, which is worth wide application.

Decreased expression of insulin-like growth factor binding protein 6 is associated with gastric adenocarcinoma prognosis.

The present study aimed to investigate the expression and prognostic significance of insulin-like growth factor binding protein 6 (IGFBP-6) in gastric adenocarcinoma. The expression of IGFBP-6 was examined in 263 specimens from gastric adenocarcinoma patients using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunohistochemical (IHC) staining. The association between IGFBP-6 expression, clinicopathological factors and clinical outcomes was investigated. Akaike information criterion (AIC) and Harrell's concordance index (c-index) were used to evaluate the accuracy of the predictive prognosis. RT-qPCR and western blotting results showed that IGFBP-6 mRNA expression was lower in the tumors compared with that in adjacent non-tumor tissues. IGFBP-6 showed significantly decreased expression in 170 out of 263 patients based on IHC data and this was associated with a larger tumor size (P<0.001) and poorly-differentiated adenocarcinoma (P=0.001), as well as with palliative gastrectomy (P=0.015). Additionally, decreased expression of IGFBP-6 was associated with stage T3/4a/4b disease and lymph node-positive metastasis (P<0.001). The association between decreased expression and a poor prognosis was revealed by Kaplan-Meier curves. Cox regression model identified IGFBP-6 as an independent prognostic factor. The prognostic value of the model with IGFBP-6 expression (AIC, 924.881; c-index, 0.878) was superior to that without IGFBP-6 expression (AIC, 947.164; c-index, 0.825). In conclusion, IGFBP-6 involves the development and progression of gastric adenocarcinoma, and its decreased expression predicts poor clinical outcomes.

[Expression of transcriptional coactivator with PDZ-binding motif (TAZ) in colon cancer tissues and its clinical significance].

OBJECTIVE: To investigate the expression of transcriptional coactivator with PDZ-binding motif(TAZ) in colon cancer tissues and its association with clinicopathological parameters and prognosis of patients. METHODS: The expression of TAZ protein was detected in 56 resected colon cancer tissues and matched tumor-adjacent tissues using immunohistochemistry. The positive expression rate of TAZ was compared between patients with different clinicopathological features. The association between TAZ expression and prognosis was analyzed. RESULTS: Expression of TAZ protein located in the nucleolus. The positive expression rate of TAZ in colon cancer tissues was significantly higher than that in matched tumor-adjacent tissues(73.2% vs. 12.5%, P=0.000). Clinicopathological evaluation suggested that the expression of TAZ protein was associated with tumor size(P=0.009), depth of infiltration(P=0.026), lymph node metastasis (P=0.007) and TNM staging(P=0.004). Colon cancer patients with negative expression of TAZ showed a better 5-year survival as compared with those with positive expression of TAZ (66.7% vs. 22.9%, P=0.0017). Multivariate Cox regression analysis revealed that positive TAZ expression was an independent factor for predicting poor prognosis in colon cancer (HR:3.532, 95% CI: 1.3-9.9, P=0.016). CONCLUSION: The expression of TAZ protein is up-regulated in colon cancer tissues and its high expression is associated with poor prognosis of colon cancer patients.

[Comparison of short- and long-term efficacy of three procedures in postoperative digestive tract reconstruction for upper gastric cancer].

OBJECTIVE: To compare the short- and long-term efficacy of three different procedures used for digestive tract reconstruction after radical gastrectomy for upper gastric cancer. METHODS: Clinical data of 191 patients with upper gastric cancer undergoing radical gastrectomy in the Fujian Provincial Hospital between January 2000 and December 2012 were analyzed retrospectively. Surgical procedures were classified as total gastrectomy followed by Roux-en-Y esophagojejunostomy (TG-RY, n=123), proximal gastrectomy followed by esophagogastrostomy (PG-EG, n=40), and proximal gastrectomy followed by jejunal interposition (PG-JI, n=28). Clinicopathological characteristics, perioperative and long-term outcomes were compared among the three groups. RESULTS: The operative time was shorter (178 vs. 248 and 224 min, P<0.05), and the intraoperative blood loss was less (194 vs. 323 and 265 ml, P<0.05) in PG-EG group than those in TG-RY and PG-JI groups. Early postoperative complications and hospital stay were comparable (both P>0.05). With respect to gastrectomy-associated symptoms, reflux and heartburn were more frequent in PG-EG patients, while dumpling syndrome was more frequent after TG-RY. Postoperative weight loss was not significantly different among three procedures (P>0.05), however, hemoglobin and serum albumin levels were lower in TG-RY patients (both P<0.05). The 5-year survival rate was similar (P>0.05). CONCLUSIONS: Surgeons need to choose the proper procedure according to tumor features and patient condition. PG-JI should be the first choice in terms of fewer complaints and better nutrition. TG-RY tends to be used for larger and more advanced tumors. PG-EG is the most minimally invasive procedure and thus may be suitable for older and high-risk patients.

