Randomised controlled trial of WISENSE, a real-time quality improving system for monitoring blind spots during esophagogastroduodenoscopy.

OBJECTIVE: Esophagogastroduodenoscopy (EGD) is the pivotal procedure in the diagnosis of upper gastrointestinal lesions. However, there are significant variations in EGD performance among endoscopists, impairing the discovery rate of gastric cancers and precursor lesions. The aim of this study was to construct a real-time quality improving system, WISENSE, to monitor blind spots, time the procedure and automatically generate photodocumentation during EGD and thus raise the quality of everyday endoscopy. DESIGN: WISENSE system was developed using the methods of deep convolutional neural networks and deep reinforcement learning. Patients referred because of health examination, symptoms, surveillance were recruited from Renmin hospital of Wuhan University. Enrolled patients were randomly assigned to groups that underwent EGD with or without the assistance of WISENSE. The primary end point was to ascertain if there was a difference in the rate of blind spots between WISENSE-assisted group and the control group. RESULTS: WISENSE monitored blind spots with an accuracy of 90.40% in real EGD videos. A total of 324 patients were recruited and randomised. 153 and 150 patients were analysed in the WISENSE and control group, respectively. Blind spot rate was lower in WISENSE group compared with the control (5.86% vs 22.46%, p<0.001), and the mean difference was -15.39% (95% CI -19.23 to -11.54). There was no significant adverse event. CONCLUSIONS: WISENSE significantly reduced blind spot rate of EGD procedure and could be used to improve the quality of everyday endoscopy. TRIAL REGISTRATION NUMBER: ChiCTR1800014809; Results.

Phosphatase and Tensin Homolog (PTEN) Represses Colon Cancer Progression through Inhibiting Paxillin Transcription via PI3K/AKT/NF-κB Pathway.

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is frequently mutated in colon cancer. However, the potential contribution of loss of PTEN to colon cancer progression remains unclear. In this study, we demonstrated that PTEN overexpression or knockdown in Lovo colon cancer cells decreased or increased paxillin expression, respectively. Moreover, paxillin reversed PTEN-mediated inhibition of Lovo cell invasion and migration. Overexpression of PTEN in an orthotropic colon cancer nude mice model inhibited tumor formation and progression. In addition, PTEN protein level was negatively correlated with that of paxillin in human colon cancer tissues. Mechanistically, we identified three NF-κB binding sites on paxillin promoter and confirmed that paxillin was a direct transcriptional target of NF-κB. Our findings reveal a novel mechanism by which PTEN inhibits the progression of colon cancer by inhibiting paxillin expression downstream of PI3K/AKT/NF-κB pathway. Thereby, PTEN/PI3K/AKT/NF-κB/paxillin signaling cascade is an attractive therapeutic target for colon cancer progression.

Sequence analysis of the HBV S protein in Chinese patients with coexisting HBsAg and anti-HBs antibodies.

The coexistence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in patients with hepatitis B virus (HBV) infection has been discovered and explained for several decades, but debate still exists. This study was to explore the relationship between this special serological pattern and mutations in S gene region. Fifteen patients with coexisting HBsAg and anti-HBs were selected as the experimental group, and 27 patients with HBsAg positive only were selected as the control group. The S gene region was amplified and sequenced. No significant differences were observed between the two groups with regard to age, gender, alanine aminotransferase level, HBsAg titer, genotype, and HBV DNA level. The patients from the two groups were infected with HBV of the genotype B and C. Compared with the control group, the experimental group showed a higher variability in amino acid within the N-terminal region and the MHR, especially the "a" determinant. The most frequent change in patients from the experimental group was located at positions s126. The coexistence of HBsAg and anti-HBs might be associated with the increased amino acid mutations in the "a" determinant. Further studies should be performed to determine the clinical implication of this serological pattern, including the binding of anti-HBs to HBsAg, escape from immune system, and efficacy of antiviral therapy.

