miR-125b reverses cisplatin resistance by regulating autophagy via targeting RORA/BNIP3L axis in lung adenocarcinoma.

The platinum-based chemotherapy is one of the most frequently used treatment protocols for lung adenocarcinoma (LUAD), and chemoresistance, however, usually results in treatment failure and limits its application in the clinic. It has been shown that microRNAs (miRNAs) play a significant role in tumor chemoresistance. In this study, miR-125b was identified as a specific cisplatin (DDP)-resistant gene in LUAD, as indicated by the bioinformatics analysis and the real-time quantitative PCR assay. The decreased serum level of miR-125b in LUAD patients was correlated with the poor treatment response rate and short survival time. MiR-125b decreased the A549/DDP proliferation, and the multiple drug resistance- and autophagy-related protein expression levels, which were all reversed by the inhibition of miR-125b. In addition, xenografts of human tumors in nude mice were suppressed by miR-125b, demonstrating that through autophagy regulation, miR-125b could reverse the DDP resistance in LUAD cells, both in vitro and in vivo. Further mechanistic studies indicated that miR-125b directly repressed the expression levels of RORA and its downstream BNIP3L, which in turn inhibited autophagy and reversed chemoresistance. Based on these findings, miR-125b in combination with DDP might be an effective treatment option to overcome DDP resistance in LUAD.

The Epstein-Barr virus-miRNA-BART6-5p regulates TGF-ß/SMAD4 pathway to induce glycolysis and enhance proliferation and metastasis of gastric cancer cells.

Background: EBV-miR-BARTs exhibit significant relevance in epithelial tumors, particularly in EBV-associated gastric and nasopharyngeal cancers. However, their specific mechanisms in the initiation and progression of gastric cancer remain insufficiently explored. Material and Methods: Initially, EBV-miRNA-BART6-5p and its target gene SMAD4 expression were assessed in EBV-associated gastric cancer tissues and cell lines. Subsequent transfection induced overexpression of EBV-miRNA-BART6-5p in AGS and MKN-45, and downregulation in EBV-positive cells (SUN-719). The subsequent evaluation aimed to observe their impact on gastric cancer cell proliferation, migration, and glycolytic processes, with the TGF-ß/SMAD4 signaling pathway value clarified using a TGF-ß inhibitor. Results: EBV-miRNA-BART6-5p exhibits pronounced upregulation in EBV-associated gastric cancer tissues and EBV-positive cells, while its target gene SMAD4 demonstrates downregulated expression. Upregulation of it can promote the proliferation and migration of gastric cancer cells. Additionally, We found EBV-miRNA-BART6-5p promotes glycolysis of gastric cancer cells. Inhibition of the TGF-ß/SMAD4 signaling pathway resulted in suppressed proliferation and migration of gastric cancer cells, concomitant with a diminished glycolytic capacity. Conclusion: In this study, we found that EBV-miRNA-BART6-5p can target SMAD4, effectively increasing glycolysis in gastric cancer cells by regulating the TGF-ß/SMAD4 signaling pathway, thereby enhancing the proliferation and metastasis of gastric cancer cells. Our findings may offer new insights into the metabolic aspects of gastric cancer.

Glutamate-cysteine ligase catalytic and its modifier function as novel immunotargets in gastric adenocarcinoma.

OBJECTIVES: To determine the expression and function of glutamate-cysteine ligase catalytic (GCLC) and glutamate-cysteine ligase catalytic modifier (GCLM) in gastric adenocarcinoma. METHODS: Bioinformatics was used to analyze the expression of GCLC and GCLM. We download and analyzed the expression of gastric adenocarcinoma patients from TCGA database. Moreover, the method of immunochemistry was used to verify the expression of GCLC and GCLM in gastric adenocarcinoma. RESULTS: At first, the expression of GCLC and GCLM in gastric adenocarcinoma tissues were both significantly higher compared with normal tissues analyzed via TCGA database. Then, gastric adenocarcinoma tissues were collected and performed with immunochemistry. The gastric adenocarcinoma with positive staining for GCLC and GCLM was 77% and 80%, respectively, which was significantly higher compared with adjacent normal tissues (9% and 11%, respectively). CONCLUSIONS: The disordered expression of GCLC and GCLM in gastric adenocarcinoma suggested that these factors may induce tumorigenesis and may be a novel target for diagnosis and treatment of gastric adenocarcinoma.

Tumor suppressor genes are reactivated by miR-26A1 via enhancer reprogramming in NSCLC.

