miR-99b/let-7e/miR-125a cluster suppresses pancreatic cancer through regulation of NR6A1.

This experiment investigates how the miR-99b/let-7e/miR-125a cluster regulates the mechanism of NR6A1 involved in the invasive and metastatic effects of pancreatic cancer (PCa). Bioinformatics prediction and dual luciferase reporter gene assay were applied to verify the targeted relationship between miR-99b/let-7e/miR-125a and NR6A1. ASPC1 cells underwent transfection with lentiviruses to overexpress miR-99b/let-7e/miR-125a (individual or together) to explore functions of miR-99b/let-7e/miR-125a cluster governing NR6A1 in PCa. The detection of tumorigenesis was verified by tumor formation assay in nude mice in vivo, and mouse models of liver metastasis of PCa observed cell metastasis of PCa. MiR-99b/let-7e/miR-125a cluster was screened for differential expression in PCa. NR6A1 was confirmed as a target gene of the miR-99b/let-7e/miR-125a cluster. Findings demonstrated that overexpression of the miR-99b/let-7e/miR-125a cluster inhibited cell invasion, metastasis, proliferation, and tumorigenesis in PCa. Conversely, overexpressed NR6A1, a crucial gene in the miR-99b/let-7e/miR-125a cluster, promoted cell invasion, migration, and proliferation in PCa. Moreover, the overexpression of the miR-99b/let-7e/miR-125a cluster inhibited liver metastases and tumor formation. Thus, the study concludes that the miR-99b/let-7e/miR-125a cluster impedes the invasion and metastasis of PCa cells via targeting the NR6A1 gene.

Key Roles of p53 Signaling Pathway-Related Factors GADD45B and SERPINE1 in the Occurrence and Development of Gastric Cancer.

p53 can function as an independent and unfavorable prognosis biomarker in cancer patients. We tried to identify the key factors of the p53 signaling pathway involved in gastric cancer (GC) occurrence and development based on the genotype-tissue expression (GTEx) and the Cancer Genome Atlas (TCGA) screening. We downloaded gene expression data and clinical data of GC included in the GTEx and TCGA databases, followed by differential analysis. Then, the key factors in the p53 signaling pathway were identified, followed by an analysis of the correlation between key factors and the prognosis of GC patients. Human GC cell lines were selected for in vitro cell experiments to verify the effects of key prognostic factors on the proliferation, migration, invasion, and apoptosis of GC cells. We found 4,944 significantly differentially expressed genes (DEGs), of which 2,465 were upregulated and 2,479 downregulated in GC. Then, 27 DEGs were found to be involved in the p53 signaling pathway. GADD45B and SERPINE1 genes were prognostic high-risk genes. The regression coefficients of GADD45B and SERPINE1 were positive. GADD45B was poorly expressed, while SERPINE1 was highly expressed in GC tissues, highlighting their prognostic role in GC. The in vitro cell experiments confirmed that overexpression of GADD45B or silencing of SERPINE1 could inhibit the proliferation, migration, and invasion and augment the apoptosis of GC cells. Collectively, the p53 signaling pathway-related factors GADD45B and SERPINE1 may be key genes that participate in the development of GC.

TIMP-2 as a predictive biomarker in 5-Fu-resistant colorectal cancer.

PURPOSE: This study aims to evaluate the value of tissue inhibitors of MMPs-2 (TIMP-2) to indicate 5-Fluorouracil (5-Fu) resistance status in colorectal cancer. METHODS: The 5-Fu resistance of colorectal cancer cell lines was detected using Cell-Counting Kit-8 (CCK-8) and calculated using IC50. Enzyme-linked immunosorbent assay (ELISA) and real time-quantitative polymerase chain reaction (RT-qPCR) were used to detect TIMP-2 expression level in the culture supernatant and serum. Twenty-two colorectal cancer patients' TIMP-2 levels and clinical characteristics were analyzed before and after chemotherapy. Additionally, the patient-derived xenograft (PDX) model of 5-Fu resistance was used to evaluate the feasibility of TIMP-2 as a predictive biomarker of 5-Fu resistance. RESULTS: Our experimental results display that TIMP-2 expression is elevated in colorectal cancer drug-resistant cell lines, and its expression level is closely related to 5-Fu resistance. Moreover, TIMP-2 in colorectal cancer patient serum undergoing 5-Fu-based chemotherapy could indicate their drug resistance status, and its efficacy is higher than CEA and CA19-9. Finally, PDX model animal experiments reveal that TIMP-2 can detect 5-Fu resistance in colorectal cancer earlier than tumor volume. CONCLUSION: TIMP-2 is a good indicator of 5-Fu resistance in colorectal cancer. Monitoring the serum TIMP-2 level can help the clinician identify 5-Fu resistance in colorectal cancer patients earlier during chemotherapy.

