LncRNA EBLN3P/miR-144-3p轴通过调控cAMP/PKA/CREB通路影响非小细胞肺癌放疗敏感性和线粒体损伤的机制研究

doi:10.3877/cma.j.issn.1674-6902.2026.05.005

目的

探讨长链非编码RNA EBLN3P(long non-coding RNA EBLN3P, LncRNA EBLN3P)通过靶向微小RNA(microRNA, miR)-144-3p调控环磷酸腺苷(cyclic adenosine monophosphate, cAMP)/蛋白激酶A(protein kinase A, PKA)/cAMP反应元件结合蛋白(cAMP-response element binding protein, CREB)信号通路对非小细胞肺癌(non-small cell lung cancer, NSCLC)细胞放疗敏感性及线粒体损伤的影响及分子机制。

方法

采用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)检测NSCLC细胞系(A549，H460，H1299，Calu-1)及正常肺上皮细胞BEAS-2B中LncRNA EBLN3P与miR-144-3p表达水平。选取A549细胞，构建LncRNA EBLN3P过表达载体，分组为Control组(空白对照)、IR组(单纯X射线照射)、IR＋oe-EBLN3P组(过表达LncRNA EBLN3P后照射)及IR＋oe-EBLN3P＋miR-144-3p mimic组(共转染LncRNA EBLN3P过表达载体与miR-144-3p模拟物后照射)。通过细胞计数试剂盒-8(cell counting kit-8, CCK-8)法、Transwell实验、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL)法、JC-1染色、蛋白质印迹(Western blot)及酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)等技术，检测各组细胞活力、侵袭、凋亡、线粒体膜电位、DNA损伤相关蛋白[共济失调毛细血管扩张突变基因(ataxia telangiectasia mutated, ATM)、尼曼-匹克病C型蛋白样1(nijmegen breakage syndrome protein 1, NBS1)、磷酸化组蛋白H2AX(phosphorylated H2AX, γ-H2AX)]及cAMP/PKA/CREB通路关键分子表达水平。采用双荧光素酶报告基因实验验证LncRNA EBLN3P与miR-144-3p靶向关系。

结果

与Control组相比，IR组A549细胞中细胞活力[(100.00±2.47)%比(57.92±8.65)%]与侵袭数目[(93.86±15.02)比(25.31±4.19)]降低，细胞凋亡率升高[(3.16±0.54)%比(15.43±2.79)%]，线粒体膜电位[红/绿荧光比值：(1.00±0.16)比(0.27±0.05)]及cAMP/PKA/CREB通路表达[cAMP：(18.25±2.67)mmol/L vs(11.93±1.86)mmol/L；p-PKA/PKA：(1.00±0.18)比(0.17±0.03)；p-CREB/CREB：(1.00±0.15)比(0.34±0.06)]下降(均P<0.001)，DNA损伤加剧[ATM：(1.00±0.15)比(0.57±0.09)；NBS1：(1.00±0.13)比(0.41±0.07)；γ-H2AX：(1.00±0.16)比(2.74±0.49)]。与IR组比较，IR＋oe-EBLN3P组细胞活力[(57.92±8.65)%比(72.15±10.84)%]、侵袭[(25.31±4.19)比(78.63±12.76)]、线粒体膜电位[红/绿荧光比值：(0.27±0.05)比(0.79±0.14)]、cAMP/PKA/CREB通路活性[cAMP：(11.93±1.86)mmol/L比(16.47±2.58)mmol/L；p-PKA/PKA：(0.17±0.03)比(0.95±0.17)；p-CREB/CREB：(0.34±0.06)比(0.71±0.13)]及ATM/NBS1蛋白表达[ATM：(0.57±0.09)比(0.82±0.14)；NBS1：(0.41±0.07)比(0.88±0.11)]回升，细胞凋亡[(15.43±2.79)%比(6.27±1.08)%]及γ-H2AX水平[(2.74±0.49)比(1.69±0.28)]降低(P<0.01)。双荧光素酶报告基因实验证实LncRNA EBLN3P可靶向结合miR-144-3p，过表达LncRNA EBLN3P可抑制miR-144-3p表达。与IR＋oe-EBLN3P组比较，过表达miR-144-3p可逆转LncRNA EBLN3P保护作用，表现为细胞凋亡增加、线粒体膜电位降低及cAMP/PKA/CREB通路活性被抑制。

结论

LncRNA EBLN3P在NSCLC中高表达，可通过竞争性结合miR-144-3p影响cAMP/PKA/CREB信号通路活性，降低肿瘤细胞放疗敏感性，机制与减轻线粒体损伤有关。

关键词: 非小细胞肺癌, 长链非编码RNA EBLN3P, miR-144-3p, cAMP/PKA/CREB通路, 放疗敏感, 线粒体损伤

Objective

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.

