Research of the mechanism on miRNA193 in exosomes promotes cisplatin resistance in esophageal cancer cells.

PURPOSE: Chemotherapy resistance of esophageal cancer is a key factor affecting the postoperative treatment of esophageal cancer. Among the media that transmit signals between cells, the exosomes secreted by tumor cells mediate information transmission between tumor cells, which can make sensitive cells obtain resistance. Although some cellular exosomes play an important role in tumor's acquired drug resistance, the related action mechanism is still not explored specifically. METHODS: To elucidate this process, we constructed a cisplatin-resistant esophageal cancer cell line, and proved that exosomes conferring cellular resistance in esophageal cancer can promote cisplatin resistance in sensitive cells. Through high-throughput sequencing analysis of the exosome and of cells after stimulation by exosomes, we determined that the miRNA193 in exosomes conferring cellular resistance played a key role in sensitive cells acquiring resistance to cisplatin. In vitro experiments showed that miRNA193 can regulate the cell cycle of esophageal cancer cells and inhibit apoptosis, so that sensitive cells can acquire resistance to cisplatin. An in vivo experiment proved that miRNA193 can promote tumor proliferation through the exosomes, and provide sensitive cells with slight resistance to cisplatin. RESULTS: Small RNA sequencing of exosomes showed that exosomes in drug-resistant cells have 189 up-regulated and 304 down-regulated miRNAs; transcriptome results showed that drug-sensitive cells treated with drug-resistant cellular exosomes have 3446 high-expression and 1709 low-expression genes; correlation analysis showed that drug-resistant cellular exosomes mainly affect the drug resistance of sensitive cells through paths such as cytokine-cytokine receptor interaction, and the VEGF and Jak-STAT signaling pathways; miRNA193, one of the high-expression miRNAs in drug-resistant cellular exosomes, can promote drug resistance by removing cisplatin's inhibition of the cell cycle of sensitive cells. CONCLUSION: Sensitive cells can become resistant to cisplatin through acquired drug-resistant cellular exosomes, and miRNA193 can make tumor cells acquire cisplatin resistance by regulating the cell cycle.

Knocking Out SST Gene of BGC823 Gastric Cancer Cell by CRISPR/Cas9 Enhances Migration, Invasion and Expression of SEMA5A and KLF2.

BACKGROUND: The impact and potential molecular mechanisms of SST in the occurrence and development of GC have not been determined. MATERIALS AND METHODS: Two pairs of sgRNA and reporter were designed according to targeting sequence of SST gene for double-nicking. Plasmids were transfected into 293T for selecting sgRNA with higher cutting efficiency. The subline which has knocked-out SST gene were selected by FACS and verified by sequencing and expression level. Moreover, the migration and invasion ability was evaluated by wound healing and transwell after knocking out SST. Besides, the protein expression of SEMA5A and KLF2 were observed by Western blotting and LSCM. Last, we detected the expression levels of SST, SEMA5A, and KLF2 in GC tissues by Western blotting. RESULTS: The results revealed that the new subline 1E9, which had knocked out SST gene, was established by CRISPR/Cas9. In addition, the knockout of SST in GC cells markedly increased migration and invasion ability. The results also demonstrated that the knockout of SST increased the expression of SEMA5A and KLF2. The expression level of SST was decreased in GC tissues, and its decrease was associated with overexpression of SEMA5A and KLF2. CONCLUSION: SST plays an inhibitory role in the migration and invasion of GC cell BGC823. The protein expression levels of SEMA5A and KLF2 were enhanced in GC cells and tissues lacking SST expression.

A Virus-Infected, Reprogrammed Somatic Cell-Derived Tumor Cell (VIReST) Vaccination Regime Can Prevent Initiation and Progression of Pancreatic Cancer.

PURPOSE: Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses. EXPERIMENTAL DESIGN: We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using in situ gene editing via stable provision of KRas G12D and p53 R172H tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development. RESULTS: Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic. CONCLUSIONS: These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals.

Exosomal miRNA-107 induces myeloid-derived suppressor cell expansion in gastric cancer.

