Hsa_circRNA_103809 regulated the cell proliferation and migration in colorectal cancer via miR-532-3p / FOXO4 axis.

Circular RNAs(circRNAs) are a class of non-coding RNAs that are widely expressed in a variety of cell species. The role they play in cancers is poorly understood, especially in colorectal cancer (CRC). Hsa_circRNA_103809 (hsa_circ_0072088, circZFR)has been demonstrated to be lowly expressed in colorectal cancer tissues and is associated with stage and lymph node metastasis of cancer tissues. Real-time quantitative PCR (qRT-PCR) was used to verify the relationship of hsa_circRNA_103809 between colorectal cancer and paired adjacent tissue in clinical tissue samples. Then, the proliferative capacity, migration ability, cell cycle, and apoptosis were measured using wound-healing assay, CCK8, transwell assay, flow cytometry, and the like, when hsa_circRNA_103809 expression in SW620 and COCA-2. The qRT-PCR, western bolt and other experiments verify that the expression of hsa_circRNA_103809 can regulate the expression of miR-532-3P and FOXO4. Hsa_circRNA_103809 was found to be significantly down regulated in CRC tissues and cell lines and compared with paired adjacent non-tumorous tissues and normal FHC cells. Hsa_circRNA_103809 participates in the regulation of biological functions through the miR-532-3P/FOXO4 axis in the CRC. Hsa_circRNA_103809 may be a potential novel gene target for the diagnosis and treatment of CRC.

Overexpression of GRK6 associates with the progression and prognosis of colorectal carcinoma.

There are a limited number of studies reporting on the expression of G protein-coupled receptor kinase 6 (GRK6) in colorectal carcinoma (CRC). The aim of the present study was to investigate and examine the clinical value of GRK6 expression in human CRC. The expression of the GRK6 protein was determined in CRC tissues (n=83) and in normal colorectal tissues (n=19) by immunohistochemical (IHC) analysis. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted to investigate GRK6 mRNA levels in matched pairs of cancerous and non-cancerous fresh frozen tissues from 19 patients with CRC. Furthermore, GRK6 protein levels were evaluated in matched pairs of cancerous and non-cancerous fresh frozen tissues from 19 other patients with CRC by western blot analysis. The expression of GRK6 was significantly upregulated in patients with CRC as indicated by IHC analysis (P=0.028). The results of RT-qPCR and western blotting confirmed that GRK6 mRNA and protein levels were upregulated in CRC tissues compared with matched adjacent non-cancerous tissues (P<0.05). Additionally, GRK6 protein expression was significantly associated with histological differentiation (P=0.001), lymph node invasion (P=0.45), venous invasion (P=0.009), depth of invasion (P=0.026), distant metastasis (P<0.0001) and TNM stages (P=0.020). Survival analysis using the Kaplan-Meier method indicated that patients with high GRK6 expression levels exhibited lower overall survival rates compared with patients with low GRK6 expression. Multivariate analysis using the Cox proportional hazards model indicated that the expression levels of GRK6 (P=0.003) were independent prognostic factors for overall survival in patients. The overexpression of GRK6 in patients with CRC may serve as an independent predictor of patient outcome.

SPOCK1 is up-regulated and promotes tumor growth via the PI3K/AKT signaling pathway in colorectal cancer.

SPOCK1 encodes a Ca2+-binding matricellular glycoprotein which plays an oncogenic role in cancer cells. However, the role of SPOCK1 in the pathogenesis of colorectal cancer (CRC) has not been determined. Here, SPOCK1 was found higher expressed in CRC tissues than that of adjacent normal tissues. Furthermore, up-regulated expression of SPOCK1 in CRC patients was associated with tumor size and TNM stage. In addition, we observed that the depletion of SPOCK1 inhibited proliferation in vitro and tumorigenicity in vivo and promoted apoptosis in cell culture. Our data suggest that inactivation of PI3K/Akt signaling pathway was involved in down-regulation of SPOCK1-mediated suppression of tumor cell proliferation. These results suggest that SPOCK1 expression is correlated with malignant features of CRC and may serve as potential therapeutic and preventive strategies for CRC.

[A Chinese multi-center study on the significance of monitoring imatinib plasma concentration in patients with gastrointestinal stromal tumor before and after administration].

