MiR-590 Suppresses Proliferation and Induces Apoptosis in Pancreatic Cancer by Targeting High Mobility Group A2.

BACKGROUND: Pancreatic ductal adenocarcinoma is a common malignancy with high morbidity. MicroRNAs have been demonstrated to be critical posttranscriptional regulators in tumorigenesis. This study aimed to investigate the effect of microRNA-590 on the proliferation and apoptosis of pancreatic ductal adenocarcinoma. MATERIAL AND METHODS: The expression of microRNA-590 and high mobility group AT-hook 2 were examined in clinical pancreatic ductal adenocarcinoma tissues. Pancreatic ductal adenocarcinoma cell line Capan-2 was employed and transfected with microRNA-590 mimics or inhibitor. The correlation between microRNA-590 and high mobility group AT-hook 2 was verified by luciferase reporter assay. Cell viability and apoptosis were detected by MTT and flow cytometry assay. The protein level of high mobility group AT-hook 2, AKT, p-AKT, mTOR, and phosphorylated mTOR were analyzed by Western blotting. RESULTS: MicroRNA-590 was found to be negatively correlated with the expression of high mobility group AT-hook 2 in pancreatic ductal adenocarcinoma tissues. Further studies identified high mobility group AT-hook 2 as a direct target of microRNA-590. Moreover, overexpression of microRNA-590 downregulated expression of high mobility group AT-hook 2, reduced cell viability, and promoted cell apoptosis, while knockdown of miR-590 led to an inverse result. MicroRNA-590 also suppressed the phosphorylation of AKT and mTOR without altering total AKT and mTOR levels. CONCLUSION: Our study indicated that microRNA-590 negatively regulates the expression of high mobility group AT-hook 2 in clinical specimens and in vitro. MicroRNA-590 can inhibit cell proliferation and induce cell apoptosis in pancreatic ductal adenocarcinoma cells. This regulatory effect of microRNA-590 may be associated with AKT signaling pathway. Therefore, microRNA-590 has the potential to be used as a biomarker for predicting the progression of pancreatic ductal adenocarcinoma.

The utility of long non-coding RNA ZEB1-AS1 as a prognostic biomarker in human solid tumors: A meta-analysis.

PURPOSE: This meta-analysis aims to assess the prognostic value of long non-coding RNA ZEB1-AS1 in human solid tumors. METHODS: We searched the available databases up to January 2018. Pooled hazard ratios (HRs) and the corresponding 95% confidence intervals (CIs) were used to examine the prognostic impact of ZEB1-AS1 on patient survival. RESULTS: Eight eligible studies with a total of 586 patients were enrolled. A significant association was observed between ZEB1-AS1 overexpression and poor overall survival (OS; HR = 2.195, 95% CI: 1.749-2.755) as well as unfavorable recurrence-free survival (pooled HR = 2.205, 95% CI: 1.486-3.270), and no heterogeneity was found across these studies (p = .962, I2 = 0%). Subsequent subgroup analyses showed that cancer type, sample size, follow up months, and HR estimation method did not alter the significant prognostic value of ZEB1-AS1. ZEB1-AS1 expression was indicated to be an independent prognostic factor for tumor OS (pooled HR = 2.177, 95% CI:1.545-3.069). Furthermore, we found that increased ZEB1-AS1 expression was significantly associated with tumor stage [III-IV vs. I-II: odds ratio (OR) = 1.644, 95% CI: 1.201-2.249] and lymph node metastasis (Positive vs. Negative: OR = 2.413, 95% CI: 1.504-3.873). CONCLUSION: High expression level of ZEB1-AS1 was associated with unfavorable survival outcome for cancer patients, and ZEB1-AS1 could be used as a prognostic predictor for cancers.

Role of ERK-MAPK signaling pathway in pentagastrin-regulated growth of large intestinal carcinoma.