Thymoquinone Pretreatment Overcomes the Insensitivity and Potentiates the Antitumor Effect of Gemcitabine Through Abrogation of Notch1, PI3K/Akt/mTOR Regulated Signaling Pathways in Pancreatic Cancer.

BACKGROUND: The gemcitabine-insensitivity remains the main challenge for pancreatic cancer treatment. Thymoquinone, the predominant bioactive ingredient of Nigella sativa, has been shown to possess promising anti-cancer and chemo-sensitizing effects on pancreatic cancer, however, its meticulous mechanism is still indistinct. AIM: The objective of the present study was to investigate the potency of thymoquinone in combination with gemcitabine in inducing apoptosis and preventing the development of gemcitabine-insensitivity in pancreatic cancer cells. METHODS: The anti-tumor effects of thymoquinone and gemcitabine were analyzed via evaluation of alterations of cell viability, tumor weight, apoptosis-related proteins, caspase-3, -9 activities and NF-κB DNA binding activity in pancreatic cancer cells in vitro and PANC-1 cells orthotopic xenograft in vivo. RESULTS: Thymoquinone pretreatment following gemcitabine treatment synergistically caused an increase in pancreatic cancer cells apoptosis and tumor growth inhibition both in vitro and in vivo. The novel combinational regimen also contributes to alterations of multiple molecular signaling targets, such as the suppression of Notch1, NICD accompanying with up-regulation of PTEN, the inactivation of Akt/mTOR/S6 signaling pathways, and the suppression of phosphorylation and nuclear translocation of p65 induced by TNF-α. Thymoquinone pretreatment and gemcitabine also induced down-regulation of anti-apoptotic Bcl-2, Bcl-xL, XIAP and up-regulation and activation of pro-apoptotic molecules including Caspase-3, Caspase-9, Bax and increased release of cytochrome c. CONCLUSIONS: This novel modality of thymoquinone pretreatment can enhance the anti-cancer activity of gemcitabine and may be a promising option in the treatment of pancreatic cancer.

Deep learning assists detection of esophageal cancer and precursor lesions in a prospective, randomized controlled study.

Endoscopy is the primary modality for detecting asymptomatic esophageal squamous cell carcinoma (ESCC) and precancerous lesions. Improving detection rate remains challenging. We developed a system based on deep convolutional neural networks (CNNs) for detecting esophageal cancer and precancerous lesions [high-risk esophageal lesions (HrELs)] and validated its efficacy in improving HrEL detection rate in clinical practice (trial registration ChiCTR2100044126 at www.chictr.org.cn). Between April 2021 and March 2022, 3117 patients ≥50 years old were consecutively recruited from Taizhou Hospital, Zhejiang Province, and randomly assigned 1:1 to an experimental group (CNN-assisted endoscopy) or a control group (unassisted endoscopy) based on block randomization. The primary endpoint was the HrEL detection rate. In the intention-to-treat population, the HrEL detection rate [28 of 1556 (1.8%)] was significantly higher in the experimental group than in the control group [14 of 1561 (0.9%), P = 0.029], and the experimental group detection rate was twice that of the control group. Similar findings were observed between the experimental and control groups [28 of 1524 (1.9%) versus 13 of 1534 (0.9%), respectively; P = 0.021]. The system's sensitivity, specificity, and accuracy for detecting HrELs were 89.7, 98.5, and 98.2%, respectively. No adverse events occurred. The proposed system thus improved HrEL detection rate during endoscopy and was safe. Deep learning assistance may enhance early diagnosis and treatment of esophageal cancer and may become a useful tool for esophageal cancer screening.

The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo.

DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 µM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.

Preoperative automatic reminder systems and impact on quality and compliance with colonoscopy preparation: A multicenter randomized controlled trial.