OBJECTIVE: To investigate the factors which may influence the imatinib plasma concentration in Chinese patients with gastrointestinal stromal tumor(GIST), and to illuminate the significance of monitoring imatinib plasma concentration in adjuvant therapy for patients with GIST. METHODS: A cross-sectional study with 60 GIST patients who accepted the imatinib therapy after surgery was conducted. They were respectively administrated in 10 domestic hospitals from December 2014 to April 2016, including The First Affiliated Hospital of Nanjing Medical University(n=28), The Affiliated Hospital of Nantong University(n=9), The Affiliated Hospital of Xuzhou Medical College(n=6), Nanjing Drum Tower Hospital(n=5), The Second Affiliated Hospital of Nanjing Medical University (n=2), Jingling Hospital (n=2), The Second People's Hospital of Lianyungang(n=2), Shandong Provincial Hospital(n=2), Jiangsu Province Tumor Hospital(n=2), and The First Affiliated Hospital of Zhejiang University(n=2). Some specific time points for collecting blood sample before and after taking imatinib were determined, then liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for monitoring imatinib plasma concentration in patients with GIST. Linear regression analysis was used for the correlation analysis of imatinib plasma concentration with dosage, clinicopathologic feature and side effect. RESULTS: Patients who could not tolerate 400 mg imatinib per day(n=3) received 300 mg per day. There was no significant difference in imatinib plasma concentration between patients with 300 mg and those with 400 mg imatinib(n=53)(P=0.527). However, the imatinib plasma concentration in patients with 600 mg imatinib per day (n=4) was significantly higher as compared to those with 400 mg(P=0.000). Linear regression analysis indicated a negative correlation between the imatinib plasma concentration in patients with 400mg imatinib per day for 90 days continuously and body surface area(R2=0.074, P=0.035), but no significant correlations of with age, creatinine clearance and serum albumin concentration were observed (all P>0.05). The differences in imatinib plasma concentration were not statistically significant between patients of different gender and those taking proton-pump inhibitor (PPI) or not (both P>0.05). Difference in imatinib plasma concentration between patients with different surgery was significant (P=0.026). Compared to patients who underwent wedge resection, enterectomy and other surgeries, the imatinib plasma concentration of patients with subtotal gastrectomy or total gastrectomy decreased significantly (all P<0.05). After 90 days of taking imatinib continuously, linear regression analysis revealed a negative correlation between imatinib plasma concentration in patients with 400 mg imatinib per day and white blood cell count (R2=0.103, P=0.013), and a positive correlation with serum alanine aminotransferase (ALT) concentration (R2=0.076, P=0.033). CONCLUSIONS: The imatinib plasma concentration in patients with larger body surface area, subtotal gastrectomy or total gastrectomy may be lower. For these patients, dosage of imatinib should be considered to increase in order to achieve effective plasma concentration. Excessive imatinib plasma concentration can result in some side effects, such as decrease of white blood cells and liver damage. Therefore, it is significant for receiving optimal clinical therapeutic efficacy to monitor imatinib plasma concentration, adjust imatinib dosage timely and keep imatinib plasma concentration in effective and safe range.

Silencing speckle-type POZ protein by promoter hypermethylation decreases cell apoptosis through upregulating Hedgehog signaling pathway in colorectal cancer.

Epigenetic silencing of tumor suppressors contributes to the development and progression of colorectal cancer (CRC). We recently found that speckle-type POZ protein (SPOP) was significantly downregulated and the inactivation of SPOP promoted metastasis in CRC. This study aimed to clarify its epigenetic alteration, molecular mechanisms and clinical significance in CRC. Our results revealed that the core region of SPOP promoter was hypermethylated in CRC tissues and its methylation was correlated with poor survival. Transcription factor RXRA had a vital role in the regulation of SPOP gene. The data indicated that DNA methylation at -167 bp of the SPOP gene altered the binding affinity between transcription factor RXRA and SPOP promoter. Moreover, SPOP was found to associate with Gli2 and promoted its ubiquitination and degradation in CRC. Consequently, the expression level of Hh/Gli2 pathway-related apoptotic protein Bcl-2 was decreased and the function of resisting cell death was inhibited in CRC. It suggests that methylation status of SPOP promoter can be used as a novel epigenetic biomarker and a therapeutic target in CRC.