AIM: To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase (ERK-MAPK) signaling in pentagastrin-regulated growth of large intestinal carcinoma. METHODS: HT-29 cells were incubated in different media and divided into the control group, pentagastrin group, proglumide group, and pentagastrin + proglumide group. No reagent was added to the control group, and other groups were incubated with reagent at different concentrations. Changes in proliferation of HT-29 cells were detected by MTT assay, and the optimal concentrations of pentagastrin and proglumide were determined. The changes in proliferation index (PI) and apoptosis rate (AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry. mRNA expression of pentagastrin receptor/cholecystokinin-B receptor (CCK-BR), ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction. The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting. All data were analyzed by analysis of variance and SNK-q test. RESULTS: The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L, and the optimal concentration of pentagastrin was 25.0 mg/L (F = 31.36, P < 0.05). Proglumide had no obvious effect on the proliferation of HT-29 cells, while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L, and the optimal concentration was 32.0 mg/L (F = 24.31, P < 0.05). The PI of the pentagastrin (25.0 mg/L) group was 37.5% ± 5.2%, which was significantly higher than 27.7% ± 5.0% of the control group and 27.3% ± 5.8% of the pentagastrin (25.0 mg/L) + proglumide (32.0 mg/L) group (Q = 4.56-4.75, P < 0.05). The AR of the pentagastrin (25.0 mg/L) group was 1.9% ± 0.4%, which was significantly lower than 2.5% ± 0.4% of the control group and 2.4% ± 0.3% of the pentagastrin (25.0 mg/L) + proglumide (32.0 mg/L) group (Q = 4.23-4.06, P < 0.05). mRNA expression of CCK-BR was detected in HT-29 cells. The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group were 0.43% ± 0.04% and 0.45% ± 0.06%, which were significantly higher than 0.32% ± 0.02% and 0.31% ± 0.05% of the control group (Q = 7.78-4.95, P < 0.05), and 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group (Q = 5.72-4.08, P < 0.05). There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control, pentagastrin, proglumide and pentagastrin + proglumide groups (F = 0.52, 0.72, 0.78, 0.28; P > 0.05). CONCLUSION: Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway, and this is restrained by proglumide.

[Relationships of the mRNA and protein expression of gastrin with Fas/FasL and caspases in colorectal carcinoma].

OBJECTIVE: To examine the correlation between the mRNA and proteins expressions of gastrin(GAS), and the association of protein expression of GAS with apoptosis index(AI) and apoptosis regulation gene Fas/FasL, caspases in colorectal cancer. METHODS: The expressions of GAS mRNA in tumor tissues of 79 cases with colorectal cancer were detected by nested RT-PCR. Cell apoptosis was detected by molecular biology in situ apoptosis detecting technic(TUNEL). Protein expressions of GAS, Fas/FasL, and caspases were detected by immunohistochemical staining (SP method). RESULTS: The positive correlation was found between the mRNA and proteins expressions of GAS(rGAS=0.99, P<0.01). The mRNA and protein expressions of GAS in well and moderately differentiated cancers were significantly lower than those in poorly differentiated cancers (chi(2)(high vs low)=10.47, 10.23, P<0.01, chi(2)(middle vs low)=6.68, 4.95, P<0.05). The mRNA and protein expressions of GAS in papillary and tubular adenocarcinomas were significantly lower than those in mucinous adenocarcinomas, signet-ring cell carcinoma and undifferentiated carcinoma (chi(2)(papillary vs mucinous and signet-ring)=4.80, 6.22, chi(2)(papillary vs undifferentiation)=5.44, 8.43, chi(2)(tubular vs mucinous and signet-ring)=4.40, 4.38, chi(2)(tubular vs undifferentiation)=4.92, 6.43, P<0.05, respectively). The mRNA and protein expressions of GAS in Dukes' stages A, B were significantly lower than those in Dukes stages C, D (chi(2)=4.84, 4.45, P<0.01). The AI in GAS high and moderate expression groups of colorectal cancer were significantly lower than that in low expression group (q(high vs low)=6.71, q(middle vs low)=4.60, P<0.01). The positive expression rate of FasL was significantly different among GAS high, moderate and low expression groups of colorectal cancer (chi(2)=9.35, P<0.01). The positive expression rate of FasL in GAS high and moderate expression groups was higher than that in low expression group (chi(2)high vs low=6.24, chi(2)(middle vs low)=4.74, P<0.05). CONCLUSIONS: GAS plays an important role in the regulation of cell apoptosis in colorectal carcinoma, whose mechanism may be related to the aberrant expression of Fas/FasL. GAS will be one of the indicators of the biological behavior in colorectal carcinoma.

Relationship between expression of gastrin, somatostatin, Fas/FasL and caspases in large intestinal carcinoma.