OBJECTIVES: Traditional preoperative reminding services have been applied to enhance the quality of bowel preparation for colonoscopy. In this study we aimed to evaluate the effectiveness of an automated electronic reminder system (E-reminder) on improving bowel preparation and the quality of preoperative education before colonoscopy. METHODS: From August 2021 to March 2022, 833 outpatients aged 50-75 years who underwent colonoscopy were included and randomly assigned to the E-reminder group and the control group. While the control group received routine preoperative education. The E-reminder group received automatic phone call, text message reminders and web services regarding the details of bowel preparation before the colonoscopic examination. The quality of bowel preparation was evaluated by the Boston Bowel Preparation Scale (BBPS) score and the previously validated objective evaluation scale of automatic BBPS (e-BBPS). RESULTS: In manual assessment, the rate of adequate bowel preparation was improved in the E-reminder group of intention-to-treat population using BBPS (60.7% vs 54.5%, P = 0.01). The percentage of objective evaluated adequate bowel preparation using e-BBPS in the E-reminder group of per-protocol population was significantly higher than that in the control group (76.9% vs 69.2%, P = 0.02). CONCLUSIONS: E-reminder was an effective tool to improve the quality of bowel preparation and compliance with medical instructions. It may be regarded as an efficient and convenient education tool, improving the quality of medical service.

Validation of a real-time biliopancreatic endoscopic ultrasonography analytical device in China: a prospective, single-centre, randomised, controlled trial.

BACKGROUND: Endoscopic ultrasonography (EUS) is a key procedure for the diagnosis of biliopancreatic diseases. However, the performance among EUS endoscopists varies greatly and leads to blind spots during the operation, which can impair the health outcomes of patients. We previously developed an artificial intelligence (AI) device that accurately identified EUS standard stations and significantly reduced the difficulty of ultrasonography image interpretation. In this study, we updated the device (named EUS-IREAD) and validated its performance in improving the quality of EUS procedures. METHODS: In this single-centre, randomised, controlled trial, we updated EUS-IREAD so it consisted of five learning models to identify eight EUS stations and 24 anatomical structures. The trial was done at the Renmin Hospital of Wuhan University (Wuhan, China) and included patients aged 18 years or older with suspected biliopancreatic (pancreas and biliopancreatic duct) lesions due to clinical symptoms, radiological findings, or laboratory findings, and with a high risk of pancreatic cancer. Patients were randomly assigned (1:1) by a dedicated research assistant using a computer-generated random number series (with a block size of four) to undergo the EUS procedure with or without the assistance of EUS-IREAD. Endoscopists in the EUS-IREAD-assisted group were required to observe all standard stations and anatomical structures according to the prompts by the AI device. Data collectors, the independent data anaylsis team, and patients were masked to group allocation. The primary outcome was the missed scanning rate of standard stations between the two groups, which was assessed in patients who underwent EUS procedure in accordance with the assigned intervention (per protocol). This trial is registered with ClinicalTrials.gov, NCT05457101. FINDINGS: Between July 9, 2022, and Feb 28, 2023, 290 patients (mean age 55·93 years [SD 14·06], 152 [52%] male, and 138 [48%] female) were randomly assigned and analysed, including 144 in the EUS-IREAD-assisted group and 146 in the control group. The EUS-IREAD-assisted group had a lower missed scanning rate of stations than the control group (4·5% [SD 0·8] vs 14·3% [1·0], -9·8% [95% CI -12·2 to -7·5]; odds ratio 3·6 [95% Cl 2·6 to 4·9]; p<0·0001). No significant adverse event was found during the study. INTERPRETATION: Our study confirms the capability of EUS-IREAD to monitor the blind spots and reduce the missed rate of stations and structures during EUS procedures. The EUS-IREAD has the potential to play an essential part in EUS quality control. FUNDING: Innovation Team Project of Health Commission of Hubei Province and College-enterprise Deepening Reform Project of Wuhan University.

Achieving Total Enteroscopy by Consecutive Bidirectional Double-balloon Enteroscopy Procedures.

