Autophagy Related Proteins Beclin-1 and LC3-ⅡExpression in Lung Cancer Metaststic Pleural Effusion

doi:10.12114/j.issn.1007-9572.2023.0812

Abstract

Abstract:

Background

Malignant pleural effusion, a critical clinical indicator of advanced-stage malignancy and metastatic progression. The existing methods for the diagnosis of the nature of pleural effusion have defects and shortcomings, and it is impossible to timely and effectively determine the nature of pleural effusion. Metastatic lung cancer constitutes the primary etiology of malignant pleural effusion. While the roles of autophagy-related proteins Beclin-1 and LC3-Ⅱ in lung cancer pathogenesis have been well established through multiple studies, their expression patterns and potential as diagnostic biomarkers in MPE urgently require further investigation.

Objective

To analyze the expression of autophagy-related proteins Beclin-1 and LC3-Ⅱ in lung cancer-associated malignant pleural effusion, and to evaluate their potential value in early clinical diagnosis.

Methods

From May 2022 to July 2023, we performed bioinformatics analysis of Beclin-1 and LC3 using GEO, GEPIA2, and GeneMANIA databases. Pleural effusion samples were collected from patients who had not received any clinical treatment at the Clinical Laboratory Cell Room of the First Affiliated Hospital of Baotou Medical College, Inner Mongolia University of Science and Technology, from 2018 to 2023. The collected pleural effusion samples were grouped through liquid-based thin-layer cytology smears combined with clinical data, dividing them into a malignant group (95 cases) and a benign control group (190 cases). The expression of Beclin-1 and LC3-Ⅱat the gene and protein levels was detected by RT-PCR and Western Blotting methods, respectively, and the expression level of LC3-Ⅱ protein was confirmed using immunofluorescence assay.

Results

Bioinformatics analysis confirmed the inclusion of Beclin-1 and LC3 in the differentially expressed gene profile of lung cancer. RT-PCR revealed significantly higher mRNA expression levels of both Beclin-1 and LC3-Ⅱ in benign controls versus malignant cases (P<0.05). Western Blotting analysis showed elevated Beclin-1 and LC3-Ⅱ protein abundance in benign specimens compared to malignant group (P<0.05). Immunofluorescence microscopy identified increased FITC-labeled LC3-Ⅱ puncta in benign controls.

Conclusion

By analyzing the expression differences of autophagy-related proteins Beclin-1 and LC3-Ⅱ in benign pleural effusion and metastatic malignant pleural effusion of lung cancer, may facilitate early diagnosis of MPE and provide novel perspectives for differential diagnosis and targeted therapeutic strategies.

Key words: Lung neoplasms, Pleural effusion, Autophgy, Beclin-1, LC3-Ⅱ

摘要：

背景

恶性胸腔积液是肿瘤晚期以及转移的重要标志之一，现有诊断胸腔积液性质的相关方法存在缺陷与不足，无法对胸腔积液的性质进行及时有效的判断。多数恶性胸腔积液是由转移性肺癌引起，自噬相关蛋白1（Beclin-1）及用于监测细胞自噬水平的微管相关蛋白1轻链3（LC3）-Ⅱ在肺癌疾病进程中有重要作用，但在转移性胸腔积液中应用的相关报道较少，仍需进一步研究。

目的

分析自噬相关蛋白Beclin-1及LC3-Ⅱ在肺癌转移性胸腔积液中的表达情况，探讨其在临床疾病早期诊断中的价值。

方法

2022年5月—2023年7月利用GEO数据库、GEPIA2及GeneMANIA数据库分析获得Beclin-1及LC3的生物信息；收集包头医学院第一附属医院检验科细胞室自2018—2023年未经任何临床治疗患者的胸腔积液，通过液基薄层细胞涂片并结合其临床资料对收集的胸腔积液进行分组，将其分为恶性测定组（95例）及良性对照组（190例），通过RT-PCR方法及Western Blotting方法分别检测Beclin-1、LC3-Ⅱ基因及蛋白水平表达，并利用免疫荧光试验确认LC3-Ⅱ蛋白的表达数量。