  Background: Myeloid-derived suppressor cells (MDSCs) promote immunosuppression in the tumor microenvironment, support tumor growth and survival, and may contribute to immunotherapy resistance. Recent studies showed that tumor-derived exosomes (TDEs) can induce MDSCs accumulation and expansion, the mechanisms of which are largely unknown. Methods: The morphologies and sizes of the exosomes was observed by using a JEM-1400 transmission electron microscope. MicroRNA(miR)-107 and ARG1, DICER1, PTEN, PI3K, AKT, mTOR, and NF-kB mRNAs were quantified by quantitative reverse tanscription PCR. Dual-Luciferase Reports Assay were used to examine the expression of genes which was targeted by miR-107. The expression of proteins were analyzed by using western blot. Results: MiR-107 was not only overexpressed in gastric cancer cells but also enriched in their secreted TDEs. Also, these miR-107 enriched TDEs could be taken up by HLA-DR-CD33+MDSCs, where miR-107 was able to target and suppress expression of DICER1 and PTEN genes. Dampened DICER1 expression supported expansion of MDSCs , while decreased PTEN led to activation of the PI3K pathway, resulting in increased ARG1 expression. Furthemore, gastric cancer-derived miR-107 TDEs, when dosed intravenously into mice, were also capable of inducing expansion of CD11b+Gr1+/high MDSCs in mouse peripheral blood and altering expression of DICER1, PTEN, ARG1, and NOS2 in the MDSCs. Conclusions: Our findings demonstrate for the first time that gastric cancer-secreted exosomes are able to deliver miR-107 to the host MDSCs where they induce their expansion and activition by targeting DICER1 and PTEN genes, thereby may provide novel cancer therapeutics target for gastric cancer.

Generation of induced pluripotent stem cell line (ZZUi0012-A) from a patient with Fahr's disease caused by a novel mutation in SLC20A2 gene.

Several SLC20A2 mutations have been implicated as potential causes of Fahr's disease, a subtype of primary familial brain calcification (PFBC), but very few patient-derived induced pluripotent stem cell (iPSC) models have been established. We have identified a novel SLC20A2 mutation in a family with Fahr's disease. We subsequently obtained dermal fibroblasts from a patient in this family. These fibroblasts were successfully transformed into iPSCs by employing episomal plasmids expressing OCT3/4, SOX2, KLF4, LIN28, and L-MYC. Our approach offers a resource and a platform for further research into the mechanism of Fahr's disease and could facilitate development and screening of pharmaceutical and gene therapies.

Linc00210 drives Wnt/ß-catenin signaling activation and liver tumor progression through CTNNBIP1-dependent manner.

BACKGROUND: Liver tumor initiating cells (TICs) have self-renewal and differentiation properties, accounting for tumor initiation, metastasis and drug resistance. Long noncoding RNAs are involved in many physiological and pathological processes, including tumorigenesis. DNA copy number alterations (CNA) participate in tumor formation and progression, while the CNA of lncRNAs and their roles are largely unknown. METHODS: LncRNA CNA was determined by microarray analyses, realtime PCR and DNA FISH. Liver TICs were enriched by surface marker CD133 and oncosphere formation. TIC self-renewal was analyzed by oncosphere formation, tumor initiation and propagation. CRISPRi and ASO were used for lncRNA loss of function. RNA pulldown, western blot and double FISH were used to identify the interaction between lncRNA and CTNNBIP1. RESULTS: Using transcriptome microarray analysis, we identified a frequently amplified long noncoding RNA in liver cancer termed linc00210, which was highly expressed in liver cancer and liver TICs. Linc00210 copy number gain is associated with its high expression in liver cancer and liver TICs. Linc00210 promoted self-renewal and tumor initiating capacity of liver TICs through Wnt/ß-catenin signaling. Linc00210 interacted with CTNNBIP1 and blocked its inhibitory role in Wnt/ß-catenin activation. Linc00210 silencing cells showed enhanced interaction of ß-catenin and CTNNBIP1, and impaired interaction of ß-catenin and TCF/LEF components. We also confirmed linc00210 copy number gain using primary hepatocellular carcinoma (HCC) samples, and found the correlation between linc00210 CNA and Wnt/ß-catenin activation. Of interest, linc00210, CTNNBIP1 and Wnt/ß-catenin signaling targeting can efficiently inhibit tumor growth and progression, and liver TIC propagation. CONCLUSION: With copy-number gain in liver TICs, linc00210 is highly expressed along with liver tumorigenesis. Linc00210 drives the self-renewal and propagation of liver TICs through activating Wnt/ß-catenin signaling. Linc00210 interacts with CTNNBIP1 and blocks the combination between CTNNBIP1 and ß-catenin, driving the activation of Wnt/ß-catenin signaling. Linc00210-CTNNBIP1-Wnt/ß-catenin axis can be targeted for liver TIC elimination.

LncAPC drives Wnt/ß-catenin activation and liver TIC self-renewal through EZH2 mediated APC transcriptional inhibition.

