METTL3 contributes to slow transit constipation by regulating miR-30b-5p/PIK3R2/Akt/mTOR signaling cascade through DGCR8.

BACKGROUND: N6-methyladenosine (m6A) is the most prevalent methylation modification of eukaryotic RNA, and methyltransferase-like 3 (METTL3) plays a vital role in multiple cell functions. This study aimed to investigate the role of m6A methylase METTL3 in slow transit constipation (STC). MATERIAL AND METHOD: The expression of METTL3 and DGCR8 was measured in STC tissues and glutamic acid-induced interstitial cells of Cajal (ICCs). The effects of METTL3, miR-30b-5p, and DGCR8 on the biological characteristics of ICCs were investigated on the basis of loss-of-function analyses. Luciferase reporter assay was used to identify the direct binding sites of miR-30b-5p with PIK3R2. RESULTS: The results showed that the METTL3, DGCR8, miR-30b-5p, and the methylation level of m6A were significantly increased in STC tissues and glutamic acid-induced ICCs. Silencing of METTL3 and miR-30b-5p inhibited apoptosis, autophagy, and pyroptosis of glutamic acid-induced ICCs. Moreover, overexpression of miR-30b-5p reversed the cytoprotection of METTL3 knockdown in glutamic acid-induced ICCs. Besides, DGCR8 knockdown could facilitate cell growth and decrease apoptotic glutamic acid-induced ICCs. Mechanically, we illustrated that METTL3 in glutamic acid-induced ICCs significantly accelerated the maturation of pri-miR-30b-5p by m6A methylation modification, resulting in the reduction of PIK3R2, which results in the inhibition of PI3K/Akt/mTOR pathway and ultimately leads to the cell death of STC. CONCLUSIONS: Collectively, these data demonstrated that METTL3 promoted the apoptosis, autophagy, and pyroptosis of glutamic acid-induced ICCs by interacting with the DGCR8 and successively modulating the miR-30b-5p/PIK3R2 axis in an m6A-dependent manner, and METTL3 may be a potential therapeutic target for STC.

VSNL1 Promotes Gastric Cancer Cell Proliferation and Migration by Regulating P2X3/P2Y2 Receptors and Is a Clinical Indicator of Poor Prognosis in Gastric Cancer Patients.

PURPOSE: The aim of this study was to investigate the role of Visinin Like 1 (VSNL1) in the proliferation and migration of gastric cancer (GC) cells as well as its clinical prognostic significance. METHODS: To this end, we evaluated VSNL1 expression in GC tissues and cell lines by real-time PCR and immunohistochemistry. To further explore the effects of VSNL1, a lentiviral vector expressing a short hairpin RNA (shRNA) against VSNL1 was constructed and transduced into the GC cell lines BGC-823 and SGC-7901. The interference efficiency of VSNL1-shRNA was determined by western blot. The effects of VSNL1 on the migration and invasion of GC cells as well as the expression of P2X3/P2Y2 were explored using MTS, colony formation, migration, and western blot assays. RESULTS: VSNL1 mRNA and protein levels were increased in GC tissues and cell lines. Furthermore, VSNL1 expression was positively correlated with Lauren's classification, lymph node metastasis, distant metastasis, TNM stage, and prognosis. VSNL1 expression was inversely correlated with the 5-year survival rate of GC patients. VSNL1 expression was markedly reduced in cells transduced with lentivirus expressing shRNA against VSNL1, and inhibiting VSNL1 expression significantly suppressed cell growth, migration, and colony formation and reduced the expression of P2X3/P2Y2. CONCLUSION: VSNL1 may promote the proliferation and migration of GC cells by regulating P2X3 and P2Y2 expression. VSNL1 plays important roles in GC development and metastasis and may be correlated with patient prognosis.

Schizandrin A exhibits potent anticancer activity in colorectal cancer cells by inhibiting heat shock factor 1.

Heat shock factor 1 (HSF1) is a powerful multifaceted oncogenic modifier that plays a role in maintaining the protein balance of cancer cells under various stresses. In recent studies, there have been reports of increased expression of HSF1 in colorectal cancer (CRC) cells, and the depletion of the HSF1 gene knockdown has inhibited colon cancer growth both in vivo and in vitro. Therefore, HSF1 is a promising target for colon cancer treatment and chemoprevention. In the present study, we found that Schizandrin A (Sch A) significantly inhibited the growth of CRC cell lines by inducing cell cycle arrest, apoptosis and death. Through HSE luciferase reporter assay and quantitative PCR (qPCR), we identified Sch A as a novel HSF1 inhibitor. In addition, Sch A could effectively inhibit the induction of HSF1 target proteins such as heat-shock protein (HSP) 70 (HSP70) and HSP27, whether in heat shock or normal temperature culture. In the Surface Plasmon Resonance (SPR) experiment, Sch A showed moderate affinity with HSF1, further confirming that Sch A might be a direct HSF1 inhibitor. The molecular docking and molecular dynamic simulation results of HSF1/Sch A suggested that Sch A formed key hydrogen bond and hydrophobic interactions with HSF1, which may contribute to its potent HSF1 inhibition. These findings provide clues for the design of novel HSF1 inhibitors and drug candidates for colon cancer treatment.

[Laparoscopic versus open wedge resection for gastrointestinal stromal tumors of the stomach: a clinical controlled study].

