Zhao Sun

MicroRNA-9 enhances migration and invasion through KLF17 in hepatocellular carcinoma

Metastasis is one of the hallmarks of cancer malignancy that usually causes more detrimental effects than a primary tumor. Many microRNAs were reported to be involved in the process of tumor metastasis. Hep11 and Hep12 cells were derived from primary and recurrence (intrahepatic metastatic) sites of hepatocellular carcinoma (HCC), respectively. Hep12 exhibited a higher invasive and migratory potential than Hep11. There was also a significantly higher expression of miR‐9 in Hep12 cells than in Hep11 cells. Further studies in HCC cell lines demonstrated that miR‐9 could promote tumor cell migration and invasion. In addition, miR‐9 downregulated KLF17 protein expression by targeting the 3′UTR region of the KLF17 gene directly. As a transcription factor, KLF17 directly acted on the promoters of EMT‐related genes (ZO‐1, Vimentin and Fibronectin (FN)) in HCC cell lines. Therefore, we conclude that miR‐9 may possibly promote HCC migration and invasion through regulation of KLF17.

MicroRNA-1246 enhances migration and invasion through CADM1 in hepatocellular carcinoma

BackgroundThe aberrant expression of microRNAs has been demonstrated to play a crucial role in the initiation and progression of hepatocarcinoma. miR-1246 expression in High invasive ability cell line than significantly higher than that in low invasive ability cell line.MethodsTranswell chambers (8-uM pore size; Costar) were used in the in vitro migration and invison anssay. Dual luciferase reporter gene construct and Dual luciferase reporter assay to identify the target of miR-1246. CADM1 expression was evaluated by immunohistochemistric staining. The clinical manifestations, treatments and survival were collected for statistical analysis.ResultsInhibition of miR-1246 effectively reduced migration and invasion of hepatocellular carcinoma cell lines. Bioinformatics and luciferase reporter assay revealed that miR-1246 specifically targeted the 3′-UTR of Cell adhesion molecule 1 and regulated its expression. Down-regulation of CADM1 enhanced migration and invasion of HCC cell lines. Furthermore, in tumor tissues obtained from liver cancer patients, the expression of miR-1246 was negatively correlated with CADM1 and the high expression of miR-1246 combined with low expression of CADM1 might serve as a risk factor for stage1 liver cancer patients.ConclusionsOur study showed that miR-1246, by down-regulation CADM1, enhances migration and invasion in HCC cells.

Chromogranin A is a reliable serum diagnostic biomarker for pancreatic neuroendocrine tumors but not for insulinomas

BackgroundPancreatic neuroendocrine tumors (PNETs) are a group of rare tumors. Chromogranin A (CgA) was considered as the most practical and useful serum tumor marker in PNET patients. But peripheral blood levels of CgA are not routinely tested in Chinese patients with PNETs. This study was to assess the diagnostic value of CgA in Chinese patients with PNETs especially in patients with insulinomas.MethodsEighty-nine patients with PNETs including 57 insulinomas and 32 non-insulinoma PNETs as well as 86 healthy participants were enrolled in this study between September 2003 and June 2013. Serum levels of CgA were measured by ELISA method. Expression of CgA protein was detected in 26 PNET tissues including 14 insulinomas by immunohistochemical staining.ResultsSerum levels of CgA in 89 PNET patients were significantly higher than that in healthy controls (Pu2009=u20097.2u2009×u200910−9). Serum levels of CgA in 57 patients with insulinomas (median 64.8xa0ng/ml, range 25–164) were slightly higher than the levels in healthy controls (median 53.4xa0ng/ml, range 39–94) but much lower than the levels in 32 patients with non-insulinoma PNETs (median 193xa0ng/ml, range 27–9021), Pu2009=u20090.001. The serum CgA levels were reduced in 16 of 17 patients with insulinomas after tumor resection. ROC curve showed that CgA values at 60xa0ng/ml distinguished patients with insulinomas from healthy controls but its sensitivity and specificity were 66.7% and 73.3%, respectively. In contrast, CgA values at 74xa0ng/ml distinguished patients with non-insulinoma PNETs from healthy controls, and the sensitivity and specificity were 65.6% and 91.9%, respectively. Except for two insulinomas with negative staining of CgA, 12 insulinoma tissues showed positive staining of CgA.ConclusionCgA is a reliable serum diagnostic biomarker for PNETs but not for insulinomas.