Methods

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.

Results

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.

Conclusion

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.

Key words: Non-small cell lung cancer, Long non-coding RNA EBLN3P, miR-144-3p, cAMP/PKA/CREB pathway, Radiosensitivity, Mitochondrial damage

表1 qRT-PCR和FISH检测NSCLC细胞LncRNA EBLN3P和miR-144-3p水平比较(

±s)

组　别	LncRNA EBLN3P	miR-144-3p	LncRNA EBLN3P (FISH)
BEAS-2B	1.00±0.14	1.00±0.11	1.00±0.16
A549	4.68±0.71^b	0.35±0.06^b	3.85±0.63^b
H460	2.96±0.43^b	0.81±0.12^a	2.49±0.37^b
H1299	3.25±0.59^b	0.53±0.08^b	2.24±0.36^b
Calu-1	1.73±0.28^b	0.68±0.10^b	1.57±0.24^b

表1 qRT-PCR和FISH检测NSCLC细胞LncRNA EBLN3P和miR-144-3p水平比较(

±s)

组　别	LncRNA EBLN3P	miR-144-3p	LncRNA EBLN3P (FISH)
BEAS-2B	1.00±0.14	1.00±0.11	1.00±0.16
A549	4.68±0.71^b	0.35±0.06^b	3.85±0.63^b
H460	2.96±0.43^b	0.81±0.12^a	2.49±0.37^b
H1299	3.25±0.59^b	0.53±0.08^b	2.24±0.36^b
Calu-1	1.73±0.28^b	0.68±0.10^b	1.57±0.24^b

表2 A549细胞中DNA损伤相关蛋白表达比较(

±s)

组　别	ATM	NBS1	γ-H2AX
Control组	1.00±0.15	1.00±0.13	1.00±0.16
IR组	0.57±0.09^a	0.41±0.07^a	2.74±0.49^a
IR＋oe-EBLN3P组	0.82±0.14^c	0.88±0.11^c	1.69±0.28^c

表2 A549细胞中DNA损伤相关蛋白表达比较(

±s)

组　别	ATM	NBS1	γ-H2AX
Control组	1.00±0.15	1.00±0.13	1.00±0.16
IR组	0.57±0.09^a	0.41±0.07^a	2.74±0.49^a
IR＋oe-EBLN3P组	0.82±0.14^c	0.88±0.11^c	1.69±0.28^c

图1 starBase预测LncRNA EBLN3P和miR-144-3p结合位点关系图

图2 LncRNA EBLN3P/miR-144-3p对A549细胞线粒体损伤的影响(×100)

图3 LncRNA EBLN3P/miR-144-3p对A549细胞cAMP/PKA/CREB通路蛋白表达的影响

表3 A549细胞中cAMP/PKA/CREB通路蛋白水平比较(

±s)

组　别	cAMP(nmol/L)	p-PKA/PKA	p-CREB/CREB
Control组	18.25±2.67	1.00±0.18	1.00±0.15
IR组	11.93±1.86^a	0.17±0.03^a	0.34±0.06^a
IR＋oe-EBLN3P组	16.47±2.58^b	0.95±0.17^c	0.71±0.13^c
IR＋oe-EBLN3P＋miR-144-3p mimic组	13.02±2.14^d	0.72±0.14^d	0.56±0.09^d

表3 A549细胞中cAMP/PKA/CREB通路蛋白水平比较(

±s)

组　别	cAMP(nmol/L)	p-PKA/PKA	p-CREB/CREB
Control组	18.25±2.67	1.00±0.18	1.00±0.15
IR组	11.93±1.86^a	0.17±0.03^a	0.34±0.06^a
IR＋oe-EBLN3P组	16.47±2.58^b	0.95±0.17^c	0.71±0.13^c
IR＋oe-EBLN3P＋miR-144-3p mimic组	13.02±2.14^d	0.72±0.14^d	0.56±0.09^d

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Mechanistic study on LncRNA EBLN3P/miR-144-3p axis affects radiosensitivity and mitochondrial damage in non-small cell lung cancer by regulating the cAMP/PKA/CREB pathway

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Mechanistic study on LncRNA EBLN3P/miR-144-3p axis affects radiosensitivity and mitochondrial damage in non-small cell lung cancer by regulating the cAMP/PKA/CREB pathway