m(6)A demethylase ALKBH5 inhibits cell proliferation and the metastasis of colorectal cancer by regulating the FOXO3/miR-21/SPRY2 axis.

OBJECTIVE: Colorectal cancer is a common malignancy worldwide. This research aimed to investigate the role of α-ketoglutarate-dependent dioxygenase alkB homologue 5 (ALKBH5), a N6-methyladenosine (m(6)A) demethylase, on the cell proliferation and metastasis of colorectal cancer. METHODS: The interaction relationship between FOXO3, miR-21, and SPRY2 were predicted by starBase 2.0 and determined using RIP, CHIP, and dual-luciferase reporter assays. Quantitative reverse transcription PCR (RT-qPCR) and western blot were used to measure the gene and miRNA expressions of ALKBH5, FOXO3, miR-21, and SPRY2. The cell proliferation was determined using CCK8 and colony formation assays. The metastatic abilities were measured using wound healing and transwell assays. RESULTS: In colorectal cancer, downregulated ALKBH5 is related to poor prognosis. Rescued ALKBH5 suppresses the proliferation and metastasis of colorectal cancer cells. The role of ALKBH5 is achieved by reducing the m(6)A modification of forkhead box O3 (FOXO3), which enhances its stability. FOXO3 targets miR-21 and increases the SPRY2 expressions. The antitumor effects of ALKBH5 can be blocked by FOXO3 knockdown, which is reversed by the miR-21 inhibitor. CONCLUSION: ALKBH5 plays an antitumor role in colorectal cancer by regulating the FOXO3/miR-21/SPRY2 axis, providing a new direction for colorectal cancer therapy.

MEG3 can affect the proliferation and migration of colorectal cancer cells through regulating miR-376/PRKD1 axis.

The down-regulation of long non-coding RNA (lncRNA) MEG3 has been observed in various cancers; nonetheless, underlying mechanisms are still unclear. The current research work aims at exploring the roles of MEG3 in the pathogenesis of CRC and the associated mechanism. We observed that MEG3 was significantly down-regulated in both CRC tumor tissue and cell lines; also, the transient over-expression of MEG3 in CRC cell line SW480 and LoVo inhibited the proliferation and the migration and clone formation capability of cells; on the other hand, the knockdown of MEG3 has revealed opposite effects. Eventually, we figured it out that target miR-376 directly targeted both MEG3 and PRDK1 in SW480 and LoVo cells. To conclude, as our findings proved, MEG3 is likely to act as a tumor suppressor in the pathogenesis of CRC by means of the regulation of the miR-376/PRDK1 signal axis, suggesting that MEG3 has the potential to become a novel therapeutic target for the treatment of CRC.

Tumor-Associated Macrophages Derived TGF-ß‒Induced Epithelial to Mesenchymal Transition in Colorectal Cancer Cells through Smad2,3-4/Snail Signaling Pathway.

PURPOSE: We investigated the role of tumor-associated macrophages (TAMs) on the epithelial to mesenchymal transition (EMT) of colorectal cancer cells and determined the potential mechanism involved in the metastatic process. MATERIALS AND METHODS: In this study, flow cytometry was used to detect the expression of target proteins. We used transwell assay to evaluate the migration of cancer cells under specific conditions. Using real-time polymerase chain reaction, we examined the expressions of cytokines and EMT-related markers in mRNA level. Animal assay was performed for analysis in vivo and hematoxylin and eosin was used to visualize the effect of TAMs on tumor metastasis. We also used immunohistochemistry and Western blotting to detect the expression of target proteins. RESULTS: Here, we observed enrichment of TAMs in colorectal tumor tissues, resulting in high metastasis in clinical therapy. Moreover, those TAMs could facilitate the EMT progression of colorectal cancer cells, which is induced by the transforming growth factor-ß (TGF-ß) derived from TAMs, leading to the invasion and migration of cancer cells. CONCLUSION: Our results demonstrated that TAMs contributed the EMT progression through a TGF-ß/Smad2,3-4/Snail signaling pathway, and disrupting this pathway with TGF-ß receptor inhibitor could suppress metastasis, readjusting our focus to the connection of TAMs and cancer metastasis.