OBJECTIVE: To compare the surgical outcomes between laparoscopic and open wedge resection for gastrointestinal stromal tumors of the stomach. METHODS: Clinical data of 18 cases undergoing laparoscopic wedge resection from June 2000 to August 2009 at the Zhejiang Provincial People's Hospital were compared with 30 patients treated by open surgery. The perioperative parameters and prognosis data of the two groups were compared. RESULTS: Compared to the open group, laparoscopic group was found with longer operative time, less blood loss, less requirement of postoperative analgesia, earlier resumption of oral intake, earlier return of first flatus, and shorter postoperative hospital stay(all P<0.05). There were no postoperative deaths in both groups. Postoperative complication rate was significantly lower in the laparoscopic group(5.5% vs. 33.3%, P<0.05). The postoperative recurrence rates were 11.8%(2/17) and 10.7%(3/28); the 5-year survival rates were 78% and 63%, respectively, and the difference was not statistically significant(P>0.05). CONCLUSION: Laparoscopic wedge resection is a feasible treatment option for GISTs of the stomach.

[Oral glucose tolerance test and insulin response in Beagle dogs after subtotal distal gastrectomy].

OBJECTIVE: To evaluate the impact of reconstruction techniques after subtotal gastrectomy on postoperative glucose and insulin levels after oral glucose tolerance test (OGTT). METHODS: Distal gastrectomy was performed in 38 Beagle dogs. Reconstruction techniques used included integral continual jejunal interposition (n=9), Billroth I( (n=6), Billroth II( (n=7), and isolated jejunal interposition (n=8). Eight controls were used. OGTT was conducted to examine the changes in glucose and insulin levels. RESULTS: Compared to controls, glucose significantly increased in all the 4 operative groups and peaked at 60 min. Billroth II( was associated with the most significant increase. Insulin level significantly increased in all the experimental groups in response to food stimulus and peaked at 60 min. However, the increase of insulin in Billroth II( group was not as prominent as in other groups. CONCLUSIONS: Fluctuation of blood glucose after gastrectomy may be mitigated and insulin elevated if duodenal passage is preserved. Continual jejunal interposition should be given priority when Billroth I( reconstruction is not feasible.

[Experimental research of integral continuous jejunal interposition after subtotal distal gastrectomy].

OBJECTIVE: To evaluate nutritional status, myoelectrical activity, and gastrointestinal tract emptying capacity after integral continuous jejunal interposition following subtotal gastrectomy. METHODS: According to different re-construction techniques, 30 Beagle dogs were divided into four groups after subtotal distal gastrectomy: group 1(n=9, integral continuous jejunal interposition), group 2(n=6, Billroth I(), group 3(n=7, Billroth II(), group 4(n=8, isolated jejunal interposition). Blood cell counts, liver function, myoelectrical activity and the rate of gastrointestinal tract emptying were compared among the four groups. RESULTS: At week 12 after operation, the body weight in group 1 [(9.65±1.54) kg] was significantly higher than that in group 2[(9.25±1.76)kg], group 3[(9.31±1.54)kg] and group 4[(7.77±1.46)kg]. At week 4, the prognostic nutritional index in group 1(2671.9±49.9) was significantly higher than that in group 3(2555.9±54.7) and group 4(2440.9±54.3), but similar to that in group 2(2791.8±54.3). At week 6, the fasting and postprandial frequency of jejunal pacesetter potentials in group 1 were higher than those in group 3 and group 4(P<0.05) but comparable with those in group 2. The emptying rate of food in the four groups were 95.4%, 91.3%, 93.1% and 94.2%, respectively and there were no significant differences(P>0.05). However, as compared with group 2 and group 3, group 1 had longer operative time and later regular diet resumption, more severe abdominal adhesion(P<0.05). CONCLUSION: Continuous jejunal interposition should be considered when Billroth I( is not feasible after subtotal gastrectomy.

[Different procedures of alimentary tract reconstruction after subtotal distal gastrectomy in dogs].

OBJECTIVE: To explore an ideal procedure of alimentary tract reconstructions after subtotal distal gastrectomy. METHODS: Thirty-two healthy adult beagle dogs were randomly divided into experimental groups A, B, C and control group (n=8). Groups A, B, C operated by subtotal distal gastrectomy underwent 3 different reconstruction methods: continual jejunal interposition (CJI), Billroth II and Roux-en-Y. The control group received a sham operation. Dogs were observed for 12 weeks post-operation. The different parameters of body weight, food intake, PNI (prognostic nutritional index) and peripheral blood concentration of ghrelin were measured in 4 groups. RESULTS: The body weight, food intake and PNI in Groups A, B, C decreased significantly at post-operation versus pre-operation. There was a slow elevation of body weight, food intake and PNI at Week 12. Group A was significantly better than Groups B and C (P<0.05) while there was no significant difference between Groups B and C. The plasma ghrelin concentrations in Groups A, B, C were significantly reduced at Day 1 post-operation versus pre-operation. But no difference was observed among Groups A, B and C. However an elevated ghrelin concentration was observed at Week 1 post-operation. At Week 12 post-operation, the plasma ghrelin concentration in Group A increased significantly versus Groups B and C (both P<0.05). However, the plasma ghrelin concentration, food intake and PNI were not significantly changed in control group (P>0.05). CONCLUSIONS: The CJI reconstruction procedure is ideally suited for the preservation of duodenal passage after subtotal distal gastrectomy. Subsequently it leads to a significant elevation of circulating ghrelin concentration and a rapid post-operative recovery of food intake, body weight and PNI.