CircRNA_100565 contributes to cisplatin resistance of NSCLC cells by regulating proliferation, apoptosis and autophagy via miR-337-3p/ADAM28 axis.

Circular RNAs (circRNAs) have been revealed to involve in the chemoresistance of various cancers, including non-small cell lung cancer (NSCLC). Here, we further investigate the role of circRNA_100565 in NSCLC cisplatin (DDP) resistance. The expression of circRNA_100565 and microRNA (miR)-337-3p, and ADAM metallopeptidase domain 28 (ADAM28) mRNA was detected using quantitative real-time polymerase chain reaction. Cell viability and apoptosis were measured by cell counting kit-8 assay and flow cytometry, respectively. Western blot was used to detect the level of ADAM28 and autophagy-related protein. The interaction between miR-337-3p and circRNA_100565 or ADAM28 was confirmed by dual-luciferase reporter assay or pull-down assay. In vivo experiments were conducted via the murine xenograft model. We found CircRNA_100565 was up-regulated in NSCLC DDP-resistant tissues and cell lines, and its high expression was associated with shorter overall survival of NSCLC patients. CircRNA_100565 deletion mitigated DDP resistance, reflected by the suppression of proliferation and autophagy, the reduction of IC50 value, as well as enhancement of apoptosis in DDP-resistant NSCLC cells. MiR-377-3p was confirmed to directly bind to circRNA_100565 or ADAM28 3'-UTR. Moreover, circRNA_100565 indirectly regulated ADAM28 expression by sponging miR-377-3p in NSCLC cells. Additionally, circRNA_100565 deletion-induced sensitivity of NSCLC resistant cells to DDP could be remarkably attenuated by miR-377-3p inhibition or ADAM28 re-expression. Meanwhile, circRNA_100565 knockdown contributed to the anti-tumor effects of DDP on NSCLC in vivo.CONCLUSION: CircRNA_100565 was an independent prognostic factor for NSCLC patient survival, and enhanced the resistance of NSCLC cells to cisplatin by regulating cell proliferation, apoptosis and autophagy via miR-337-3p/ADAM28 axis, shedding light on the development of a novel therapeutic strategy to boost the effectiveness of NSCLC chemotherapy.

Downregulation of long non­coding RNA GACAT1 suppresses proliferation and induces apoptosis of NSCLC cells by sponging microRNA­422a.

Increasing evidence has demonstrated the important roles of long non­coding (lnc) RNA in non­small cell lung cancer (NSCLC). lncRNA gastric cancer­associated transcript 1 (GACAT1) has been reported to play an oncogenic role in different types of cancer; however, the function of GACAT1 in NSCLC remains unclear. The present study found that GACAT1 was overexpressed in NSCLC tissues and was associated with poor outcomes in patients with NSCLC. Functional experiments revealed that GACAT1 downregulation inhibited proliferation, induced apoptosis and cell cycle arrest of 2 NSCLC cell lines. GACAT1 was found to target microRNA(miR)­422a mechanically and negatively regulated miR­422a expression. Reduced expression of miR­422a in NSCLC tissues was inversely correlated with that of GACAT1. Furthermore, YY1 transcription factor (YY1) was identified as a downstream miR­422a target. Reduced expression of GACAT1 inactivated YY1 by sponging miR­422a in NSCLC cells. YY1 reintroduction reversed the reduced proliferation of NSCLC cells via GACAT1 knockdown. Taken together, these results revealed the novel role of the GACAT1/miR­422a pathway in the progression of NSCLC cell lines, providing a possible therapeutic strategy for NSCLC treatment.

[Effect of phosphorylation of cortactin at different sites on secretion by airway mucus 5AC].

OBJECTIVE: To explore the role of cortical actin-binding protein (cortactin) in shear stress-induced mucin (MUC) 5AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites.
 Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin (Cort), pEGFP-N1-Cort-Y421A, pEGFP-N1-Cort-Y470A and pEGFP-N1-Cort-Y486A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFP-N1-Cort-Y421A group, a shear stress + pEGFP-N1-Cort-Y470A group, and a shear stress + pEGFP-N1-Cort-Y486A group. The shear stress were set at 4 dynes/cm2. The levels of MUC5AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay (ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate (FITC)-phalloidin.
 Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5AC protein production and mRNA expression in the different experiment groups (all P<0.05). Compared with single shear stress-stimulated group, MUC5AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group (both P<0.05), while there were no changes in the MUC5AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421A group and the shear stress + pEGFP-N1-Cort-Y470A group (both P<0.05), while there was no significant effect in the shear stress + pEGFP-N1-Cort-Y486A group (P>0.05).
 Conclusion: Cortactin is involved in shear stress-mediated MUC5AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.

Polymorphisms in VEGF-A are associated with COPD risk in the Chinese population from Hainan province.

In this study, we examined and validated how common variants contribute to susceptibility to chronic obstructive pulmonary disease (COPD) in the Han Chinese population. Here, we genotyped 18 nucleotide polymorphisms and evaluated their association with COPD using chi-square test and genetic model analysis (246 COPD patients and 350 controls), and found three SNPs that might cause a predisposition to COPD. Both rs3025030 and rs3025033 are located on chromosome 6 in VEGF-A. We found one risk allele 'C' from rs3025030 and another 'G' from rs3025033 using the log-additive model (OR 1.40; 95% CI 1.05-5.96; P = 0.022), (OR 1.38; 95% CI 1.03-1.84; P = 0.03). We also found another risk allele 'A' of rs9296092 in gene region ZBTB9-BAK1 by the allele model (OR 2.63; 95% CI 1.27-5.45; P = 0.0078), (adjusted OR 3.53; 95% CI 1.12-11.11; P = 0.031).We found a risk haplotype 'CG' associated with the risk of COPD (OR 1.39; 95% CI 1.04-1.86; P = 0.028). Our results when compared with previous studies showed significant association between VEGF-A polymorphism and COPD. We also identified rs9296092 as a risk factor for COPD.

[A novel mutation of the SLC34A2 gene in a Chinese pedigree with pulmonary alveolar microlithiasis].

OBJECTIVE: To identify the mutation of solute carrier family 34 member 2 (SLC34A2) gene in a Chinese family with pulmonary alveolar microlithiasis (PAM). METHODS: Genomic DNA was extracted from the family members. DNA sequencing was carried out to confirm the mutation detected by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP). The fragments with variation were screened in 100 healthy controls by PCR-SSCP. RESULTS: In both patients of the family, a homozygous mutation of the SLC34A2 gene was identified in exon 8 (c.A910T), resulting in a premature stop codon. In addition, a homozygous single nucleotide polymorphism (SNP) was found in intron 2 in both patients and the daughter of proband. CONCLUSION: A novel homozygous mutation in SLC34A2 gene, leading to a premature stop codon therefore a truncated protein, was probably responsible for the PAM in this family. The SNP in intron 2 needs further study.