Simple instruments facilitating achievement of transanal total mesorectal excision in male patients.

AIM: To assess the efficacy of a modified approach with transanal total mesorectal excision (taTME) using simple customized instruments in male patients with low rectal cancer. METHODS: A total of 115 male patients with low rectal cancer from December 2006 to August 2015 were retrospectively studied. All patients had a bulky tumor (tumor diameter ≥ 40 mm). Forty-one patients (group A) underwent a classical approach of transabdominal total mesorectal excision (TME) and transanal intersphincteric resection (ISR), and the other 74 patients (group B) underwent a modified approach with transabdominal TME, transanal ISR, and taTME. Some simple instruments including modified retractors and an anal dilator with a papilionaceous fixture were used to perform taTME. The operative time, quality of mesorectal excision, circumferential resection margin, local recurrence, and postoperative survival were evaluated. RESULTS: All 115 patients had successful sphincter preservation. The operative time in group B (240 min, range: 160-330 min) was significantly shorter than that in group A (280 min, range: 200-360 min; P = 0.000). Compared with group A, more complete distal mesorectum and total mesorectum were achieved in group B (100% vs 75.6%, P = 0.000; 90.5% vs 70.7%, P = 0.008, respectively). After 46.1 ± 25.6 mo follow-up, group B had a lower local recurrence rate and higher disease-free survival rate compared with group A, but these differences were not statistically significant (5.4% vs 14.6%, P = 0.093; 79.5% vs 65.1%, P = 0.130). CONCLUSION: Retrograde taTME with simple customized instruments can achieve high-quality TME, and it might be an effective and economical alternative for male patients with bulky tumors.

Prognostic Value of Lymph Node Ratio in Locally Advanced Rectal Cancer Patients After Preoperative Chemoradiotherapy Followed by Total Mesorectal Excision.

Although the absolute number of positive lymph nodes (LNs) has been established as 1 of the most important prognostic factors in rectal cancers, many researchers have proposed that the lymph node ratio (LNR) may have better predicted outcomes. We conducted a retrospective study to compare the predictive ability of LNR and ypN category in rectal cancer. A total of 264 locally advanced rectal cancer (LARC) patients who underwent preoperative chemoradiotherapy (CRT) followed by total mesorectal excision (TME) between 2005 and 2012 were reviewed. All patients were categorized into 3 groups or patients with metastatic LNs were categorized into 2 groups according to the LNR. The prognostic effect on overall survival (OS) and disease-free survival (DFS) was evaluated. With a median follow-up of 45 months, the OS and DFS were 68.4% and 59.3% for the entire cohort, respectively. The respective 5-year OS and DFS rates for the 3 groups (LNR = 0, 0 < LNR ≤ 0.20, and 0.20 < LNR ≤ 1.0) were as follows: 83.2%, 72.6%, and 49.4% (P < 0.001) and 79.5%, 57.3%, and 33.5% (P < 0.001), respectively. Multivariate analysis revealed that LNR and differentiation, but not the number of positive LNs, had independent prognostic value for OS (hazard ratio [HR] = 2.328, 95% confidence interval [CI]: 1.850-4.526, P < 0.001) and DFS (HR = 3.004, 95% CI: 1.616-5.980, P < 0.001). As for patients with positive LNs, the respective 5-year OS and DFS rates for the 2 groups (0 < LNR ≤ 0.20, and 0.20 < LNR ≤ 1.0) were 72.6% and 49.4% (P < 0.001) and 57.3% and 33.5% (P < 0.001), respectively. Multivariate analysis revealed that only LNR was an independent factor for OS (HR = 3.214, 95% CI: 1.726-5.986, P < 0.001) and DFS (HR = 4.230, 95% CI: 1.825-6.458, P < 0.001). Subgroups analysis demonstrated that the ypN category had no impact on survival whereas increased LNR was a significantly prognostic indicator for worse survival in the LNs < 12 subgroup. LNR is an independent prognostic factor in LARC patients treated with preoperative CRT followed by TME. It may be a better independent staging method than the number of metastatic LNs when <12 LNs are harvested after preoperative CRT.