Circulating tumor cells as a prognostic and predictive marker in gastrointestinal stromal tumors: a prospective study.

BACKGROUND: Circulating tumor cells (CTC) are prognostic and predictive for several cancer types. Only limited data exist regarding prognostic or predictive impact of CTC on gastrointestinal stromal tumor (GIST) patients. The aim of our study was to elucidate the role of CTC in GIST patients. RESULTS: A total of 121 GIST patients and 54 non-GIST samples were enrolled in the study. The cutoff value for ANO1 positive was 3*10-5 and 65 (54%) GIST patients were defined as ANO1 positive. ANO1s were more frequently detected in unresectable patients. Tumor size, mitotic count and risk level were associated with ANO1 detection in resectable GIST patients. The presence of ANO1 significantly correlated with poor disease-free survival (15.3 versus 19.6 months, p = 0.038). Most patients turned ANO1-negative after surgery and inversely, all 21 patients with recurrence turned ANO1-positive with high ANO1 expression levels. Moreover, in the neoadjuvant setting, decline of ANO1 expression level correlated with the response of imatinib. METHODS: Cells from peripheral blood mononuclear cells tested positive for anoctamin 1, calcium activated chloride channel, ANO1 (DOG1) were considered as tumor CTC of GISTs. The expression levels of ANO1 were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The highest level of ANO1 expression in non-GIST samples was used as the "cutoff" value. CONCLUSION: ANO1 detection by qRT-PCR in peripheral blood is of clinical potential for monitoring recurrence and evaluating therapeutic efficacy of imatinib for GIST patients.

Prognostic role of microscopically positive margins for primary gastrointestinal stromal tumors: a systematic review and meta-analysis.

The impact and management of microscopically positive margins in gastrointestinal stromal tumors (GISTs) remain unclear. The aim of this study is to estimate the prognostic value of surgical margins for disease-free survival (DFS) and overall survival (OS) in patients with primary GISTs. Twelve studies with 1985 GIST patients were included. The overall recurrence rate in R1 resection and R0 resection group was 0.364 (95% CI 0.299-0.429) and 0.296 (95% CI 0.161-0.430), respectively. Meta-analysis confirmed that a microscopically positive margin could significantly impact the disease-free survival (HR 1.596, 95% CI 1.128-2.258; I(2) = 37.5%, P value = 0.091), but had no influence on overall survival (HR 1.430, 95% CI 0.608-3.363; I(2) = 60.8%, P value = 0.013). Importantly, subgroup analysis revealed that adjuvant imatinib treatment could attenuate the risk of recurrence for primary GIST patients who received R1 resection. (HR 1.308, 95% CI 0.583-2.935; I(2) = 53.2%, P value = 0.074). The level of evidence achieved in this study was "moderate" for DFS and "low" for OS. In conclusion, this study revealed that a microscopically positive margin is an unfavorable prognostic factor for GIST patients with R1 resection, and adjuvant imatinib treatment is proved to be effective.

MicroRNA expression profile of gastric cancer stem cells in the MKN-45 cancer cell line.

MicroRNAs (miRNAs) play important roles in post-transcriptional gene silencing of target messenger RNAs, which are involved in virtually all biological processes. Previously, we have demonstrated that spheroid body-forming cells from the MKN-45 cancer cell line possessed gastric cancer stem cell (CSC) properties. In this study, we aimed to determine the miRNA profile of the gastric CSCs and to explore the role of miRNAs in gastric CSCs. Human miRNA microarrays, which contain probes specific for 1887 human miRNAs were used to determine the expression profiles of the gastric CSCs. A total of 182 miRNAs with a more than 2-fold change were identified to be differentially expressed between the spheroid body-forming cells and the parental cells. Of these miRNAs, 9 miRNAs were over-expressed in the spheroid body-forming cells, while the other 173 miRNAs were all under-expressed, indicating that the role of most miRNAs in human gastric CSCs may be tumor suppressors. The results of microarray analysis were validated by quantitative real-time polymerase chain reaction, and the consistence rate is 70% (7 out of 10). The target genes for the validated miRNAs were predicted by using three online software programs, miRanda, PicTar, and TargetScan. Most of the potential targets of the miRNAs were relevant to the regulation of actin cytoskeleton, focal adhesion, extracellular matrix-receptor interaction, and the pathways in cancer. Especially, several genes are associated with some pivotal signaling pathways of the 'stem cell genes'. Evaluating the characteristic miRNAs of the gastric CSCs may be a new method for studying gastric cancer and developing therapeutic strategies, which aimed at eradicating the subpopulation of CSCs in gastric cancer.