OBJECTIVE: To investigate the feasibility and safety of achieving total enteroscopy by consecutive bidirectional double-balloon enteroscopy (DBE) procedures. METHODS: The demographic data, indication, initial insertion route, examination time for each insertion and the entire procedure, total enteroscopy rate, diagnostic yield and adverse events of patients who attempted to achieve total enteroscopy by consecutive bidirectional DBE procedures from January 2014 to December 2019 were retrospectively analyzed. RESULTS: A total of 189 patients were included, and the total enteroscopy rate was 87.3%. Initiating the DBE procedure via the retrograde approach as the initial insertion route achieved a higher total enterosocpy rate (90.9% vs. 78.9%, P=0.023), with shorter overall examination time (134.2±36.2 vs. 156.9±47.6 min, P=0.017) and shorter examination time for the opposite insertion route (23.8±19.9 vs. 53.1±27.6 min, P=0.000) compared with anteograde approach as the initial insertion route. The overall diagnostic yield was 37.6%. The diagnostic yield for successfully achieving total enteroscopy was higher, when compared to the yield for not successfully achieving total enteroscopy (39.4% vs. 25%, P=0.029). The overall rate of adverse events was 2.1% (4/189). There was no significant difference in adverse event rate between the overall examination time ≥2 h group and <2 h group (2.1% vs. 2.0%, P=0.593). CONCLUSION: Consecutive bidirectional DBE procedure is an effective and safe strategy for achieving total enteroscopy with a considerable success rate. This may be a promising option and alternative to traditional methods, and helpful to more promptly establish a definite diagnosis. The retrograde approach, as the initial insertion route, is preferred in clinical practice.

[The expression and role of PTEN in doxorubicin induced gastric cancer cell apoptosis].

OBJECTIVE: To study the expression of PTEN and its significance in doxorubicin-treated gastric cancer cells. METHODS: (1) Gastric cancer BGC-823 cells were treated with doxorubicin. Cell proliferation and apoptosis were evaluated by MTT and flow cytometry. The expression of PTEN at the mRNA and protein level were determined by RT-PCT and Western blot, respectively. (2) The gastric cancer xenografts model was constructed. The apoptosis of gastric cancer xenografts cells was determined by TUNEL. The expression of PTEN at the mRNA and protein level were detected using RT-PCR and Western blot, respectively. (3)BGC-823 cells were transfected with PTEN siRNA before addition of doxorubicin. The proliferation and apoptosis of these cells as well as the expression level of PTEN protein were determined. RESULTS: (1) After administration of doxorubicin, the proliferation of BGC-823 cells was inhibited in a time-dependent manner. (2) Doxorubicin significantly induced apoptosis of BGC-823 cells. (3) Doxorubicin treated BGC-823 cells showed a significant increase in the expression of PTEN at the mRNA and protein level in a time-dependent manner. TUNEL assay also showed a significant increase of apoptosis rate in gastric cancer xenografts treated with doxorubicin compared with control group [(28.11 ± 1.05)% vs (2.78 ± 1.63)%]. The expression of PTEN at the mRNA and protein level in the gastric cancer xenografts were significantly increased after administration of doxorubicin (0.5667 ± 0.0043 vs 0.2217 ± 0.0063, 0.14 ± 0.26 vs 0.04 ± 0.15, P < 0.05). (4) After treated with doxorubicin, the expression of PTEN in siRNA-transfected BGC-823 cells was significantly higher than that in non-transfected BGC-823 cells (P < 0.0001). The apoptosis of PTEN siRNA-transfected BGC-823 cells was significantly decreased compared with non-transfected BGC-823 cells [(10.35 ± 1.04)% vs (31.37 ± 3.58)%, P < 0.05]. CONCLUSION: Doxorubicin can effectively inhibit the growth and induce the apoptosis of BGC-823 gastric cancer cells. Increasing PTEN protein may be one of the main mechanism involved in this effect.

MFAP2 is overexpressed in gastric cancer and promotes motility via the MFAP2/integrin α5ß1/FAK/ERK pathway.