[Value of protective stoma in intersphincteric resection for ultra-low rectal cancer].

OBJECTIVE: To investigate the value of protective stoma in intersphincteric resection (ISR) for ultra-low rectal cancer. METHODS: Clinical data of 56 ultra-low rectal cancer patients without involvement of external anal sphincter treated during January 1999 to July 2009 with trans-anal ISR plus trans-abdominal total mesorectum excision and coloanal anastomosis were retrospectively analyzed. The patients were divided into two groups based on whether they received protective ostomy: ostomy group (16 cases) and ostomy-free group (40 cases). The postoperative complications as well as anal functional restoration were compared between the two groups. RESULTS: Sixteen cases (32.1%) of the 56 patients received protective stoma. The complication rate of anastomosis and anus complication rate in the ostomy-free group were significantly higher than those in ostomy group [35.0% (14/40) and 40.0% (16/40) vs. 1/16 and 1/16; P < 0.05]. In the ostomy-free group, one patient developed anastomotic dehiscence and tumor recurrence, the patients was given permanent colostomy, and the other three patients with lesions in the anastomosis and anus received ostomy and secondary surgical treatment, with a reoperation rate of 10.0% (4/40). The anal function of patients in the two groups were both decreased after the operation. The rate of patients got Kirwan grade I anal sphincter function in the 3rd, 6th and 12th month after protective stoma operation was 11/16, 13/15 and 11/13 in the ostomy group, respectively; and those were 30.0%, 37.5% and 45.0% in the ostomy-free group, respectively. Anal function was significantly better in the ostomy group than that in the ostomy-free group during the same postoperative period (P < 0.05). CONCLUSION: Protective stoma can avoid anastomotic leakage following ISR for ultra-low rectal cancer, and alleviate the suffering of anal incontinence in the early postoperative period, and is conducive to the restoration of anal function.

[Application of Altemeier procedure in the emergent management of acute incarcerated rectal prolapse].

OBJECTIVE: To evaluate the perineal rectosigmoidectomy(Altemeier procedure) in the emergent management of acute incarcerated rectal prolapse. METHODS: Clinical and follow-up data of 9 patients with acute incarcerated rectal prolapse undergone Altemeier procedure were retrospectively analyzed. RESULTS: The mean operation time was 1.7 (range 1.0-1.5) hours. The mean total blood loss during surgery was 109 (50-200) ml. The mean time to the first bowel movements was 2.8(1-6) days after surgery. The hospital stay was 5.3(3-10) days. There were no postoperative complications such as anastomotic leakage, intra-abdominal infection, or urogenital dysfunction. One patient developed thrombosis in the mesorectum and one patient had symptoms of anal discomfort. After a mean follow-up of 3.5(5 months-6.5 years) years, no patient had recurrent prolapse. Six months after operation, anal function was Kirwan grade I( in 8 cases and grade II( in 1 case. All the patients were satisfied with the result. CONCLUSION: Altemeier procedure can result in good postoperative anal function when treating incarcerated rectal prolapse, which should be the first choice in emergency treatment.

[Combination of trans-anal intersphincteric resection and trans-abdominal total mesorectal excision for anus-retained ultra-low rectal tumors].

OBJECTIVE: To study the combination of trans-anal intersphincteric resection and transabdominal total mesorectal excision for anus-retained ultra-low rectal tumors. METHODS: Clinical data of 34 ultra-low rectal tumor patients without external anal sphincter involved, who underwent the combination surgery, were retrospectively analyzed. RESULTS: The distance from the distal incisal margin of the rectum to the inferior margin of the tumor ranged from 1.8 cm to 3.0 cm on an average of 2.1 cm. For pathological types, there were 23 cases of adenocarcinoma (9 well differentiated and 14 moderately differentiated), 1 papillary carcinoma, 2 rectal stromal tumor, 5 rectal villous adenoma with neoplasia and 3 giant villous adenoma. For pathological stages, there were 18 cases at stage pTNM I, 5 at IIA, 1 at IIB, 4 at IIIA, 1 at III and for T grading, there were 15 cases at stage T1, 5 at T2, 8 at T3, 1 at T4. In these 34 patients, there were 3 cases with postoperative anastomotic stenosis, 2 with postoperative anastomotic rupture, 2 with rectovaginal fistula and no operative death. Because of the dysfunction of bowel control, bowel frequency varied from 3 to 12 in the early stage after operation, but with the recovery of anus function, bowel frequency decreased and ranged form 1 to 5 times a day and the time of formed bowel control could be more than 5 min in 6-12 months after operation. However, patients underwent total resection of internal anal sphincter still suffered from incontinence of loose stool after 1 year. After operation, anastomotic recurrence was found in 1 case in 5 months, liver metastasis in 1 case in 10 months and 28 months respectively, cardiac sudden death in 1 case in 26 months. CONCLUSION: The combination of trans-anal ISR and trans-abdominal TME for anus-retained ultra low rectal tumor is not only coincident with radical tumor principle but also retains the function of anus, on the premise of the strict indication.