NANOG Has a Role in Mesenchymal Stem Cells' Immunomodulatory Effect

It is well known that terminally differentiated cells derived from mesenchymal stem cells (MSCs) will lose the immunomodulation capacity. NANOG is known to be a core transcription factor in the maintenance of stem cell specific features or stemness. To evaluate whether NANOG was involved in the immunomodulation effects of MSCs, MSCs immunomodulation capacity on lymphocyte activation and proliferation before or after endogenous NANOG interference was investigated. We found that MSCs inhibitory effects on lymphocyte activation and proliferation was significantly weakened after NANOG knockdown. In addition, NANOG RNAi and chromatin immunoprecipitation experiments showed that NANOG suppressed the expression and secretion of DKK-1, transforming growth factor-beta1 (TGF-β1), TGF-β2, and TGF-β3, which are all important factors mediating MSCs immunomodulation capacity. Based on these data, we propose that NANOG plays an important role in maintaining the immunomodulation functions of MSCs by regulating the expression and secretion of TGF-β1, TGF-β2, TGF-β3, and DKK-1.

Polyethyleneimine-coating enhances adenoviral transduction of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential, which makes them attractive targets for regenerative medicine applications. Efficient gene transfer into MSCs is essential for basic research in developmental biology and for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors (CARs), but not into MSCs, which lack CAR expression. To overcome this problem, an Adv coated with cationic polymer polyethyleneimine (PEI) was developed. In this study, we demonstrated that PEI coating with an optimal ratio can enhance adenoviral transduction of MSCs without cytotoxicity. We also investigated the physicochemical properties and internalization mechanisms of the PEI-coated Adv. These results could help to evaluate the potentiality of the PEI-coated Adv as a prototype vector for efficient and safe transduction into MSCs.

MicroRNA-197 influences 5-fluorouracil resistance via thymidylate synthase in colorectal cancer

PurposeThe response rate of first-line fluoropyrimidine-based regimens for metastatic colorectal cancer (mCRC) is generally less than 50xa0%. The down-regulation of miR-197 in colorectal cancer cells after exposure to 5-fluorouracil might be related to the mechanism of resistance to fluoropyrimidine-based chemotherapy. So we investigated the regulatory mechanism of miR-197 on 5-FU sensitivity.MethodsDual luciferase reporter gene construct and dual luciferase reporter assay were used to identify the target of miR-197. TYMS expression was evaluated by immunohistochemistry staining. 5-Fu resistance of colorectal cancer cell lines was detected by MTS assay. The expression of miR-197 was detected by real time PCR.ResultsA luciferase assay and western blot analysis confirmed that miR-197 directly binds to and negatively regulates TYMS expression. Overexpressing miR-197 could increase the sensitivity of colorectal cancer cells to 5-fluorouracil (5-FU). The expression of miR-197 negatively correlated with TYMS expression in cancerous tissues from patients with stage IV colorectal cancer.ConclusionmiR-197 mediates the response of colorectal cancer cells to 5-FU by regulating TYMS expression.

Dynamic monitoring of EGFR mutations in circulating cell-free DNA for EGFR-mutant metastatic patients with lung cancer: Early detection of drug resistance and prognostic significance

Detecting genetic mutations in circulating cell-free DNA (cfDNA) is a promising approach of liquid biopsy. Between June 2014 and May 2015, 168 plasma samples were collected monthly from 20 patients with metastatic lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutation receiving gefitinib therapy. Clinically relevant EGFR mutations, including exon 19 deletion, L858R and T790M, were quantified using droplet digital polymerase chain reaction. In baseline samples, 19 (95.0%) patients had the same mutation with the matched tumors, and pretreatment T790M mutations were also detected in 3 (15.0%) patients. The dynamics of EGFR mutations were generally associated with treatment response for patients with or without measurable disease. For patients with immeasurable tumor deposits, monitoring disease evolution using cfDNA-based mutation quantification appeared to be more reliable compared with measuring the diameters of target tumor lesions. In addition, molecular progressive disease, defined as a ≥20% increase of EGFR mutation concentration compared with the lowest concentration recorded during treatment, was tracked up to 8 months prior to objective progression. In survival analysis, sex (P=0.005), pretreatment T790M mutation status (P=0.006), T790M mutation status at the disease progression (P=0.043) and growth rate of EGFR mutations (P=0.023), had a significant impact on median progression-free survival. In conclusion, dynamic monitoring of EGFR mutations in cfDNA is feasible and appears to be useful in early prediction of drug resistance for patients with lung cancer receiving EGFR tyrosine kinase inhibitors.