Gastric cancer (GC) is one of the most common malignancies and its prognosis is extremely poor. This study identifies a novel oncogene, microfibrillar-associated protein 2 (MFAP2) in GC. With integrative reanalysis of transcriptomic data, we found MFAP2 as a GC prognosis-related gene. And the aberrant expression of MFAP2 was explored in GC samples. Subsequent experiments indicated that silencing and exogenous MFAP2 could affect motility of cancer cells. The inhibition of silencing MFAP2 could be rescued by another FAK activator, fibronectin. This process is probably through affecting the activation of focal adhesion process via modulating ITGB1 and ITGA5. MFAP2 regulated integrin expression through ERK1/2 activation. Silencing MFAP2 by shRNA inhibited tumorigenicity and metastasis in nude mice. We also revealed that MFAP2 is a novel target of microRNA-29, and miR-29/MFAP2/integrin α5ß1/FAK/ERK1/2 could be an important oncogenic pathway in GC progression. In conclusion, our data identified MFAP2 as a novel oncogene in GC and revealed that miR-29/MFAP2/integrin α5ß1/FAK/ERK1/2 could be an important oncogenic pathway in GC progression.

Consensus on the digestive endoscopic tunnel technique.

With the digestive endoscopic tunnel technique (DETT), many diseases that previously would have been treated by surgery are now endoscopically curable by establishing a submucosal tunnel between the mucosa and muscularis propria (MP). Through the tunnel, endoscopic diagnosis or treatment is performed for lesions in the mucosa, in the MP, and even outside the gastrointestinal (GI) tract. At present, the tunnel technique application range covers the following: (1) Treatment of lesions originating from the mucosal layer, e.g., endoscopic submucosal tunnel dissection for oesophageal large or circular early-stage cancer or precancerosis; (2) treatment of lesions from the MP layer, per-oral endoscopic myotomy, submucosal tunnelling endoscopic resection, etc.; and (3) diagnosis and treatment of lesions outside the GI tract, such as resection of lymph nodes and benign tumour excision in the mediastinum or abdominal cavity. With the increasing number of DETTs performed worldwide, endoscopic tunnel therapeutics, which is based on DETT, has been gradually developed and optimized. However, there is not yet an expert consensus on DETT to regulate its indications, contraindications, surgical procedure, and postoperative treatment. The International DETT Alliance signed up this consensus to standardize the procedures of DETT. In this consensus, we describe the definition, mechanism, and significance of DETT, prevention of infection and concepts of DETT-associated complications, methods to establish a submucosal tunnel, and application of DETT for lesions in the mucosa, in the MP and outside the GI tract (indications and contraindications, procedures, pre- and postoperative treatments, effectiveness, complications and treatments, and a comparison between DETT and other operations).

Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death.

The major obstacle to successful treatment of gastric cancer is chemotherapy resistance. Our study was designed to investigate the role of phosphoinositide 3-kinase (PI3K)/Akt pathway in the development of chemoresistance in gastric cancer. In the present study, elevated Akt expression and Akt phosphorylation (Ser 473), as well as decreased PTEN expression were observed in 28 cases of gastric cancer tissues. Etoposide and doxorubicin stimulated Akt and PI3K activities in 2 gastric cancer cell lines (BGC-823 and SGC-7901), and the activities were concentration and time-dependent. Up-regulation of PTEN expression in BGC-823 cells by PEAK8-PTEN transient transfection obviously decreased the basal and anticancer drugs induced Akt activities, then sensitized BGC-823 cells to etoposide and doxorubicin. Pretreatment of BGC-823 and SGC-7901 cells with wortmannin, a PI3K inhibitor, attenuated cells's resistance to etoposide and doxorubicin. In addition, pretreatment of wortmannin blocked etoposide and doxorubicin induced IkappaB-alpha degradation, NFkappaB activation, phosphorylation of Akt, MDM-2 and forkhead transcription factors. Wortmannin pretreatment also promoted the accumulation of p27/Kip, but inhibited the Mcl-1 expression. Furthermore, wortmannin promoted etoposide and doxorubicin induced caspase-3, caspase-9 activation and poly ADP-ribose polymerase cleavage. Taken together, the observations indicate the PI3K/Akt pathway plays an important role in the chemoresistance of gastric cancer cells. A new strategy for combined chemotherapy of gastric cancer should be designed to more specifically block PI3K/Akt pathway and then decrease the amount of resistant cells.