AIM: To explore the correlation between the mRNAs and protein expression of gastrin (GAS), somatostatin (SS) and apoptosis index (AI), apoptosis regulation gene Fas/FasL and caspases in large intestinal carcinoma (LIC). METHODS: Expression of GAS and SS mRNAs were detected by nested RT-PCR in 79 cases of LIC. Cell apoptosis was detected by molecular biology in situ apoptosis detecting methods (TUNEL). Immunohistochemical staining for GAS, SS, Fas/FasL, caspase-3 and caspase-8 was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. RESULTS: There was a significant positive correlation between mRNA and protein expression of GAS and SS (GASrs = 0.99, P < 0.01; SSrs = 0.98, P < 0.01). There was significant difference in positive expression rates of GAS, SS mRNAs and protein among different histological differentiation, histological types and Dukes' stage of LIC. The AI in GAS high and moderate expression groups was significantly lower than that in low expression groups (3.75 +/- 2.38 vs 7.82 +/- 2.38, P < 0.01; 5.51 +/- 2.66 vs 7.82 +/- 2.38, P < 0.01), and the AI in SS high and moderate expression groups was significantly higher than that in low expression groups (9.03 +/- 1.76 vs 5.35 +/- 3.00, P < 0.01; 7.44 +/- 2.67 vs 5.35 +/- 3.00, P < 0.01). There was a significant negative correlation between the integral ratio of GAS to SS and the AI (r(s) = -0.41, P < 0.01). The positive expression rate of FasL in GAS high and moderate expression groups was higher than that in low expression group (90.9% and 81.0% vs 53.2%, P < 0.05). The positive expression rates of Fas, caspase-8 and caspase-3 in SS high (90.0%, 90.0% and 100%) and moderate (80.0%, 70.0%, 75.0%) expression groups were higher than that in low expression group (53.1%, 42.9%, 49.0%) (90.0% and 80.0% vs 53.1%, P < 0.05; 90.0% and 70.0% vs 42.9%, P < 0.05; 100.0% and 75.0% vs 49.0%, P < 0.05). There was a significant positive correlation between the integral ratio of GAS to SS and the semiquantitative integral of FasL (rs = 0.32, P < 0.01). CONCLUSION: GAS and SS play important roles in the regulation and control of cell apoptosis in LIC, and the mechanism may be directly related to the aberrant expression of Fas/FasL. The GAS and SS will be valuable targets of the biological behavior of LIC.

Relationship between vascular invasion and microvessel density and micrometastasis.

AIM: To evaluate the relationship between vascular invasion and microvessel density (MVD) of tissue and micrometastasis in blood. METHODS: Vascular invasion was detected by both hematoxylin and eosin staining and immunohistochemiscal staining. Blood samples were collected from 17 patients with vascular invasion and 29 patients without vascular invasion and examined for cytokeratin20 (CK20) expression by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Microvessel density of tissue samples was also determined by immunohistochemistry using antibodies to CD105. RESULTS: CK20 was detected in 12 of the 17 patients with vascular invasion and in 9 of the 29 patients without vascular invasion. Positive RT-PCR was significantly correlated with vascular invasion (70.6% vs 30.0%, P < 0.05). The average MVD was significantly higher in patients with positive vascular invasion than in patients with negative vascular invasion (29.2 +/- 3.3 vs 25.4 +/- 4.7, P < 0.05). The vascular invasion detected with hematoxylin-eosin staining was less than that with immunohistochemical staining. There was a significant difference between the two staining methods (19.6% vs 36.9%, P < 0.05). CONCLUSION: Positive CK20 RT-PCR, depth of tumor invasion, lymph node status, metastasis and MVD are significantly correlated with vascular invasion. Immunohistochemical staining is more sensitive than hematoxylin-eosin staining for detecting vascular invasion.

Correlation between expression of gastrin, somatostatin and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma.