Liver tumor initiating cells (TICs), a small subset cells in tumor bulk, are responsible for liver tumor initiation, metastasis, and relapse. However, the regulatory mechanism of liver TICs remains largely unknown. Here we found a long noncoding RNA lncAPC, locating near from APC locus, was highly expressed in liver cancer and liver TICs. LncAPC was required for liver TIC self-renewal. Silencing and overexpressing lncAPC showed impaired and enhanced sphere formation capacity of liver TICs, respectively. By recruiting EZH2 to APC promoter, LncAPC inhibits APC transcription and thus drives the activation of Wnt/ß-catenin signaling. Attenuate binding between EZH2 and APC promoter was observed upon lncAPC knockdown. What is more, lncAPC-EZH2-APC axis can be targeted to eliminate liver TICs. Altogether, LncAPC promotes liver TIC self-renewal through EZH2-dependent APC transcriptional inhibition.

Expression profile of cytokines in gastric cancer patients using proteomic antibody microarray.

Gastric cancer (GC) is often a deadly disease due to the late diagnosis and chemoresistance that characterizes many cases of this disease. The aim of this study was to explore a panel of candidate cytokines as diagnostic and predictive biomarkers for GC. Sixteen tissue samples of GC and adjacent noncancerous mucosa were selected from GC patients (n=8) for antibody microarray analysis. Proteomic chip-based analysis was performed to simultaneously identify 507 cytokines using a cytokine antibody array in the gastric tissues to screen for differential proteins related in cases of GC. Fold changes of protein expression >2.0 or <0.5 were considered significant. The proteins that showed significant differences in levels between the cancerous and non-cancerous samples were analyzed using bioinformatics analysis. One hundred and five cytokines that were significantly different (p<0.05) between GC tissues and normal gastric mucosa were identified. Gene Ontology (GO) enrichment analysis showed that these differentially expressed proteins are involved in many biological and immunological processes, mainly in response to stress, chloroplast thylakoid membrane, vacuole, photosynthesis, aspartic-type endopeptidase activity and flavin adenine dinucleotide binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that these proteins mainly were involved in the process of cytokine-cytokine receptor interaction, transforming growth factor-ß (TGF-ß) signaling pathway, pathways in cancer, tumor necrosis factor (TNF) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway. These findings suggest that the differentially expressed proteins could be associated with GC in patients. Further study of these cytokines may provide a promising approach for diagnosis, classification and prognosis of GC.

Cytokine-induced killer cell therapy-associated idiopathic thrombocytopenic purpura: rare but noteworthy.

Idiopathic thrombocytopenic purpura (ITP) is characterized by a diminished platelet count, an autoimmune condition with antibodies against platelets and an increased tendency to bleed. The association between ITP and solid tumors is uncommon. Cytokine-induced killer (CIK) cell therapy is a well tolerated and promising cancer treatment with minimal toxicity. For the first time, CIK cell therapy was reported to be followed by ITP. The mechanism through which CIK induces ITP remains unclear. Imbalanced ratio of Th cells, decreased numbers or impaired function of Treg cells and excessive secretion of cytokines inducing abnormal activation of B cells may be among the possible reasons. Therefore, a better understanding of this rare condition will require further investigation of these cases.

EGFR, ALK, RET, KRAS and BRAF alterations in never-smokers with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), caused by various mutations in a spectrum of cancer driver genes, may have distinct pathological characteristics and drug responses. Extensive genetic screening and pathological characterization is required for the design of customized therapies to improve patient outcomes. Notably, NSCLC in never-smokers exhibits distinctive clinicopathological features, which are frequently associated with tumorigenic mutations, and thus may be treated as a unique disease entity. However, to the best of our knowledge, these mutations have not been extensively and accurately characterized in an NSCLC study with a large sample size. Therefore, the present study enrolled a large cohort of NSCLC patients, which consisted of 358 never-smokers, for the screening of genetic alterations in the epidermal growth factor receptor (EGFR), ret proto-oncogene (RET), anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-Raf proto-oncogene serine/threonine kinase (BRAF) tumorigenic genes. It was identified that the mutation rate was 47.8, 7.5, 3.6, 1.4 and 0.3% for EGFR, ALK, KRAS, RET and BRAF, respectively. In addition, clinicopathological features associated with these mutations were characterized. EGFR mutations were more frequently observed in female and older patients. By contrast, KRAS mutations were more frequently detected in male patients, and ALK and RET translocations in younger patients. The cancer cells were frequently well-differentiated in carcinoma cases exhibiting EGFR mutations, however, were less differentiated in those with ALK translocations. In conclusion, the present study determined the frequency of oncogenic alterations and associated clinicopathological features in NSCLC exhibited by never-smokers using a large sample size. The results of the present study may enrich our knowledge of NSCLC in never-smokers and provide useful insights for improvement of the outcome of molecularly targeted therapies for the treatment of NSCLC.

