BackgroundTo investigate the roles of miR-17-5p/RRM2 in A549/G+. MethodsCell survival was analyzed by CCK8 kit and clone formation experiment; The mRNA expression level was detected by qRT-PCR, and the protein level was compared by Western blotting; And ow cytometry for cell cycle detection; The target gene of miR-17-5p was veri ed by double luciferase activity experiment. ResultsCCK8 assay displayed that miR-17-5p's overexpression can let A549/G + cells turn into sensitive ones; in turn, in the case of inhibition of expression, miR-17-5p can make A549/G-cells turn into resistance ones.And similar results were obtained in cell clone formation experiment. Cell cycle analysis showed that miR-17-5p's overexpression increased the number of G 1 phase cells and reduced the number of S phase cells in A549/G + cells; Conversely, lowing expression level of miR-17-5p yielded the opposite results in A549/G-cells. Western blot results showed that when miR-17-5p was expressed highly, the expression levels of cell cycle related proteins, CCNE1, CCNA2 and P21 decreased in A549/G + cells; Conversely, inhibition of miR-17-5p expression yielded the opposite results too in A549/G-cells. Western blot analysis of signal pathway proteins and the results showed that PTEN and PI3K expressed higher, but p-PTEN expressed lower in A549/G + cells. After miR-17-5p overexpressed in A549/G + cells, the level of p-PTEN increased, and the level of p-AKT decreased; when miR-17-5p expressed low in A549/G-cells, the expression of p-PTEN and p-AKT were opposite. The dual-luciferase experiment demonstrated that RRM2 was the target gene of miR-17-5p. The restorative experiments of RRM2 veri ed it. ConclusionThis article suggested that the miR-17-5p/RRM2 axis could adjust gemcitabine-resistance in A549 cells and might associate with p-PTEN/PI3K/AKT signal pathway.

Background To investigate the roles of miR-17-5p/RRM2 in A549/G+. Methods Cell survival was analyzed by CCK8 kit and clone formation experiment; The mRNA expression level was detected by qRT-PCR, and the protein level was compared by Western blotting; And flow cytometry for cell cycle detection; The target gene of miR-17-5p was verified by double luciferase activity experiment. Results CCK8 assay displayed that miR-17-5p’s overexpression can let A549/G + cells turn into sensitive ones; in turn, in the case of inhibition of expression, miR-17-5p can make A549/G- cells turn into resistance ones. And similar results were obtained in cell clone formation experiment. Cell cycle analysis showed that miR-17-5p’s overexpression increased the number of G1 phase cells and reduced the number of S phase cells in A549/G + cells; Conversely, lowing expression level of miR-17-5p yielded the opposite results in A549/G- cells. Western blot results showed that when miR-17-5p was expressed highly, the expression levels of cell cycle related proteins, CCNE1, CCNA2 and P21 decreased in A549/G + cells; Conversely, inhibition of miR-17-5p expression yielded the opposite results too in A549/G- cells. Western blot analysis of signal pathway proteins and the results showed that PTEN and PI3K expressed higher, but p-PTEN expressed lower in A549/G + cells. After miR-17-5p overexpressed in A549/G + cells, the level of p-PTEN increased, and the level of p-AKT decreased; when miR-17-5p expressed low in A549/G-cells, the expression of p-PTEN and p-AKT were opposite. The dual-luciferase experiment demonstrated that RRM2 was the target gene of miR-17-5p. The restorative experiments of RRM2 verified it. Conclusion This article suggested that the miR-17- 5p/RRM2 axis could adjust gemcitabine-resistance in A549 cells and might associate with p-PTEN/PI3K/AKT signal pathway.

Lung cancer is a very common cancer diagnosed and the leading cause of cancer death. Among them, the treatment options for non-small cell lung cancer cell (NSCLC) are very limited, mainly due to the frequent development of anti-cancer drug resistance. Targeted DNA repair mechanisms have reached clinical application, and new therapeutic strategies including targeting cell cycle checkpoint sites, such as ATR and CHK1, are currently under clinical development. In this study, NSCLC A549 cells and gemcitabine-resistant strain A549/G+ cells were used to explore the synergistic effect of CHK1 inhibition and gemcitabine, and to evaluate the potential of overcoming drug resistance and related molecular mechanisms. Our results showed that inhibition of CHK1 and gemcitabine combination significantly reduced the proliferation ability of the two cell lines. We also revealed the effect of full-length PARP degradation and reduced Bcl-2/Bax ratio on increased apoptosis. Inhibition of CHK1 expression leads to DNA damage, induces phosphorylation of γ-H2AX, and affects the repair of homologous recombination ability through Rad51. Mechanistically, gemcitabine increased phosphorylation-ATR (pATR) and phosphorylation-CHK1 (pCHK1), indicating activation of the DNA repair system and ATR-CHK1-CDC25A pathway. Inhibition of CHK1 resulted in increased synthesis of CDK2/Cyclin A2 and CDK2/Cyclin E1 complexes, and more cells entered the subsequent cell cycle, leading to S phase arrest and mitotic catastrophe. In conclusion, in our study, we identified inhibition of CHK1 as a potential treatment for NSCLC and confirmed that inhibition of this kinase can overcome acquired gemcitabine resistance.

The purpose of the study is mainly to investigate the role of CHK1 in gemcitabine-resistant lung cancer cell line A549. The mRNA and protein levels of genes were analyzed by RT-qPCR and Western blot, respectively. Cell viability was detected by CCK-8 assay and clone formation assay. The detection of the cell cycle was used by Annexin V-PE/7-AAD apoptosis detection kit. Analysis of DNA damage by immunofluorescence and alkaline comet assay. The results showed that inhibition of CHK1 and gemcitabine combination significantly reduced the proliferation ability of the two cell lines. We also revealed the degradation of full-length PARP and reduced Bcl-2/Bax ratio on increased apoptosis. Inhibition of CHK1 expression leads to DNA damage, induces phosphorylation of γ-H2AX, and affects the repair of homologous recombination ability through Rad51. Mechanistically, gemcitabine increased phosphorylation-ATR and phosphorylation-CHK1, indicating activation of the DNA repair system and ATR-CHK1-CDC25A pathway. Inhibition of CHK1 resulted in increased synthesis of CDK2/Cyclin A2 and CDK2/Cyclin E1 complexes, and more cells entered the subsequent cell cycle, leading to S phase arrest and mitotic catastrophe. We identified inhibition of CHK1 as a potential treatment for NSCLC and confirmed that inhibition of this kinase could overcome acquired gemcitabine resistance.