To investigate the effect and molecular mechanism of long non-coding RNA EBLN3P (LncRNA EBLN3P) on the radiosensitivity and mitochondrial damage of non-small cell lung cancer (NSCLC) cells by targeting microRNA (miR)-144-3p to regulate the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-response element binding protein (CREB) signaling pathway.

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of LncRNA EBLN3P and miR-144-3p in NSCLC cell lines (A549, H460, H1299, Calu-1) and normal lung epithelial cells BEAS-2B. A549 cells were selected, and a LncRNA EBLN3P overexpression vector was constructed. The cells were divided into the Control group (blank control), IR group (X-ray irradiation alone), IR+ oe-EBLN3P group (irradiation after LncRNA EBLN3P overexpression), and IR+ oe-EBLN3P+ miR-144-3p mimic group (irradiation after co-transfection with the LncRNA EBLN3P overexpression vector and miR-144-3p mimic). Techniques including the cell counting kit-8 (CCK-8) assay, Transwell assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, JC-1 staining, Western blot, and enzyme-linked immunosorbent assay (ELISA) were used to detect cell viability, invasion, apoptosis, mitochondrial membrane potential, DNA damage-related proteins [ataxia telangiectasia mutated (ATM), Nijmegen breakage syndrome protein 1 (NBS1), phosphorylated H2AX (γ-H2AX)], and the expression levels of key molecules in the cAMP/PKA/CREB pathway in each group. A dual-luciferase reporter gene assay was used to verify the targeting relationship between LncRNA EBLN3P and miR-144-3p.

Compared with the Control group, in the IR group, A549 cell viability [(100.00±2.47)% vs. (57.92±8.65)%] and invasion number [(93.86±15.02) vs. (25.31±4.19)] were decreased, apoptosis rate was increased [(3.16±0.54)% vs. (15.43±2.79)%], mitochondrial membrane potential [red/green fluorescence ratio: (1.00±0.16) vs. (0.27±0.05)] and cAMP/PKA/CREB pathway expression [cAMP: (18.25±2.67) mmol/L vs. (11.93±1.86) mmol/L; p-PKA/PKA: (1.00±0.18) vs. (0.17±0.03); p-CREB/CREB: (1.00±0.15) vs. (0.34±0.06)] were decreased (all P<0.001), and DNA damage was aggravated [ATM: (1.00±0.15) vs. (0.57±0.09); NBS1: (1.00±0.13) vs. (0.41±0.07); γ-H2AX: (1.00±0.16) vs. (2.74±0.49)]. Compared with the IR group, in the IR+ oe-EBLN3P group, cell viability [(57.92±8.65)% vs. (72.15±10.84)%], invasion [(25.31±4.19) vs. (78.63±12.76)], mitochondrial membrane potential [red/green fluorescence ratio: (0.27±0.05) vs. (0.79±0.14)], cAMP/PKA/CREB pathway activity [cAMP: (11.93±1.86) mmol/L vs. (16.47±2.58) mmol/L; p-PKA/PKA: (0.17±0.03) vs. (0.95±0.17); p-CREB/CREB: (0.34±0.06) vs. (0.71±0.13)] and ATM/NBS1 protein expression [ATM: (0.57±0.09) vs. (0.82±0.14); NBS1: (0.41±0.07) vs. (0.88±0.11)] were increased, while apoptosis [(15.43 ± 2.79)% vs. (6.27±1.08)%] and γ-H2AX levels [(2.74±0.49) vs. (1.69±0.28)] were decreased (P<0.01). The dual-luciferase reporter gene assay confirmed that LncRNA EBLN3P can target and bind miR-144-3p, and overexpression of LncRNA EBLN3P inhibited miR-144-3p expression. Compared with the IR+ oe-EBLN3P group, overexpression of miR-144-3p reversed the protective effect of LncRNA EBLN3P, manifested as increased apoptosis, decreased mitochondrial membrane potential, and inhibited cAMP/PKA/CREB pathway activity.

LncRNA EBLN3P is highly expressed in NSCLC and may reduce the radiosensitivity of tumor cells by competitively binding to miR-144-3p and affecting the activity of the cAMP/PKA/CREB signaling pathway; the mechanism is related to alleviating mitochondrial damage.