AIM: To explore the correlation between expression of somatostatin (SS), gastrin (GAS) and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma. METHODS: Sixty-two large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma. Immunohistochemical staining for bcl-2, bax, GAS, SS was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. According to the semi-quantitative integral evaluation, SS and GAS were divided into three groups as follows. Scores 1-3 were defined as the low expression group, 4-8 as the intermediate expression group, 9-16 as the high expression group. Bax and bcl-2 protein expressions in different GAS and SS expression groups of large intestine carcinoma were assessed. RESULTS: The positive expression rate of bax had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, chi(2)(SS) = 9.246; P<0.05, chi(2)(GAS) = 6.981). The positive expression rate of bax in SS high (80.0%, 8/10) and intermediate (76.5%, 13/17) expression groups was higher than that in low expression group (40.0%, 14/35) (P<0.05, chi(2)( high vs low ) = 5.242; P<0.05, chi(2)( middle vs low ) = 6.097). The positive expression rate of bax in GAS high expression group (27.3%, 3/8) was lower than that in low expression group (69.4%, 25/36) (P<0.05, chi(2) = 4.594). However, bax expression in GAS intermediate expression group (46.7%, 7/15) was lower than that in low expression group, but not statistically significant. The positive expression rate of bcl-2 had a prominent difference between SS and GAS high, intermediate and low expression groups (P<0.05, chi(2)(SS) = 7.178; P<0.05, chi(2)( GAS ) = 13.831). The positive expression rate of bcl-2 in GAS high (90.9%, 10/11) and intermediate (86.7%, 13/15) expression groups was higher than that in low expression group (44.4%, 16/36) (P<0.05, chi(2)( high vs low ) = 5.600; P<0.05, chi(2)( middle vs low ) = 7.695). However, the positive expression rate of bcl-2 in SS high (40.0%, 4/10) and intermediate (47.1%, 8/9) expression groups was lower than that in low expression group (77.1%, 27/35) (P<0.05, chi(2)( high vs low ) = 4.710; P<0.05, chi(2)( middle vs low ) = 4.706). There was a significant positive correlation between the integral ratio of GAS to SS and the integral of bcl-2 (P<0.01, r = 0.340). However, there was a negative correlation between the integral ratio of GAS to the SS and bax the integral of (P<0.05, r = -0.299). CONCLUSION: The regulation and control of gastrin, somatostatin in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of bcl-2, bax.

Correlation between the expressions of gastrin, somatostatin and cyclin and cyclin-depend kinase in colorectal cancer.

AIM: To explore the correlation between the expressions of gastrin (GAS), somatostatin (SS) and cyclin, cyclin-dependent kinase (CDK) in colorectal cancer, and to detect the specific regulatory sites where gastrointestinal hormone regulates cell proliferation. METHODS: Seventy-nine resected large intestine carcinomatous specimens were randomly selected. Immunohistochemical staining for GAS, SS, cyclin D1, cyclin E, cyclin A, cyclin B1, CDK2 and CDK4 was performed according to the standard streptavidin-biotin-peroxidase (S-P) method. According to the semi-quantitative integral evaluation, SS and GAS were divided into high, middle and low groups. Cyclin D1, cyclin E, cyclin A, cyclin B1, CDK2, CDK4 expressions in the three GAS and SS groups were assessed. RESULTS: The positive expression rate of cyclin D1 was significantly higher in high (78.6%, 11/14) and middle GAS groups (73.9%, 17/23) than in low GAS group (45.2%, 19/42) (P<0.05, c2(high vs low) = 4.691; P<0.05, c2(middle vs low) = 4.945). The positive expression rate of cyclin A was significantly higher in high (100%, 14/14) and middle GAS groups (82.6%, 19/23) than in low GAS group (54.8%, 23/42) (P<0.01, c2(high vs low) = 9.586; P<0.05, c2(middle vs low) = 5.040). The positive expression rate of CDK2 was significantly higher in high (92.9%, 13/14) and middle GAS groups (87.0%, 20/23) than in low GAS group (50.0%, 21/42) (P<0.01, c2(high vs low) = 8.086; P<0.01, c2(middle vs low) = 8.715). The positive expression rate of CDK4 was significantly higher in high (78.6%, 11/14) and middle GAS groups (78.3%, 18/23) than in low GAS group (42.9%, 18/42) (P<0.05, c2(high vs low) = 5.364; P<0.01, c2(middle vs low) = 7.539). The positive expression rate of cyclin E was prominently higher in low SS group (53.3%, 24/45) than in high (9.1%, 1/11) and middle (21.7%, 5/23) SS groups (P<0.05, c2(high vs low) = 5.325; P<0.05, c2(middle vs low) = 6.212). The positive expression rate of CDK2 was significantly higher in low SS group (77.8%, 35/45) than in high SS group (27.3%, 3/11) (P<0.01, c2(high vs low) = 8.151). There was a significant positive correlation between the integral ratio of GAS to SS and the semi-quantitative integral of cyclin D1, cyclin E, cyclin A, CDK2, CDK4 (P<0.05, (D1)r(s) = 0.252; P<0.01, (E)r(s) = 0.387; P<0.01, (A)r(s) = 0.466; P<0.01, (K2)r(s) = 0.519; P<0.01, (K4)r(s) = 0.434). CONCLUSION: The regulation and control of gastrin, SS in colorectal cancer cell growth may be directly related to the abnormal expressions of cyclins D1, A, E, and CDK2, CDK4. The regulatory site of GAS in the cell cycle of colorectal carcinoma may be at the G(1), S and G(2) phases. The regulatory site of SS may be at the entrance of S phase.