Integrated characterisation of cancer genes identifies key molecular biomarkers in stomach adenocarcinoma.

AIMS: Gastric cancer is one of the leading causes for cancer mortality. Recent studies have defined the landscape of genomic alterations of gastric cancer and their association with clinical outcomes. However, the pathogenesis of gastric cancer has not been completely characterised. METHODS: Driver genes were detected by five computational tools, MutSigCV, OncodriveCLUST, OncodriveFM, dendrix and edriver, using mutation data of stomach adenocarcinoma (STAD) from the cancer genome altas database, followed by an integrative investigation. RESULTS: TTN, TP53, LRP1B, CSMD3, OBSCN, ARID1A, FAT4, FLG, PCLO and CSMD1 were the 10 most frequently mutated genes. PIK3CD, NLRC3, FMNL1, TRAF3IP3 and CR1 were the top five hub genes of the blue coexpression module positively correlated with pathological tumour stage and lymph node stage (p values <0.05 for all cases). Hierarchical clustering analysis of copy number variations of driver genes revealed three subgroups of STAD patients, and cluster 2 tumours were significantly associated with lower lymph node stage, less number of positive lymph nodes and higher microsatellite instability and better overall survival than cluster 1 and cluster 3 tumours (p values <0.05 for all cases, Wilcoxon rank-sum test or log rank test). High expression in one or more of DNER, LHCGR, NLRP14, OR4N2, PSG6, TTC29 and ZNF568 genes was associated with increased mortality (p values <0.05 for all cases, log rank test). CONCLUSIONS: The driver genes shed insights into the tumourigenesis of gastric cancer and the genes DNER, LHCGR, NLRP14, OR4N2, PSG6, TTC29 and ZNF568 pave the way for developing prognostic biomarkers for the disease.

Development of NKG2D-based chimeric antigen receptor-T cells for gastric cancer treatment.

Gastric cancer is the third leading cause of cancer-related mortalities worldwide and mostly incurable. It remains an urgent need for novel strategies in the management of patients with advanced gastric cancer. Chimeric antigen receptor (CAR) T therapy has shown unprecedented clinical success in hematological malignancies and potential utility is going on various solid tumors like gastric cancer. In this study, a broad expression of NKG2D ligands was observed in gastric cancer cell lines, making them suitable targets for gastric cancer therapy. T cells were engineered with an NKG2D-based second-generation CAR and the resulting NKG2D-CAR-T cells showed significantly increased cytolytic activity against gastric cancer compared to untransduced T cells. In vivo, these cells can significantly suppressed the growth of established gastric cancer xenografts. Besides, cisplatin was shown to upregulate NKG2D ligand expression in gastric cancer cells and enhance the susceptibility to NKG2D-CAR-T-cell-mediated cytotoxicity. In conclusion, NKG2D-based CAR-T cells have potent in vivo and in vitro anti-tumor activities against gastric cancer and could be a new paradigm for patients with gastric cancer, either used alone or combined with chemotherapy.

Metastasis-associated in colon cancer-1 is associated with poor prognosis in hepatocellular carcinoma, partly by promoting proliferation through enhanced glucose metabolism.

Metastasis-associated in colon cancer-1 (MACC1) is a newly identified gene that is involved in the development and progression of hepatocellular carcinoma (HCC), however its investigation has not been comprehensive. In the present study, in vitro techniques, including immunohistochemistry, western blotting, reverse transcription quantitative polymerase chain reaction, metabolic assay, MTT assay, colony formation assay and prognostic analysis were used to confirm the involvement of MACC1 in HCC. Histological examination confirmed that the protein expression of MACC1 was upregulated in HCC and was associated with the hexokinase-2 (HK2) protein, which also indicates a poor prognosis. Knockdown of MACC1 induced the reduction of glycogen consumption and lactate production, which then lead to a marked reduction of proliferation in the MHCC-97H cells. However, the overexpression of MACC1 produced the opposite results in the HepG2 cells. These results suggested that MACC1 leads to a poor prognosis in HCC, partly by promoting proliferation via enhancement in glucose metabolism by HK2. Therefore, this pathway has the potential to become an important therapeutic target in HCC.