Non-small cell lung cancer (NSCLC) is one of the most malignant epithelial tumors. Studies have suggested that DNA hypermethylation of promoters and abnormal histone modifications could induce tumor suppressor genes (TSGs) downregulation in NSCLC. However, the exact mechanism of TSGs downregulation remains unclear. In this study, we found that there is no difference in the regions of most TSGs promoters in NSCLC. Moreover, we found that there is no DNA methylation difference in the region of VILL promoter in NSCLC compared with adjacent tissue samples by pyrosequencing. We further demonstrated that VILL was markedly reactivated in A549 and H1703 cells infected with miR-26A1 lentivirus while this activation was inhibited by JQ1, an enhancer inhibitor. In addition, we identified that miR-26A1 could function as a tumor suppressor to inhibit proliferation and metastasis of NSCLC cells. Chromatin immunoprecipitation assays revealed that overexpression of miR-26A1 could significantly induce the enrichment of H3K27ac at the enhancer regions in A549 cells. To sum up, our findings revealed that enhancer-mediated TSGs regulation occured in NSCLC, suggesting that miR-26A1 could serve as a key regulator and may be a potential therapeutic target for NSCLC.

VALD-3 inhibits proliferation and induces apoptosis of colorectal cancer cells via upregulating tumor suppressor activity of p53 to inhibit Wnt/ß-catenin signal pathway.

Colorectal cancer is the third most common malignant tumor and a leading cause of cancer death. Currently lacks effective therapies available to improve the prognosis. In the present study, VALD-3, an important Schiff base ligand from o-vanillin derivatives was evaluated for its anti-cancer activity in vitro and in vivo against colorectal cancer. The effect of VALD-3 on colorectal cancer cells proliferation was assessed using MTT assay and the cell migration was evaluated using wound healing scratch assay. The appearance of apoptotic colorectal cancer cells was detected by flowcytometry analysis. Morphological changes caused by VALD-3 induced apoptosis were also observed by Hoechst 33258 staining. The flow cytometry assay was also used to measure cell cycle arrest. The expression levels of TP53 and Bad were analyzed using quantitative real-time PCR. Protein expression of P53, Wnt/ß-catenin signaling pathway proteins, apoptosis proteins and cell cycle-related protein were viewed by Western blotting. In addition, HT-29 cells xenograft tumor model was used for the study in vivo. Immunohistochemistry (IHC) staining was employed to detect the P53 protein expression. The results showed that VALD-3 obviously inhibited the proliferation and migration for colorectal cancer cells. In addition, flow cytometry analysis demonstrated that VALD-3 markedly increased early and late apoptosis on colorectal cancer cells, respectively. VALD-3 induced cell cycle arrest at the G0/G1 phase. Most importantly, tumor growth in HT-29 xenograft mice was suppressed by VALD-3, but no significant change in body weight. As confirmed by IHC staining from tumor tissue, the P53 proteins expression increased. These results suggested that VALD-3 represses cell proliferation and induces apoptosis associated with upregulating tumor suppressor activity of p53 to inhibit Wnt/ß-catenin signal pathway, and it is a potential anticancer agent for colorectal cancer.

VALD-3, a Schiff base ligand synthesized from o-vanillin derivatives, induces cell cycle arrest and apoptosis in breast cancer cells by inhibiting the Wnt/ß-catenin pathway.

Schiff base compounds and their metal complexes have become important synthetic organic drugs due to their extensive biological activities, which include anticancer, antibacterial and antiviral effects. In this study, we investigated the cytotoxic and apoptotic effects of VALD-3, a Schiff base ligand synthesized from o-vanillin derivatives, on human breast cancer cells and the possible underlying mechanisms. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-test was used to observe the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells induced by VALD-3. Flow cytometry analysis showed that VALD-3 triggered cell cycle arrest and induced apoptosis of breast cancer cells. Western blot analysis revealed that VALD-3 upregulated pro-apoptotic proteins (Bad and Bax), downregulated anti-apoptotic proteins (Bcl-2, Bcl-xl, survivin and XIAP) and increased the expression of cleaved caspase-3, cleaved caspase-8, Cyto-c and cleaved PARP. VALD-3 also regulated the Wnt/ß-catenin signaling pathway in breast cancer cells, inhibiting the activation of downstream molecules. By xenografting human breast cancer cells into nude mice, we found that VALD-3 significantly suppressed tumor cell growth while showing low toxicity against major organs. In addition, survival analysis showed that VALD-3 can significantly prolong the survival time of mice (P = 0.036). This study is the first to show that VALD-3 induces apoptosis and cell cycle arrest in human breast cancer cells by suppressing Wnt/ß-catenin signaling, indicating that it could be a potential drug for the treatment of breast cancer.