结果

生物信息学分析结果显示，Beclin-1与LC3-Ⅱ均存在于肺癌差异表达基因数据集中，在肺癌组织与正常组织中表达具有差异性；RT-PCR结果显示Beclin-1与LC3-Ⅱ的基因表达在良性对照组中均高于恶性测定组（P<0.05）；Western Blotting结果中Beclin-1与LC3-Ⅱ的蛋白表达在良性对照组较恶性测定组高（P<0.05）；此外在良性对照组中异硫氰酸荧光素标记的代表自噬体数量的LC3-Ⅱ荧光斑点较恶性测定组高。

结论

通过分析自噬相关蛋白Beclin-1及LC3-Ⅱ在良性胸腔积液及肺癌转移性恶性胸腔积液中的表达差异，可用于恶性胸腔积液的早期诊断，为良恶性胸腔积液的鉴别诊断与针对性治疗提供新的视角。

关键词: 肺肿瘤, 胸腔积液, 自噬, Beclin-1, LC3-Ⅱ

CLC Number:

R 734.2

YAO Wenjing,WANG Cuifeng,GAO Jinliang, et al. Autophagy Related Proteins Beclin-1 and LC3-ⅡExpression in Lung Cancer Metaststic Pleural Effusion[J]. Chinese General Practice, 2026, 29(05): 623-630. DOI: 10.12114/j.issn.1007-9572.2023.0812.
姚文静,王翠峰,高金亮等. 自噬相关蛋白Beclin-1及微管相关蛋白1轻链3-Ⅱ在肺癌转移性胸腔积液中的表达研究[J]. 中国全科医学, 2026, 29(05): 623-630. DOI: 10.12114/j.issn.1007-9572.2023.0812.

Figures/Tables 9

Figure 1 Diagram of the interacting protein network of Beclin-1 and LC3

Table 1 Beclin-1 and LC3 related parameters in the GSE19188 database

基因名称	校正后P值	P值
Beclin-1	1.49×10^-2	8.45×10^-3
LC3	6.44×10^-3	3.32×10^-3

Figure 2 Volcano map of differential genes

Figure 3 Microscopic cell morphology of benign and malignant pleural effusion

Table 2 PCR primer sequence table

基因名称	引物序列
Beclin-1	F	5'-CTGGACACTCAGCTCAACGTCA-3'
Beclin-1	R	5'-CTCTAGTGCCAGCTCCTTTAGC-3'
LC3	F	5'-GCTACAAGGGTGAGAAGCAGCT-3'
LC3	R	5'-CTGGTTCACCAGCAGGAAGAAG-3'
β-actin	F	5'-TCCCTGGAGAAGAGCTACGA-3'
β-actin	R	5'-AGCACTGTGTTGGCGTACAG-3'

Figure 4 GEPIA2 analyzed Beclin-1，LC3 mRNA expression in the TCGA database

Figure 5 The expression levels of Beclin-1/LC3-Ⅱ mRNA in benign/malignant pleural effusion

Figure 6 The expression levels of Beclin-1/LC3-Ⅱ protein in benign/malignant pleural effusion

Figure 7 Expression of LC3-Ⅱ of benign /malignant pleural effusion under fluorescence microscopy