[miR-143 inhibits proliferation and invasion of hepatocellular carcinoma cells via down-regulation of TLR2 expression].

OBJECTIVE: To detect the expression of microRNA-143 (miR-143) in hepatocellular carcinoma and investigate whether transfection of miR-143 could influence the biological behaviors of hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma tissues and its corresponding adjacent normal tissues were obtained from patients undergoing radical surgical resection. Real-time quantitative PCR was utilized to detect the relative expression of miR-143 in hepatocellular carcinoma tissues and corresponding adjacent normal tissues. MiR-143 mimics and negative control oligonucleotides were synthesized and transfected into HepG-2 cells in vitro. Proliferation, apoptosis and invasion of the transfected cells were measured by MTT assay, flow cytometry (FCM) combined with annexin V-FITC/PI staining, and Transwell(TM) assay, respectively. The protein levels of Toll-like receptor 2 (TLR2), nuclear factor kappa B (NF-κB), matrix metalloproteinase 2 (MMP-2) and MMP-9 were detected by Western blotting. RESULTS: Expression of miR-143 was significantly lower in hepatocellular carcinoma tissues than in paired adjacent normal tissues. The level of miR-143 was significantly up-regulated after transfection of miR-143 mimics. And proliferation and invasion were significantly inhibited, but apoptosis was promoted after transfection. Expressions of TLR2, NF-κB, MMP-2 and MMP-9 were reduced by miR-143 mimics' transfection. CONCLUSION: The miR-143 expression was low in hepatic carcinoma and its over-expression could down-regulate the expressions of TLR2, NF-κB, MMP-2 and MMP-9.

MACC1 expression correlates with PFKFB2 and survival in hepatocellular carcinoma.

OBJECTIVE: To validate the relationship between MACC1 and 6-phosphofructo-2-kinase/fructose 2, 6 bisphosphatase (PFKFB2) expression as well as its clinicopathological features and prognostic significance in hepatocellular carcinoma. METHODS: By using immunohistochemistry, we investigated the MACC1 and PFKFB2 protein expression in 60 pairs of hepatocellular carcinoma and corresponding non-tumor tissues. Using the Mann-Whitney U test, the Chi-square test, Kaplan-Meier survival analysis, Cox proportional hazard regression analysis and Spearman analysis, we studied the relationship between MACC1 and PFKFB2 protein expression and postoperative overall survival (OS) of the HCC patients. RESULTS: MACC1 and PFKFB2 positive staining rates were significantly higher in hepatocellular carcinoma than in the corresponding nontumor tissues (P=0.012 and 0.04, respectively). The clinicopathological features evaluation revealed that positive expression of MACC1 was associated with a high Edmondson classification (P=0.007) and advanced TNM stage (P=0.027). Similar findings were evident for PFKFB2 expression (P=0.002 and P=0.027). MACC1 and PFKFB2 positive expression was associated with a lower OS rate (P=0.004 and 0.03, respectively). Kaplan-Meier survival and Cox proportional hazard regression analyses revealed MACC1 positive expression to be a prognostic factor for postoperative OS, but PFKFB was not. CONCLUSION: Highly expressed MACC1 and PFKFB2 protein were associated with TNM stage, Edmondson -Steier classification and overall survival. MACC1 may affect tumor metabolism partly through expression and phophorylation of PFKFB2.

[Establishment of experimental ventricular septal defect in pigs].

OBJECTIVE: This is a study on the method for establishing experimental animal model of ventricular septal defect(VSD) in pigs. METHODS: Thoracotomy was performed on 15 young pigs under general anaesthesia. A spool-like ring amounted on a specially designed centesis tool was put into right ventricle via an incision on right auricle which was pushed to puncture through the septum. The ring was then left on the septum to create the artificial VSD. 3 days after the operation, echocardiography was conducted to verify the existence of VSDs. The animals were catheterized one month later to obtain hemodynamic data. RESULTS: Ten pigs survived the operation with successful acquisition of VSDs. Doppler echocardiography demonstrated the transseptal shunts with peak velocities ranging from 1.7-4.4 m/sec. Cardiac catheterization showed the Qp/Qs of 1.68-2.12 although the pulmonary pressures remained in normal limits. CONCLUSION: The authors report their experience in the establishment of experimental VSD model in pigs using revised Synhorst method. The preliminary result shows that the VSD model using pigs is feasible and worth improving further in order to provide a useful animal model for the pathophysiological study of intracardiac left to right shunt in the future.