WWP1 as a potential tumor oncogene regulates PTEN-Akt signaling pathway in human gastric carcinoma.

Whelming evidence has demonstrated that WW domain containing E3 ubiquitin protein ligase 1 (WWP1) participates in a wide variety of biological processes and is tightly related to the initiation and progression of many tumors. Currently, although mounting evidence supports a role of WWP1 in tumor promotion and tumorigenesis, the potential roles of WWP1 and its biological functions in gastric carcinoma are not fully understood. Here, we found that WWP1 messenger RNA (mRNA) and protein were highly expressed in gastric carcinoma tissues and cells. High WWP1 mRNA and protein levels were tightly related to differentiation status, TNM stage, invasive depth, lymph node metastasis, and poor prognosis in gastric carcinoma. Furthermore, WWP1 siRNA significantly decreased WWP1 protein level in MKN-45 and AGS cells; meanwhile, WWP1 depletion markedly inhibited tumor proliferation in vitro and in vivo, arrested cell cycle at G0/G1 phase, and induced cell apoptosis in MKN-45 and AGS cells. Most notably, WWP1 downregulation both inactivated PTEN-Akt signaling pathway in MKN-45 and AGS cells. Taken altogether, our findings suggest that WWP1 acts as an oncogenic factor and should be considered as a novel interfering molecular target for gastric carcinoma.

EML4-ALK translocation is associated with early onset of disease and other clinicopathological features in Chinese female never-smokers with non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) translocation is resistant to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, but responds to the ALK-TKI crizotinib. Characterization of EML4-ALK translocation may provide invaluable information to facilitate disease diagnosis and improve the outcome of customized treatment. Although the occurrence of EML4-ALK translocation is likely to be affected by the smoking habits and gender of patients, the translocation has not been characterized extensively in female never-smokers with NSCLC. Therefore, 280 female never-smokers that were diagnosed with NSCLC were enrolled in the present study, and characteristics of EML4-ALK translocation, including the frequency, were determined in these NSCLC patients. EML4-ALK fusion variants were detected using Multiplex one-step reverse transcription-polymerase chain reaction and subsequently confirmed by DNA sequencing and Vysis ALK Break Apart fluorescence in situ hybridization analysis. The EML4-ALK fusion variants were detected in 21 carcinoma tissue specimens, accounting for 7.5% of the enrolled patients. Out of these patients with EML4-ALK fusion variants, EML4-ALK fusion variant 1 was identified in 12 patients, indicating that variant 1 is the most common type of EML4-ALK fusion gene in the present cohort of patients. ALK mRNA was aberrantly expressed in all the tissues with EML4-ALK translocation, but not in the carcinoma tissues without EML4-ALK translocation. In addition, the EML4-ALK translocation was more frequently found in younger patients. The median age of patients with EML4-ALK translocation was 50.95±2.29 years, which was significantly younger (P<0.01) than the median age of the patients without EML4-ALK translocation (57.15±0.56). The EML4-ALK translocation was detected exclusively in undifferentiated tumors that were graded as poorly- or moderately-differentiated carcinomas and suspected to be more malignant compared with well-differentiated tumors. In summary, the present study found that 7.5% of patients with NSCLC that are female never-smokers harbor EML4-ALK translocations, which are associated with the aberrant expression of ALK mRNA, early onset of disease and undifferentiated carcinomas.

Both gene deletion and promoter hyper-methylation contribute to the down-regulation of ZAC/PLAGL1 gene in gastric adenocarcinomas: a case control study.

BACKGROUND AND OBJECTIVE: Pleiomorphic adenoma gene-like 1 (PLAGL1, also known as LOT1 and ZAC) is a zinc-finger nuclear transcription factor, which possesses antiproliferative effects and is frequently epigenetically silenced during tumorigenesis. PLAGL1 gene is located on 6q24-25, a chromosomal region that is frequently deleted in various kinds of cancers. Both promoter hyper-methylation and loss of heterozygosity may lead to the down-regulation of PLAGL1 in human somatic cancers. Here we aimed to investigate the abnormalities of PLAGL1 in gastric cancers. METHODS: We collected 153 case-matched gastric adenocarcinoma (GAC) cases. Quantitative real-time PCR method was applied to evaluate the expression levels as well as gene copy numbers of PLAGL1 in the collected samples. Methylation-specific PCR (MSP) assay was performed to analyze the methylation status of PLAGL1 P1 promoter. RESULTS: Decreased expression of PLAGL1 mRNA was observed in GAC tissues, especially in advanced GACs. Copy number decrease of PLAGL1 gene in GACs was observed in 9.15% (19 out of 153) of the GAC samples and was closely correlated with gene expression. Methylation status of PLAGL1 promoter in GAC tissues was higher than in normal controls, which was inversely correlated with the expression levels of PLAGL1 mRNA. CONCLUSION: DNA deletion and promoter hyper-methylation both contribute to the down-regulation of PLAGL1 in GACs.