[Impact of PI3K /Akt /mdm2 signaling pathway on the sensitivity of gastric cancer cell line SGC7901 to doxorubicin].

OBJECTIVE: To explore whether PI3K/Akt/mdm2 signalling pathway affect the sensitivity of gastric cancer cell line SGC7901 cells to doxorubicin. METHODS: Gastric cancer cell line SGC7901 cells were exposed to doxorubicin and specific PI3K inhibitor wortmannin. Cell apoptosis was detected using flow cytometry. PI3K activity was detected by radioactive immunoprecipitation-kinase assay. Western blotting was employed to evaluate the expressions of PI3K-p85, pAkt-S473, Akt, pmdm2-S166 and p53. RESULTS: The level of apoptosis in gastric cancer SGC7901 cells treated with doxorubicin was gradually increasing. wortmannin enhanced its effects significantly. PI3K activity and the expression of pAkt-S473 increased in a time-dependent manner, pmdm2-S166, p53 were also increased wortmannin inhibited phosphorylation of mdm2 and improved the p53 expression. CONCLUSION: PI3K/Akt/mdm2 signalling pathway can be activated by doxorubicin and suppress apoptosis by promoting phosphorylation of mdm2. PI3K inhibitor wortmannin can enhance sensitivity of gastric cancer cells to chemotherapy.

Wnt6 contributes tumorigenesis and development of colon cancer via its effects on cell proliferation, apoptosis, cell-cycle and migration.

Wnt proteins have been reported to contribute to the progression of various types of cancer. Wnt6 is a member of the Wnt family and may promote tumorigenesis in gastrointestinal cancer and cervical cancer. In the present study, the expression of Wnt6 in human colon cancer cell lines was evaluated, in order to investigate the role of Wnt6 in the development of colon cancer. Additionally, the effects of Wnt6 upregulation or downregulation on proliferation, apoptosis, cell cycle and cell migration of colon cancer cells have been investigated. Furthermore, western blot analysis was employed to evaluate the expression of Wnt6, B-cell lymphoma 2-associated X protein (Bax), caspase-3 and matrix metalloproteinase (MMP)2. The results of the present study demonstrated that the expression of Wnt6 was increased in HCT116 and SW480 cells compared with the remaining colon cancer cell lines. Furthermore, overexpression Wnt6 resulting from transfection of pGPU6/GFP/Neo-Wnt6-Homo-1 plasmid promoted the proliferation, cell cycle and migration of HCT116 and SW480 cells, but inhibited cell apoptosis in vitro. The expression of caspase-3 and MMP2 was increased, whereas the expression of Bax was decreased in response to upregulation of Wnt6. These results suggested that Wnt6 may serve a vital function in the development of colon cancer.

Doxorubicin combined with Notch1-targeting siRNA for the treatment of gastric cancer.

Notch1, a transmembrane receptor that has a notable role in gastric cancer (GC) as an oncogene, has been reported to be involved in doxorubicin resistance. Thus, Notch1 is a potential therapeutic target for GC. In the present study, the protein levels of Notch1 intracellular domain (NICD; a marker of Notch1 activation) in human GC cell lines and tumor tissues was measured by western blotting. Next, the effects of Notch1 depletion in SGC7901 cells were evaluated. Finally, the efficacy of Notch1 small interfering RNA (siRNA) combined with doxorubicin therapy for GC was examined in vitro and in vivo. The results revealed that NICD levels were high in GC cells, and that the inhibition of NICD by transfection with Notch1 siRNA induced apoptosis and inhibited proliferation. Ectopic downregulation of Notch1 expression enhanced the sensitivity of GC tumors to doxorubicin, which suppressed the development of GC. These data demonstrated that Notch1 was a significant regulator of cell proliferation and apoptosis in GC. Thus, the combination of doxorubicin with Notch1 siRNA is a potential strategy for the treatment of GC.