[Clinical application of intersphincteric resection in the anal-preserving operation for ultra-low rectal carcinoma].

OBJECTIVE: To investigate the clinical application of intersphincter resection (ISR) combined with total mesorectal excision (TME) and colon-anal anastomosis in the treatment for ultra-low rectal carcinoma. METHODS: To review and analyze retrospectively the data of 34 patients with ultra-low rectal carcinoma (without external anal sphincter involvement) who received treatment of ISR, TME and colon-anal anastomosis. RESULTS: Partial resection of internal sphincter was performed in the patients with a distal edge of the tumor greater than or equal to 2 cm from the dentate line. Subtotal removal of the rectum was performed between 1 cm and 2 cm. Total resection was performed in less than 1 cm or involvement of dentate line. Reconstruction of digestive tract was done by manual colon-anal anastomosis. The average distance from distal excised margin to the tumor was 2.3 (1.8 - 3.2) cm among 34 patients. The pathological types were as follows: 28 cases of adenocarcinoma (11 were well differentiated, 17 moderately differentiated), 1 case of papillary carcinoma and 5 cases of villous adenoma with malignant change. The postoperative pathological stages were: Dukes stage A in 28 cases, stage B in 1 and stage C in 5 cases. The pTNM staging was 28 cases in phase I, 1 in phase IIa, 4 in phase IIIa and 1 in phase IIIb. The T stages of the patients were as following: 16 Tl, 17 T2 and 1 T3. Postoperative anastomotic stenosis occurred in 3 cases, anastomotic dehiscence in 2 cases and rectovaginal fistula in 2 cases. The ability of controlling feces of patients decreased significantly in the early postoperative period, and restored gradually at 6 to 12 months after operation. Anastomotic recurrence occurred in 1 case at 5 months after operation and liver metastasis in 1 case at 40 months. CONCLUSION: With strictly grasping indications, radical resection can be attained and anal sphincter preserved by ISR combined with TME and colon-anal anastomosis. It is an effective sphincter-preserving operation.

[Effects of fuzheng quxie granule on immune cells and cytokines in populations with respiratory viral infection].

OBJECTIVE: To investigate the effect of fuzheng quxie granule (FQG) on immune cells and cytokines in populations with respiratory viral infection. METHODS: Fifty-nine patients were randomly divided into 3 groups, that is, 19 patients treated with conventional western medicine (WM) plus FQG in the treated group, 19 patients treated with conventional western medicine alone in the WM group, and 21 patients treated with FQG alone in the TCM group. The levels of T lymphocyte subsets, interleukine-2,4,6,10 (IL-2, IL-4, IL-6, IL-10), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma) and Th1/Th2 were determined before treatment, and at the end of 1st and 2nd week of treatment respectively. RESULTS: Before treatment, levels of TNF-alpha, IL-2, IL-6, IL-10 and INF-gamma in all patients were significantly higher than normal range (P < 0.05). After being treated for 1 week, the levels of serum TNF-alpha, IL-6, and IL-10 were significantly decreased in all groups (P < 0.05), serum IL-2 and INF-gamma decreased to the normal level in the WM group, but in the treated and the FQG group by the end of the 2nd week, the two indexes still remained at the rather higher level (P < 0.05). The ratio of Th1 and Th2 in the treated group and the FQG group increased significantly by the end of 2nd week, reached the level higher than that in the WM group and that before treatment (P < 0.05). No significant difference in, T lymphocytes subsets (CD3+ , CD4+ , CD8+) and percentage of B and NK cells before and after treatment was found in all the 3 groups. CONCLUSION: FQG can positively regulate the immune function of patients with respiratory tract viral infection in certain degree.