Significantly increased expression of OCT4 and ABCG2 in spheroid body-forming cells of the human gastric cancer MKN-45 cell line.

The cancer stem cell (CSC) theory hypothesizes that CSCs are the cause of tumor formation, recurrence and metastasis. Key to the study of CSCs is their isolation and identification. The present study investigated whether spheroid body-forming cells in the human gastric cancer (GC) MKN-45 cell line are enriched for CSC properties, and also assessed the expression of the candidate CSC markers, octamer-binding transcription factor-4 (OCT4) and adenosine triphosphate-binding cassette transporter G2 (ABCG2) in the MKN-45 spheroid body cells. The MKN-45 cells were plated in a stem cell-conditioned culture system to allow for spheroid body formation. The expression levels of OCT4 and ABCG2 in the spheroid body cells were assessed by qPCR, western blot analysis and immunofluorescence staining, while the tumorigenicity of the spheroid body-forming cells was assessed by in vivo xenograft studies in nude mice. The MKN-45 cells were able to form spheroid bodies when cultured in stem cell-conditioned medium. The spheroid body-forming cells showed a significantly higher (P<0.01) expression of OCT4 and ABCG2 compared with the parental cells. These data suggest that the spheroid body cells from the MKN-45 GC cell line cultured in stem cell-conditioned medium possessed gastric CSC properties. The co-expression of OCT4 and ABCG2 by these cells may represent the presence of a subpopulation of gastric CSCs.

Co-expression of CD44 and ABCG2 in spheroid body-forming cells of gastric cancer cell line MKN45.

BACKGROUND/AIMS: The cancer stem cell (CSC) theory hypothesizes that CSCs are regarded as the cause of tumor formation, recurrence and metastasis. This study aimed to investigate whether spheroid body-forming cells in human gastric cancer cell were enriched for CSC properties, and to assess the expression of candidate CSC markers, cluster of differentiation 44 (CD44) and adenosine triphosphate binding cassette transporter G 2 (ABCG2) in the MKN45 spheroid body cells. METHODOLOGY: Human gastric cancer cell line MKN45 were plated in stem cell conditioned culture system allowed for spheroid body forming. The expression levels of CD44 and ABCG2 in the spheroid body cells were assessed by quantitative real-time PCR, western blot analysis and immunofluorescence staining, and the tumorigenicity of the spheroid body-forming cells were assessed by in vivo xenograft studies in nude mice. RESULTS: The MKN45 cells could form spheroid bodies cultured in stem cell conditioned medium. The spheroid body-forming cells showed a significantly greater (p <0.05) expression of CD44 and ABCG2 than the parental cells. CONCLUSIONS: Spheroid body cells from gastric cancer cell line MKN45 cultured in stem cell conditioned medium possessed gastric CSC properties. The cells co-expressed of CD44 and ABCG2 might represent a subpopulation of gastric CSCs.

Spheroid body-forming cells in the human gastric cancer cell line MKN-45 possess cancer stem cell properties.

The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.

[Comparison of bowel function pre and post-sigmoid augmentation cystoplasty in spinal cord injury patients].

OBJECTIVE: To explore the changes of bowel function in spinal cord injury (SCI) patients undergoing sigmoid augmentation cystoplasty. METHODS: From September 2005 to January 2011, 30 SCI patients undergoing sigmoid augmentation cystoplasty were surveyed by follow-up questionnaires at Beijing Charity hospital and Affiliated Hospital of Nantong University. RESULTS: Among them, 18 cases (60.0%) believed their defecation became softer and 18 cases (60.0%) thought their defecation time became shorter. The postoperative profiles of patient defecation traits and defecation time were better (P < 0.05), especially traumatic SCI patients (P < 0.05). CONCLUSION: The subtotal resection of sigmoid colon improves the defecation of spinal cord injury patients. The SCI patients undergoing sigmoid augmentation cystoplasty may avoid urinary tract dysfunctions and improve bowel dysfunction.