References 35

[1]	BRAY F, FERLAY J, SOERJOMATARAM I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6): 394-424. DOI: 10.3322/caac.21492.
[2]	CARTER J, MILLER J A, FELLER-KOPMAN D, et al. Molecular profiling of malignant pleural effusion in metastatic non-small-cell lung carcinoma. the effect of preanalytical factors[J]. Ann Am Thorac Soc, 2017, 14(7): 1169-1176.
[3]	XIA H W, ZHANG Z Q, YUAN J, et al. Human RECQL5 promotes metastasis and resistance to cisplatin in non-small cell lung cancer[J]. Life Sci, 2021, 265: 118768. DOI: 10.1016/j.lfs.2020.118768.
[4]	HOWLADER N, FORJAZ G, MOORADIAN M J, et al. The effect of advances in lung-cancer treatment on population mortality[J]. N Engl J Med, 2020, 383(7): 640-649.
[5]	CHEN R Q, MANOCHAKIAN R, JAMES L, et al. Emerging therapeutic agents for advanced non-small cell lung cancer[J]. J Hematol Oncol, 2020, 13(1): 58.
[6]	PSALLIDAS I, KALOMENIDIS I, PORCEL J M, et al. Malignant pleural effusion: from bench to bedside[J]. Eur Respir Rev, 2016, 25(140): 189-198. DOI: 10.1183/16000617.0019-2016.
[7]	GAYEN S. Malignant pleural effusion: presentation, diagnosis, and management[J]. Am J Med, 2022, 135(10): 1188-1192.
[8]	BIBBY A C, DORN P, PSALLIDAS I, et al. ERS/EACTS statement on the management of malignant pleural effusions[J]. Eur Respir J, 2018, 52(1): 1800349. DOI: 10.1183/13993003.00349-2018.
[9]	FENG Y C, HE D, YAO Z Y, et al. The machinery of macroautophagy[J]. Cell Res, 2014, 24(1): 24-41.
[10]	HANAHAN D, WEINBERG R A. Hallmarks of cancer: the next generation[J]. Cell, 2011, 144(5): 646-674.
[11]	ALZAHRANI A S. PI3K/Akt/mTOR inhibitors in cancer: At the bench and bedside[J]. Semin Cancer Biol, 2019, 59: 125-132. DOI: 10.1016/j.semcancer.2019.07.009.
[12]	LI X H, HE S K, MA B Y. Autophagy and autophagy-related proteins in cancer[J]. Mol Cancer, 2020, 19(1): 12.
[13]	JACQUET M, GUITTAUT M, FRAICHARD A, et al. The functions of Atg8-family proteins in autophagy and cancer: linked or unrelated [J]. Autophagy, 2021, 17(3): 599-611.
[14]	POON I K, CHAN R C K, CHOI J S H, et al. A comparative study of diagnostic accuracy in 3026 pleural biopsies and matched pleural effusion cytology with clinical correlation[J]. Cancer Med, 2023, 12(2): 1471-1481. DOI: 10.1002/cam4.5038.
[15]	ARNOLD D T, DE FONSEKA D, PERRY S, et al. Investigating unilateral pleural effusions: the role of cytology[J]. Eur Respir J, 2018, 52(5): 1801254. DOI: 10.1183/13993003.01254-2018.
[16]	WANG S F, TIAN S, LI Y, et al. Development and validation of a novel scoring system developed from a nomogram to identify malignant pleural effusion[J]. EBioMedicine, 2020, 58: 102924. DOI: 10.1016/j.ebiom.2020.102924.
[17]	WOO C G, SON S M, HAN H S, et al. Diagnostic benefits of the combined use of liquid-based cytology, cell block, and carcinoembryonic antigen immunocytochemistry in malignant pleural effusion[J]. J Thorac Dis, 2018, 10(8): 4931-4939.
[18]	HAN Y H, MUN J G, JEON H D, et al. Betulin inhibits lung metastasis by inducing cell cycle arrest, autophagy, and apoptosis of metastatic colorectal cancer cells[J]. Nutrients, 2019, 12(1): 66. DOI: 10.3390/nu12010066.
[19]	ZHANG S Q, QIAN Y, YE L H. Delineating the twin role of autophagy in lung cancer[J]. Biol Futura, 2023, 74(1): 119-135. DOI: 10.1007/s42977-023-00165-4.