Clinicopathological and molecular genetic analysis of HNPCC in China.

AIM: To explore the clinicopathological and molecular genetic features of hereditary nonpolyposis colorectal cancer (HNPCC) in Chinese population. METHODS: We collected 16 Chinese HNPCC families from Wenzhou, Zhejiang Province, China. Tumor tissues and peripheral white blood cells were studied using microdissection, microsatellite analysis, immunostaining of hMSH2 and hMLH1 proteins and direct DNA sequencing of hMSH2 and hMLH1 genes. RESULTS: (1) A total of 50 patients had CRC. Average age at diagnosis of the first CRC was 45.7 years; 40.9% and 28.7% of the CRCs were located proximal to the splenic flexure and in the rectum, respectively. Thirty-eight percent of the colorectal cancer patients had synchronous and metachronous CRC. 34.4% and 25% of the CRCs were poor differentiation cancer and mucinous adenocarcinoma, respectively. Fourteen extracoloni tumors were found, and the hepatic cancer was the most common tumor type. Twenty-one patients whose median survival time was 5.7 years died during 1-23 years. Twenty-nine patients have survived for 1-28 years, 58.6%, 41.4% and 24.1% patients have survived for more than 5, 10 and 15 years, respectively; (2) All nine tumor-tissues showed microsatellite instability (MSI) at more than two loci. Four tumor-tissues lost hMSH2 protein expression and one lost hMLH1 protein expression. Three pathological germline mutations were identified from five genetically analyzed families; two of three mutations had not been reported previously as they were a transition from C to A in exon 14 (codon 743) of hMSH2 and a TTC deletion in exon 14 (codon 530) of hMLH1. CONCLUSION: Chinese HNPCC have specific clinicopathological features, such as early onset, propensity to involve the proximal colon, and high frequency of multiple CRCs, liver cancer more frequent than endometrial cancer. Chinese HNPCC showed relatively frequent germline mutation of mismatch repair (MMR) genes that correlated closely with high-level MSI and loss of expression of MMR genes protein.

[Etiologic fraction and interaction of risk factors for primary hepatocellular carcinoma in Wenzhou, Zhejiang Province].

OBJECTIVE: To explore etiologic fraction (EF) and interaction of serum hepatitis B surface antigen (HBsAg) carriage and other risk factors for primary hepatocellular carcinoma (PHC) in Wenzhou, Zhejiang, China. METHODS: 1:1 matched case-control study was carried out in Wenzhou, with 180 cases of PHC and 180 controls. EF and interactions of serum positive HBsAg [HBsAg(+)] and other risk factors for PHC were analyzed by Mantel-Haenszel stratified method and conditional multiple logistic regression. RESULTS: Serum HBsAg(+), poor economic status during the past five years, preferring intake of pickled vegetables, history of PHC in their first-degree relatives, and negative life events all were risk factors for PHC, with EFs of 0.728, 0.245, 0.224, 0.084, and 0.234, respectively. There existed interactions of HBsAg(+) with other risk factors, including poor economic status during the past five years, preferring intake of pickled vegetables, history of PHC in their first-degree relatives, and negative life events, with etiologic fractions attributable to interaction [EF (A x B)] of 0.770, 0.630, 0.848, and 0.627, and indices of interaction of 0.789, 0.638, 0.852, and 0.634, respectively. CONCLUSIONS: Main risk factor for PHC in Wenzhou, Zhejiang, China could include HBsAg(+), poor economic status during the past five years, preferring intake of pickled vegetables, history of PHC in their first-degree relatives, and negative life events. HBsAg(+) plus any of the following factors, such as poor economic status during the past five years, preferring intake of pickled vegetables, history of PHC in their first-degree relatives, and negative life events, could increase the risk of PHC.