B, miR\26b\5p expression in SPC\A1, HCC827, NCI\H1395 and A549 LUAD cell lines determined by RT\qPCR
B, miR\26b\5p expression in SPC\A1, HCC827, NCI\H1395 and A549 LUAD cell lines determined by RT\qPCR. assay, followed by co\transfection with radiation\resistant A549R cells. LUAD tissues and serum were collected, followed by miR\26b\5p relative expression quantification using RT\qPCR. miR\26b\5p was identified as the most differentially expressed miRNA and was down\regulated in LUAD. Radiation\resistant cells were more resistant to X\radiation compared with parent cells. miR\26b\5p WIN 55,212-2 mesylate overexpression and X\irradiation led to enhanced radiosensitivity of LUAD cells. ATF2 was negatively targeted by miR\26b\5p. Exosomal miR\26b\5p derived from A549 cells could be transported to irradiation\resistant LUAD cells and inhibit ATF2 expression to promote DNA damage, apoptosis and radiosensitivity of LUAD cells, which was verified using serum\based miR\26b\5p. Our results WIN 55,212-2 mesylate show a regulatory network of miR\26b\5p on radiosensitivity of LUAD cells, which may serve as a non\invasive biomarker for LUAD. for 10?minutes; 2000?for 15?minutes; 12?000?for 30?minutes) to discard floating cells and cell debris, followed by filtering using 0.22\m filter. Supernatants were ultracentrifuged for 2?hours at 4C (1??106?(L?=?length; W?=?width). 2.13. Statistical analysis All data were processed and analysed using SPSS 21.0 statistical software (IBM Corp., Armonk). Measurement data were expressed as mean??standard deviation. Paired/unpaired test was used to analyse differences between normally distributed values of two experimental groups. Differences among normally distributed values of three or more experimental groups were analysed by one\way analysis of variance (ANOVA), followed by a Tukey’s post hoc test. Comparisons between time\based measurements within each group were performed using ANOVA of repeated measurements, followed by Bonferroni’s post\test. Pearson’s correlation analysis was adopted to analyse the correlation between two indicators. The criterion for statistical significance was set at test was used to analyse differences between two groups. ANOVA of repeated measurements was used in panel A, followed by Bonferroni’s post\test. Experiments were repeated in triplicates Western blot assay (Figure?1B) was performed to determine expression of Cleaved\PARP, Cleaved\Caspase 3 and H2AX in parent cells and irradiation\resistant cells following irradiation. The data demonstrated that Cleaved\PARP, Cleaved\Caspase 3 and H2AX expression increased over time during the irradiation treatment. In addition, significantly lower expression WIN 55,212-2 mesylate of Cleaved\PARP, Cleaved\Caspase 3 and H2AX was observed in irradiation\resistant cells compared to their parent cells. Thus, irradiation\resistant cells exhibit reduced Caspase\3 and RARP protease activity in the DNA damage signalling in vitro. To better elucidate the function of miRNAs in radiation sensitivity, miR\21\5p, miR\206, miR\191\5p and miR\26\5p were selected as potential miRNAs that might affect the progression of non\small cell lung cancer based on a previous study. 11 Expression of these miRNAs was determined by RT\qPCR in A549 and radiation\resistant A549 (A549R) cells (Figure?1C). miR\26b\5p was identified as the most differentially expressed miRNA in A549R cells. The function of miRNA in cell apoptosis was further tested by transfecting miRNAs into A549 cells, followed by exposure to 6.0?Gy X\radiation. In Figure?1D, the results showed that overexpression of miRNAs led to enhanced Caspase\3 and RARP protease activity in response to DNA damage and overexpression of miR\26b\5p contributed to the greatest up\regulation of Cleaved\PARP, Cleaved\Caspase 3 and H2AX, suggesting overexpression of miR\26b\5p can induce cell apoptosis via these genes, and therefore, miR\26b\5p was used for the subsequent experiment. WIN 55,212-2 mesylate 3.2. miR\26b\5p overexpression restored radiosensitivity of A549 cells Until now, the modulatory roles of miR\26b\5p on LUAD cells to radiosensitivity are not clear. To address this, we measured miR\26b\5p expression in LUAD tissues and cells. Down\regulation of miR\26b\5p was found both in LUAD tissues and LUAD cell lines compared to cancer tissues and HBE, respectively (Figure?2A,B). Next, we overexpressed miR\26b\5p in Tmem34 A549 cells and performed miR\26b\5p knockdown in HCC827 cells to further investigate the relationship between radiosensitivity and miR\26b\5p (Figure?2C\E). The results indicated that miR\26b\5p overexpression restored radiosensitivity of A549 cells, and knockdown of miR\26b\5p resulted in radioresistance..