Next-generation sequencing-based identification of EGFR and NOTCH2 complementary mutations in non-small cell lung cancer.

Although targeted therapy has emerged as an effective treatment strategy for non-small cell lung cancer (NSCLC), some patients cannot benefit from such therapy due to the limited number of therapeutic targets. The present study aimed to identify mutated genes associated with clinicopathological characteristics and prognosis and to screen for mutations that are not concurrent with applicable drug target sites in patients with NSCLC. Tumor tissue and blood samples were obtained from 97 patients with NSCLC. A lung cancer-specific panel of 55 genes was established and analyzed using next-generation sequencing (NGS). The results obtained from the clinical cohort were compared with the NSCLC dataset from The Cancer Genome Atlas (TCGA). Subsequently, 25 driver genes were identified by taking the intersection of the 55 lung-cancer-specific genes with three databases, namely, the Catalog of Somatic Mutations in Cancer database, the Network of Cancer Genes database and Vogelstein's list. Functional annotation and protein-protein interaction analysis were conducted on these 25 driver genes. The χ2 test and logistic regression were used to evaluate the association between mutations in the 25 driver genes and the clinicopathological characteristics of 97 patients, and phosphatase and tensin homolog (PTEN) and kirsten rat sarcoma viral oncogene homolog (KRAS) were associated with stage at diagnosis and sex, respectively, while epidermal growth factor receptor (EGFR) was associated with sex, stage at diagnosis, metastasis, CEA and CYFRA21-1. Moreover, the association between the 25 driver gene mutations and overall survival were examined using Cox regression analysis. Age and Notch homolog 2 (NOTCH2) mutations were independent prognostic factors in TCGA dataset. The correlations between statistically significant mutations in EGFR, KRAS, PTEN and NOTCH2 were further examined, both in the clinical data and TCGA dataset. There was a negative correlation between EGFR and NOTCH2 mutations (correlation coefficient, -0.078; P=0.027). Thus, the present study highlights the importance of NOTCH2 mutations and might provide novel therapeutic options for patients with NSCLC who do not harbor EGFR mutations.

Deep-sequencing transcriptome analysis of chilling tolerance mechanisms of a subnival alpine plant, Chorispora bungeana.

BACKGROUND: The plant tolerance mechanisms to low temperature have been studied extensively in the model plant Arabidopsis at the transcriptional level. However, few studies were carried out in plants with strong inherited cold tolerance. Chorispora bungeana is a subnival alpine plant possessing strong cold tolerance mechanisms. To get a deeper insight into its cold tolerance mechanisms, the transcriptome profiles of chilling-treated C. bungeana seedlings were analyzed by Illumina deep-sequencing and compared with Arabidopsis. RESULTS: Two cDNA libraries constructed from mRNAs of control and chilling-treated seedlings were sequenced by Illumina technology. A total of 54,870 unigenes were obtained by de novo assembly, and 3,484 chilling up-regulated and 4,571 down-regulated unigenes were identified. The expressions of 18 out of top 20 up-regulated unigenes were confirmed by qPCR analysis. Functional network analysis of the up-regulated genes revealed some common biological processes, including cold responses, and molecular functions in C. bungeana and Arabidopsis responding to chilling. Karrikins were found as new plant growth regulators involved in chilling responses of C. bungeana and Arabidopsis. However, genes involved in cold acclimation were enriched in chilling up-regulated genes in Arabidopsis but not in C. bungeana. In addition, although transcription activations were stimulated in both C. bungeana and Arabidopsis, no CBF putative ortholog was up-regulated in C. bungeana while CBF2 and CBF3 were chilling up-regulated in Arabidopsis. On the other hand, up-regulated genes related to protein phosphorylation and auto-ubiquitination processes were over-represented in C. bungeana but not in Arabidopsis. CONCLUSIONS: We conducted the first deep-sequencing transcriptome profiling and chilling stress regulatory network analysis of C. bungeana, a subnival alpine plant with inherited cold tolerance. Comparative transcriptome analysis suggests that cold acclimation is not a major chilling tolerance mechanism of C. bungeana. Activation of protein phosphorylation and ubiquitination may confer chilling tolerance to C. bungeana in a more rapid and flexible way than cold acclimation. Such differences may have contributed to the differences in cold tolerance between C. bungeana and Arabidopsis. The results presented in this paper will be informative for gene discovery and the molecular mechanisms related to plant cold tolerance.