Detection and treatment of lung cancer still remain a clinical challenge. This study aims to validate exosomal microRNA‐96 (miR‐96) as a serum biomarker for lung cancer and understand the underlying mechanism in lung cancer progression. MiR‐96 expressions in normal and lung cancer patients were characterized by qPCR analysis. Changes in cell viability, migration and cisplatin resistance were monitored after incubation with isolated miR‐96‐containing exosomes, anti‐miR‐96 and anti‐miR negative control (anti‐miR‐NC) transfections. Dual‐luciferase reporter assay was used to study interaction between miR‐96 and LIM‐domain only protein 7 (LMO7). Changes induced by miR‐96 transfection and LMO7 overexpression were also evaluated. MiR‐96 expression was positively correlated with high‐grade and metastatic lung cancers. While anti‐miR‐96 transfection exhibited a tumour‐suppressing function, exosomes isolated from H1299 enhanced cell viability, migration and cisplatin resistance. Potential miR‐96 binding sites were found within the 3′‐UTR of wild‐type LMO7 gene, but not of mutant LMO7 gene. LMO7 expression was inversely correlated with lung cancer grades, and LMO7 overexpression reversed promoting effect of miR‐96. We have identified exosomal miR‐96 as a serum biomarker of malignant lung cancer. MiR‐96 promotes lung cancer progression by targeting LMO7. The miR‐96‐LMO7 axis may be a therapeutic target for lung cancer patients, and new diagnostic or therapeutic strategies could be developed by targeting the miR‐96‐LMO7 axis.
Curcumin is a novel drug for lung cancer treatment. However, the mechanism underlying the anti-tumor effect of curcumin remains elusive. Previous evidences indicated that, the methylating transferase DNMT1 is downregulated by curcumin, and the transcription factor 21 (TCF21) is suppressed by DNMT1. We hereby attempt to elucidate the correlation between curcumin treatment and TCF21 expression. Exosomes derived from curcumin-pretreated H1299 cells were used to treat BEAS-2B cells, which induced proliferation, colony formation and migration of BEAS-2B cells. An increase in TCF21 expression in response to curcumin was also seen, as revealed by real-time PCR (RT-PCR) and western blot. Analysis using the GEO database (access #GSE21210) indicated that a positive correlation existed between TCF21 levels and lung cancer patient survival. TCF21 overexpression and knockdown was introduced to H1299 cells through lentiviral system, which led to suppression and promotion of tumor growth, respectively. We also demonstrated that DNMT1 expression was downregulated by curcumin. Therefore, curcumin exerts its anti-cancer function by downregulating DNMT1, thereby upregulating TCF21.
Transcription factor 21 (TCF21) has been identified as a candidate tumor suppressor at 6q23-q24 that is epigenetically inactivated in many types of human cancers. We recently found that TCF21 methylation level was significantly increased in clear cell renal cell carcinoma (ccRCC). The purpose of this study was to investigate the prognostic impact of TCF21 expression in ccRCC and analyze the relationship between TCF21 expression and methylation level. We used real-time PCR and immunohistochemical staining to detect the expression of TCF21, and used methylation specific-PCR (MS-PCR) to determine the methylation status of TCF21 in ccRCC samples and cell line 786-O. The results showed that TCF21 expression level in ccRCC samples was significantly lower than in normal adjacent tissue samples (NAT samples). The Kaplan-Meier survival analysis demonstrated that TCF21 was a significant prognosticator of cancer-specific survival (p=0.001). Furthermore, the DNA demethylating agent 5'-azacytidine restored part of TCF21 expression by suppressing TCF21 methylation in 786-O. The methylation level of TCF21 in ccRCC samples was much higher than in NAT samples. These results suggest that the expression of TCF21 was an independent prognostic factor for poor survival in patients with ccRCC. Aberrant methylation was an important reason for the down-regulation the expression of TCF21, and may be associated with tumorigenesis in ccRCC.
Lung cancer is a leading cause of cancer mortality worldwide. Promoter methylation of transcription factor 21 (TCF21) was frequently observed in the early stage of nonsmall cell lung cancer (NSCLC). However, clinical relevance and molecular functions of TCF21 in NSCLC progression remain unclear. In this study, we analyzed the associations between TCF21 expression and clinicopathological features in 100 patients with NSCLC and revealed the underlying molecular mechanisms of TCF21 methylation on cell viability, apoptosis and invasion of H1299 cells. We found that the expression of TCF21 was significantly regulated by its methylation level in patients with NSCLC and was associated with tumor stage, metastasis and invasion. Demethylation of H1299 cells by 5-aza-2′-deoxycytine (5-Aza) demonstrated that a higher level of TCF21 expression led to remarkable decreases of cell viability and invasion ability but an increase of cell apoptosis. Accordingly, TCF21 knockdown showed converse results to high expression of TCF21. TCF21 knockdown cells exhibited significantly upregulated ATG-9, BECLIN-1, and LC3-I/II expressions but decreased p62 expression compared to wildtype cells. Inhibition of autophagy by 3-methyladenine (3-MA) elevated TCF21 expression and increased cell apoptosis. TCF21 expression is clinically related to the progress of lung cancer and may inhibit autophagy by suppressing ATG-9 and BECLIN-1. In turn, autophagy may also play an important role in regulation TCF21 expression. Keywords Transcription factor 21 (TCF21). Methylation. Non-small cell lung cancer (NSCLC). Autophagy Abbreviations TCF21 transcription factor 21 NSCLC non-small cell lung cancer 5-Aza 5-aza-2′-deoxycytine 3-MA 3-methyladenine Electronic supplementary material The online version of this article (
Background Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template. Methodology/Principal Findings The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl 2 , the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition. Conclusions/Significance The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.
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