[Expression of miR-146a in colon cancer and its significance].

OBJECTIVE: To investigate miR-146a expression in colonic cancer and its clinical implications. METHODS: Quantitative real-time PCR was employed to detect the levels of miR-146a expression in colonic cancer tissues, pair-matched adjacent normal tissues and different colonic cancer cell lines. MTT essay was used to evaluate the proliferation of colonic cancer SW260 cells transfected with miR-146a mimics, and the cell cycle and apoptosis of the cells were analyzed with flow cytometry. RESULTS: Compared with the normal tissues, 38 of the 43 colonic cancer samples showed down-regulated miR-146a expression, which was associated with poor tumor differentiation. The expression of miR-146a in the tumor tissues was significantly correlated with tumor size and clinical stages. The patients with high miR-146a expression levels had significantly longer total survival time than those with low expression of miR-146a. In SW260 cell cultures, transfection with miR-146a mimics significantly inhibited cell growth (P<0.05) and increased the cell apoptosis rate (11.9% vs 5.9%) but produced no obvious effect on cell cycle. CONCLUSIONS: miR-146a may serve as a potential therapeutic target for colonic cancer for its role in inhibiting colonic cancer cell proliferation.

[Pattern of lymph node metastasis and extent of lymphadenectomy for distal gastric cancer].

OBJECTIVE: To analyze lymph node (LN) metastasis patterns and determine the appropriate extent of LN dissection in distal-third gastric cancer. METHODS: Clinical data of 545 patients with distal third gastric cancer undergoing radical operation in the Fujian Provincial Hospital between 2001 and 2010 were analyzed retrospectively. The metastasis rate for each LN station was analyzed stratified by the depth of tumor invasion. RESULTS: The incidence of LN metastasis in this cohort was 38.2% (208/545). LN metastasis rate in mucosal cancer was 2.0% (2/99) and involved LNs were limited to station 1 LN stations. LN metastasis rate in submucosal cancer was 18.9% (18/95), significantly higher than that in mucosal cancer (P<0.01). The metastasis rates to groups No.7, 8 and 9 in station 2 were 5.3% (5/94), 3.2% (3/94), and 1.1% (1/89) respectively. In addition, 3 cases (3.2%) had metastasis in station 2 outside the range of groups 7, 8 and 9 including groups No.1, 11p and 12. Gastric cancer invading the muscularis propria or deeper layers showed an significant increased rate of metastasis (P<0.01). CONCLUSION: D1 dissection seems to be sufficient for mucosal cancer. Standard D2 dissection should be performed for cancers of the muscularis propria or deeper. For submucosal cancer, an extended D1+ dissection is required for complete removal of metastatic nodes.

[Analysis of prognostic factors in lymph node-negative advanced gastric cancer patients].

OBJECTIVE: To investigate the prognostic factors of lymph node-negative advanced gastric cancer patients in order to guide adjunctive therapy and surveillance tragedy. METHODS: A total of 236 advanced gastric cancer patients with no less than 12 retrieved lymph nodes and without lymph node metastasis from Fujian Provincial Hospital between 1998 and 2008 were collected retrospectively. Univariate and multivariate prognostic analysis were performed. RESULTS: Two hundred and twenty-four patients(94.9%) were followed up and 5-year overall and disease-free survival rates were 75.2% and 66.4% respectively. Univariate prognostic analysis showed that depth of infiltration, Lauren histotype and retrieved lymph nodes were associated with 5-year overall survival(all P<0.05). Multivariate prognostic analysis testified that depth of infiltration was independent prognostic predictor(P<0.05). Recurrent rates of T2 and T3 patients were 5.8%(8/138) and 14.0%(12/86),5-year overall survival rates were 82.5% and 59.0%, 5-year disease-free survival rates were 70.4% and 52.2% respectively. These differences were all statistically significant (all P<0.05). CONCLUSIONS: T2N0 gastric cancer patients have a better prognosis than T3N0 patients. Depth of infiltration should be considered to stratify lymph node-negative gastric cancer patients for an adjunctive treatment and follow-up scheduling.