Aberrant high expression level of MORC2 is a common character in multiple cancers.

Microrchidia 2 (MORC2) plays important roles in DNA damage repair and lipogenesis, but the clinical and functional role of MORC2 in cancer remains largely unexplored. In this study, we showed that MORC2 was widely expressed in human tissues while significantly up-regulated in most cancer types using immunohistochemical staining and analysis of messenger RNA expression profile of more than 2000 human tissue samples from 15 different organs (lung, prostate, liver, breast, brain, stomach, colon/rectum, pancreas, ovary, endometrium, skin, nasopharynx, kidney, esophagus, and bladder). We also found that the MORC2 expression level in high-grade cancer tissues was much more elevated and associated with unfavorable pathological characteristics, poor overall survival, and disease-free survival in several kinds of cancers such as non-small cell lung cancer and breast cancer. Gene set enrichment analysis was used to predict the genes modulated by MORC2, and the results showed that dysregulation of MORC2 in tumor may take part in the cell cycle regulation and genomic instability. We observed that MORC2 knockdown would arrest the cell cycle progress, and the genome of tumors with high MORC2 expression contained more point mutations and gene copy number variation, which validates our gene set enrichment analysis results. The results also showed that MORC2 knockdown would significantly inhibit the proliferation, colony forming, migration, and invasion in multiple cancer cell lines. Taken together, these results highlight the importance of MORC2 in tumorigenesis and cancer progression, and it may act as a potential diagnostic marker and therapeutic target for these diseases.

The CCK-2/gastrin splice variant receptor retaining intron 4 transactivates the COX-2 promoter in vitro.

The expression of the human cholecystokinin-2/gastrin receptor (CCK-2R) has been widely reported in human colorectal cancers. Recently, a splice variant of the CCK-2R retaining intron 4 (CCK-2i4svR) has been cloned from human colorectal cancers and postulated to exhibit constitutive activity. But its role in mediating carcinogenic effects of mature-amidated gastrin in colorectal cancers has not been fully explored. The purpose of the present study was to determine whether the activation of CCK-2i4svR by gastrin transactivates the COX-2 promoter in human colon cancer cells and in COS-7 cells. In this study, Colo320 cells and COS-7 cells were transfected with the human CCK-2R wild type (CCK-2wtR) (COS-7WT, Colo320WT) and the human CCK-2i4svR (COS-7SV, Colo320SV) cDNA. After stimulation with gastrin-17 (G-17), transactivation of the COX-2 promoter was determined by luciferase reporter gene assay. 5'deletions of the COX-2 promoter were transfected into Colo320 cells to narrow down the minimally required regulatory element. Induction of COX-2 expression was further explored at the mRNA level using real time RT-PCR. The effects of CCK-2i4svR activation on phosphorylation of ERK1/2, p38(MAPK) and JNK were examined by using immunoblotting. Prostaglandin E(2) (PGE(2)) secretion was measured by ELISA. Our results showed that gastrin transactivates the COX-2 promoter in both Colo320 cells and COS-7 cells expressing the CCK-2i4svR cDNA. Inhibition of p38(MAPK) pathway using specific inhibitor significantly blocked the gastrin-induced COX-2 transactivation. Gastrin time-dependently increased COX-2 mRNA expression, the peak mRNA levels appeared at 10 h after stimulation. PGE(2) secretion from gastrin-treated cells increased significantly 8 h after stimulation. Treatment with gastrin also stimulated PGE(2) secretion in the Colo320 cells expressing CCK-2i4svR. In conclusion, the CCK-2i4svR mediates transactivation of the COX-2 promoter and MAPK pathway is involved in this process.