Artemisinin inhibits gastric cancer cell proliferation through upregulation of p53.

Recent population studies suggest that the use of artemisinin is associated with reduced incidence and improved prognosis of certain cancers. In the current study, we assessed the effect of artemisinin on gastric cancer cells (AGS and MKN74 cells). We found that artemisinin inhibited growth and modulated expression of cell-cycle regulators in these cells. Treatment with artemisinin was also associated with induction of p27 kip1 and p21 kip1, two negative cell-cycle regulators. Furthermore, we revealed that artemisinin treatment led to an increased expression of p53. Taken together, these results provide evidence for a mechanism that may contribute to the antineoplastic effects of artemisinin suggested by recent population studies and justify further work to explore potential roles for it in gastric cancer prevention and treatment.

Endoplasmic reticulum stress-mediated signaling pathway of gastric cancer apoptosis.

BACKGROUND/AIMS: To investigate ER stress-mediated CHOP-signaling pathway of gastric cancer apoptosis in vitro and in vivo. METHODOLOGY: Based on the dose-and time-response experiments about tunicamycin (TM),gastric cancer cell line BGC823 was treated with 10tg/mL of TM for 24h. BGC823 apoptosis was detected with TUNEL assay and ultrastructural changes in BGC823 cells under ER stress were observed with transmission electron microscopy (TEM). RT-PCR and western blot-ting were used to determine the expression of ERS-related proteins, glucose-regulated protein 78 (GRP78) and CHOP and apoptosis-associated protein B-cell lympho-ma 2 (Bcl-2). After the knockdown of CHOP, the changes were also observed in vitro and in vivo. RESULTS: TEM assay showed that after treatment with TM, BGC823 cell size became smaller with ER dilation, vacuolization and karyopyknosis. RT-PCR and western blotting indicated that TM up-regulated GRP78 and CHOP expression and down-regulated Bcl-2 expression. The knock-down of CHOP could convert Bcl-2 expression and reduce BGC823 apoptosis caused by ERS in vitro and in vivo, but failed to influence GRP78. CONCLUSIONS: TM can induce ESR and regulate downstream molecules CHOP up-regulation and Bcl-2 down-regulation which lead to BGC823 apoptosis. This study may provide a new theoretical basis for the pathogenesis of gastric cancer.

Amplifications of NCOA3 gene in colorectal cancers in a Chinese population.

AIM: To investigate the copy number variation of NACO3 gene in colorectal cancer (CRC) and its correlation with tumor progression. METHODS: A total of 142 samples of case-matched CRC tissues and adjacent normal tissues were obtained from patients undergoing bowel resection. Quantitative real-time polymerase chain reaction method was used to investigate the copy number variations of NCOA3 as well as gene expression in the collected tissues. RESULTS: Copy number gains of NCOA3 were detected in 39 CRC samples (27.5%) and were correlated with tumor progression (χ2 = 6.42, P = 0.0112). Moreover, there was a positive correlation between copy number gain and mRNA over-expression of NCOA3 in CRCs (P = 0.0023). Expression level of NCOA3 mRNA was also enhanced in the CRC samples with unaltered copy numbers (3.85 ± 1.23 vs. 2.71 ± 0.64, P < 0.01). CONCLUSION: Sporadic colorectal cancers exhibit different mechanisms of NCOA3 regulation.

Lentiviral vector-mediated siRNA knockdown and concurrent rescue of Murine CIN85.

RNA interference (RNAi), an evolutionarily conserved process of gene silencing, is now a common tool in gene functional studies. However, potential "off-target effects" is one of major concerns in RNAi experiment associated with false positive results. Apart from continuing improvement in small interfering RNA (siRNA) designs, there is no method available to prevent the generation of "off-target effects" resulted from possible identity between siRNA and abundant cellular mRNA transcripts. In the present study, we have developed a lentiviral vector-mediated system that allows efficient siRNA silencing and concurrent rescue of targeted genes. While this approach does not eliminate potential "off-target effects," concurrent rescue of a target gene allows a definite judgment with regard to a phenotype change, either from expected siRNA silencing or "off-target effects." The system has been validated with murine CIN85 gene and may be generally applicable in molecular studies from broad fields.