Gefitinib-Integrated Regimen versus Chemotherapy Alone in Heavily Pretreated Patients with Epidermal Growth Factor Receptor–Mutated Lung Adenocarcinoma: A Case-Control Study

BACKGROUND: The study aimed to compare the tolerability and efficacy of gefitinib combined with chemotherapy agents versus chemotherapy alone for the treatment of epidermal growth factor receptor (EGFR)–mutated lung adenocarcinoma in heavily pretreated patients. METHODS: The study was designed as a matched-pair case-control investigation to minimize intergroup heterogeneity. Patients were stratified into gefitinib plus chemotherapy and chemotherapy alone groups with matching for sex, age, ECOG performance status, progress-free survival (PFS) from previous EGFR tyrosine kinase inhibitor treatment, EGFR mutation types, and tumor metastasis status. RESULTS: Sixty-six patients were selected from our database using the matched-pair method. The median age was 61 years (95% confidence interval, 57-65 years). During a follow-up period of 14.5 months on average, the overall response rates of the gefitinib-integrated and chemotherapy alone groups were 9.1% and 6.5%, respectively (P > .05), whereas the corresponding disease-control rates were 39.4% and 30.3%, respectively (P > .05). No statistically significant differences in PFS (median, 4.2 vs 3.3 months; P = .06) and overall survival (median, 10.4 vs 7.9 months; P = .44) were observed between two groups. The 6-month survival rates of the gefitinib-integrated and chemotherapy alone groups were 21.2% and 12.1%, respectively (P < .05). Side effects were mild, and all treatments were well tolerated. CONCLUSIONS: Our results indicated that gefitinib-integrated therapy offered a trend to better PFS and an improved 6-month survival rate in heavily pretreated patients with metastatic EGFR-mutated lung adenocarcinoma. All treatments were well tolerated. Future prospective studies are warranted to confirm our findings.

Impact of mismatch-repair deficiency on the colorectal cancer immune microenvironment

Colorectal cancer patients respond inconsistently to immunotherapies, likely due to the immune microenvironments around their tumors. We analyzed the relationship between deficient mismatch repair (dMMR) and the colorectal cancer immune microenvironment to identify predictors of effective immunotherapy. Colorectal cancer patients (n=113) who had undergone surgical resection were divided into dMMR and proficient mismatch repair (pMMR) groups. The levels of immune checkpoint proteins, including programmed cell death 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), indoleamine 2,3 dioxygenase and CD8 were assessed immunohistochemically. The percentage of tumor-infiltrating lymphocytes strongly positive for PD-1 (score=3) was higher in the dMMR than pMMR group (79.3% vs. 41.7%; p=0.003). The groups showed similar tumor cell PD-L1 positivity rates (34.5% vs. 35.7%, p=0.905) and PD-L1 intensity levels on immune cell infiltrates (86.2% vs. 84.5%, p=0.964). However, when a cut-off value of 80% was used for PD-L1 positivity, the rate of PD-L1 positivity on immune cell infiltrates differed between the groups (51.7% vs. 22.6%, p=0.003). The rate of high indoleamine 2,3 dioxygenase expression was greater in the dMMR than pMMR group (55.2% vs. 36.9%, p=0.026). CD8+ T cells were elevated in the dMMR group in both compartments (p=0.017 for tumor-infiltrating lymphocytes and stroma; p=0.038 for invasive front). Thus the immune microenvironment of dMMR colorectal cancer differs from that of pMMR colorectal cancer.

miRNA-125b regulates apoptosis of human non-small cell lung cancer via the PI3K/Akt/GSK3β signaling pathway

The present investigation demonstrated that regulation of microRNA (miR)-125b affected the apoptosis of human non-small cell lung cancer (NSCLC) through targeting of the PI3K/Akt and Wnt/β-catenin signaling pathways. The expression of miR-125b was assessed in patients with NSCLC, which demonstrated that miR-125b expression in NSCLC tissue was higher than that in para-carcinoma tissue. Furthermore, survival analysis of patients with NSCLC over 3xa0years indicated that the overall survival (OS) and disease-free survival (DFS) rates of patients with low miR-125b expression were higher than those of patients with high miR-125b expression. Proliferation and apoptosis assays were subsequently conducted in the human NSCLC cell line A549 using MTT assay and Annexinxa0V-FITC/PI kits, respectively. Caspase-3 activity ELISA and western blot analysis were also used to assess caspase-3 activity and the protein expression of Bax, Akt, phosphorylatedxa0(p)-Akt, p-GSK3β, Wnt and β-catenin. It was observed that downregulation of miR-125b inhibited the proliferation and induced the apoptosis of A549 cells. Downregulation of miR-125b also suppressed the protein expression of p-Akt, Wnt and β-catenin, and increased caspase-3 activity and Bax protein expression in A549 cells. In addition, downregulation of miR-125b combined with the PI3K inhibitor LY294002 enhanced cell growth inhibition, suppression of p-GSK3β, Wnt and β-catenin protein expression and promotion of caspase-3 activity in A549 cells. These results revealed that the downregulation of miR-125b regulates apoptosis in human NSCLC through the suppression of the PI3K/Akt/GSK3β and Wnt/β-catenin signaling pathways.