[The role of p38 MAPK in gastrin-induced u-PA expression in human colon cancer cells].

OBJECTIVE: To study the effect of gastrin on the mRNA and protein expression of urokinase-type plasminogen activator (u-PA) in human colon cancer cells and detect the role of p38 MAPK in this process. METHODS: Lipofectin method was used to transfect pCR3. 1/CCK2R vector expressing gastrin receptor into a colon cancer cell line colo320. Gastrin and gastrin antagonist were used to up-regulate and down-regulate the signaling pathway, respectively. Human colon cancer colo320 cells and colo320/ CCK2,R cells were cultured and then stimulated with gastrin for different time; SB203580 was added into culture medium to prevent p38 kinase pathway before incubating with gastrin; Western blot and RT-PCR were used to examine the u-PA expression. Western blot was employed to detect p38 kinase phosphorylation. RESULTS: Gastrin increased evidently the mRNA and protein expressions of u-PA and induced p38 kinase phosphorylation in colo320/CCK,R cells time-dependently. However, the extent of enhancement of u-PA and p38 MAPK expression in colo320 cells was much less than that in colo320/CCK2R cells. The gastrin antagonist L-365, 260 showed an effect of competitive inhibition on gastrin-induced u-PA expression and p38 kinase phosphorylation. The inhibitor SB203580 could sufficiently suppress gastrin-induced p38 kinase phosphorylation and significantly attenuate gastrin-induced u-PA mRNA and protein expressions in colo320/ CCK2 R cells in a dose-dependent manner. CONCLUSION: Gastrin-gastrin receptor signal transduction pathway can obviously induce u-PA expression in human colon cancer cells via activating the phosphorylation of p38 kinase.

[Effects of hFRNK on E-cadherin/beta-catenin in colon cancer cells in vitro].

OBJECTIVE: To explore the effects of human FRNK gene on E-cadherin/beta-catenin complex in colon cancer cell line Colo320WT cells stimulated with extrinsic gastrinl7. METHODS: AdEasy system was used to construct pAdhFRNK expressing human FRNK gene by recombination in E. coli. BJ5283. pCR3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colon cancer cell line Colo320 cells by Lipofectamine 2000 and expressing stably CCK-2R clones were screened by G418 (500 pg/ml). The expression levels of gastrin receptor in Colo320 cells and the transfected Colo320WT cells were assayed by RT-PCR. Colo320WT cells were treated by 10(-8) mol/L gastrinl7 for 12 h; and after Colo320WT cells were infected by pAdhFRNK (MOI: 100) for 2 d the cells were treated by gastrin17 for 12 h again. The expression levels of E-cadherin and beta-catenin in TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells were assayed by co-immunoprecipation and Western blot. E-cadherin and beta-catenin's distribution in Colo320WT cells were detected by immunocytochemistry. RESULTS: When 10(-8) mol/L gastrin17 stimulated Colo320WT cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction decreased apparently, while the expression levels of E-cadherin and beta-catenin in TX-100-insoluble fraction increased markedly. When pAdhFRNK infected Colo320WT cells for 2 d and 10(-8) mol/L gastrin17 treated the cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction increased apparently again, and the expression levels of E-cadherin and beta-catenin in TX-100-insolutble fraction decreased markedly. Immunocytochemistry showed that the distribution of E-cadherin and beta-catenin was translocated from plasma membrane into cytoplasm and nucleus in the cells stimulated with gastrinl7, and after the cells were infected with pAdhFRNK and stimulated by gastrinl7 again. beta-catenin was mainly observed in cytoplasm and little nuclear immunoreactivity. CONCLUSION: An adenovirus vector pAdhFRNK can inhibit abnormal distribution of E-cadherin and beta-catenin in the gastrin17-stimulated cells. The mechanism is probably that hFRNK can disphosphorylate phosphorylated FAK and block FAK pathway.