[20]	YU J E, KIM Y, HONG D E, et al. Bee venom triggers autophagy-induced apoptosis in human lung cancer cells via the mTOR signaling pathway[J]. J Oncol, 2022, 2022: 8916464.
[21]	RAGUSA M A, NASELLI F, CRUCIATA I, et al. Indicaxanthin induces autophagy in intestinal epithelial cancer cells by epigenetic mechanisms involving DNA methylation[J]. Nutrients, 2023, 15(15): 3495. DOI: 10.3390/nu15153495.
[22]	LI X, YANG K B, CHEN W, et al. CUL3(cullin 3)-mediated ubiquitination and degradation of BECN1(beclin 1)inhibit autophagy and promote tumor progression[J]. Autophagy, 2021, 17(12): 4323-4340. DOI: 10.1080/15548627.2021.1912270.
[23]	NICCO C, THOMAS M, GUILLERMET J, et al. Mechanistic target of rapamycin (mTOR) regulates self-sustained quiescence, tumor indolence, and late clinical metastasis in a Beclin-1-dependent manner[J]. Cell Cycle, 2023, 22(5): 542-564.
[24]	DI LEO L, BODEMEYER V, BOSISIO F M, et al. Loss of Ambra1 promotes melanoma growth and invasion[J]. Nat Commun, 2021, 12(1): 2550.
[25]	WAN Y H, QIAN Y H, WANG Y Y, et al. Prognostic value of Beclin 1, EGFR and ALK in non-squamous non-small cell lung cancer[J]. Discov Oncol, 2022, 13(1): 127.
[26]	刘俊英, 杨会钗, 李诗, 等. 肺腺癌中CALHM2、PD-L1的表达及其预后分析[J]. 临床与实验病理学杂志, 2023, 39(8): 957-962. DOI: 10.13315/j.cnki.cjcep.2023.08.013.
[27]	LIANG L J, HUI K Y, HU C X, et al. Autophagy inhibition potentiates the anti-angiogenic property of multikinase inhibitor anlotinib through JAK2/STAT3/VEGFA signaling in non-small cell lung cancer cells[J]. J Exp Clin Cancer Res, 2019, 38(1): 71. DOI: 10.1186/s13046-019-1093-3.
[28]	ICHIMURA Y, KOMINAMI E, TANAKA K, et al. Selective turnover of p62/A170/SQSTM1 by autophagy[J]. Autophagy, 2008, 4(8): 1063-1066. DOI: 10.4161/auto.6826.
[29]	SUN X D, LI J, LI Y Z, et al. Apatinib, a novel tyrosine kinase inhibitor, promotes ROS-dependent apoptosis and autophagy via the Nrf2/HO-1 pathway in ovarian cancer cells[J]. Oxid Med Cell Longev, 2020, 2020: 3145182. DOI: 10.1155/2020/3145182.
[30]	XING Y H, WEI X Q, LIU Y C, et al. Autophagy inhibition mediated by MCOLN1/TRPML1 suppresses cancer metastasis via regulating a ROS-driven TP53/p53 pathway[J]. Autophagy, 2022, 18(8): 1932-1954.
[31]	JIANG X W, LU W Q, SHEN X Y, et al. Repurposing sertraline sensitizes non-small cell lung cancer cells to erlotinib by inducing autophagy[J]. JCI Insight, 2018, 3(11): e98921.
[32]	LV Y S, ZHANG W J, ZHAO J Y, et al. SRSF1 inhibits autophagy through regulating Bcl-x splicing and interacting with PIK3C3 in lung cancer[J]. Signal Transduct Target Ther, 2021, 6(1): 108. DOI: 10.1038/s41392-021-00495-6.
[33]	王婧喆, 王翠峰. Beclin-1、LC3和mTOR在肺癌中的表达及意义[J]. 包头医学院学报, 2022, 38(1): 45-49. DOI: 10.16833/j.cnki.jbmc.2022.01.011.
[34]	TORII S, HONDA S, MUROHASHI M, et al. Autophagy involvement in oncogenesis[J]. Cancer Sci, 2020, 111(11): 3993-3999. DOI: 10.1111/cas.14646.
[35]	WU H, LU X X, WANG J R, et al. TRAF6 inhibits colorectal cancer metastasis through regulating selective autophagic CTNNB1/β-catenin degradation and is targeted for GSK3B/GSK3β-mediated phosphorylation and degradation[J]. Autophagy, 2019, 15(9): 1506-1522. DOI: 10.1080/15548627.2019.1586250.