[Interference of invasion and metastasis of colorectal cancer cells by EMS1-siRNA encoded by a recombinant lentiviral vector].

OBJECTIVE: To explore the effect of Cortactin on invasion and metastasis of human colorectal cancer cells through silencing the expression of Cortactin by transfecting colorectal cancer cells with EMS1-siRNA encoded by a recombinant lentiviral vector. METHODS: EMS1-siRNA lentiviral vector and negative control lentiviral vector were both transfected into HCT8 cells after virus packing, then the expression of Cortactin of the cells was examined by Western blot to determine the inhibited level of Cortactin. The cells were defined as three groups: EMS1-siRNA transfecting cells, negative control transfecting cells and un-transfection cells. Cell invasion was evaluated with Transwell body. In addition, cells metastasis was observed in animal models. Thirty nude mice were evenly divided into three groups and all the mice were injected through tail vein with 1×10(7) cells/0.2 ml per animal for each group with the three kinds of cells. The mice were sacrificed 6 weeks after injection and examined for lung metastases development. RESULTS: The expression of Cortactin and the ability of invasion of the EMS1-siRNA transfecting cells were all decreased. Lung metastasis rates of EMS1-siRNA transfecting group, negative control group and un-transfection group were 20%, 80% and 80%, respectively. CONCLUSION: siRNA silencing of Cortactin expression can decrease the colorectal cancer cells invasion, and can also decrease the cells' implantation metastasis in animals.

[A pivotal role of cortactin, a CTTN encoding protein, in endocytosis of human colon cancer].

OBJECTIVE: To explore the role of cortactin in endocytosis of colon cancer cells and clarify the significance of its over-expression in colon cancer tissues. METHODS: Immunohistochemistry and Western blot were employed to detect the expression of cortactin in benign and malignant tissues and cells. Cell endocytosis was examined in cancer cells after siRNA treatment and DNA transfection with plasmid encoding cortactin wild type and domain deletion mutants. RESULTS: Cortactin was over-expressed in colon cancer tissues than in adjacent normal tissues. The expression rate was 77.5% in cancer tissues and 47.5% in normal tissues (P < 0.05). The value of transferrin uptake was 0.61 ± 0.02 in siRNA treated cancer cells and 1.01 ± 0.16 in the control cells (P < 0.05). Intact molecule and sufficient level of cortactin was required for an optimal endocytosis of cancer cells. Cortactin was involved in coated-vesicle transportation in cells. CONCLUSION: Endocytosis in colon cancer cells is dependent on an intact expression of CTTN. An over-expression of cortactin facilitates the signaling in invasion and metastasis related to endocytosis.

JAB1 expression is associated with inverse expression of p27(kip1) in hepatocellular carcinoma.

BACKGROUND/AIMS: Recent studies have shown that overexpression of c-jun activation domain binding protein 1 (JAB1) and reduced expression of p27(kip1) are associated with advanced tumor stage and poor prognosis in several human cancers. Here, We investigated the functional role and correlation of JAB1 and p27(kip1) in hepatocellular carcinoma (HCC). METHODOLOGY: Immunohistochemical study for JAB1, p27(kip1) was performed on 76 cases of HCC and adjacent nontumorous tissues. 6 Fresh specimens of HCC and the adjacent liver tissue were collected for Western blot analysis. The influence of As2O3 on HCC SMMC-7721 cells, was detected by flow cytometry and Hochest staining. The expression and subcellular localization of p27(kip1) and JAB1 were investigated by Western blot and immunofluorescence. RESULTS: The expression of JAB1 was higher but p27(kip1) was lower in HCC than that in adjacent liver tissue. As2O3 treatment inhibited the growth of SMMC-7721 cells. In As2O3-treated cells, p27(kip1) expression was increased while JAB1 was decreased. The location of p27(kip1) and JAB1 were transferred from cytoplasm to nucleus. CONCLUSIONS: In HCC, JAB1 was inversely correlated with p27(kip1). As2O3 attenuated the expression of JAB1, disturbed the location and expression of p27(kip1), which may participate in regulating the growth of human hepatoma cells.