Expression, prognostic value of CBX3 in lung adenocarcinoma and its effect on biological behavior of cancer cells

doi:10.19401/j.cnki.1007-3639.2022.02.007

Abstract

Abstract:

Background and purpose: Chromobox 3 (CBX3), an important component of chromatin, is involved in the development and progression of various cancers. However, the expression and role of CBX3 in lung adenocarcinoma are still unclear. This study aimed to investigate the relationship between the expression of CBX3 in lung adenocarcinoma and prognosis of lung adenocarcinoma, and to study the effects of CBX3 on proliferation, migration and invasion of A549 lung adenocarcinoma cells. Methods: Transcriptome data of lung adenocarcinoma and corresponding clinical data were downloaded from the Cancer Genome Atlas (TCGA), and R software was used to analyze the expression difference of CBX3 and the correlation of immune infiltration in lung adenocarcinoma. The prognostic significance of CBX3 in lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve and Kaplan-Meier analysis. Twenty lung adenocarcinoma samples from patients (with informed consent obtained) who underwent palliative or radical lung cancer resection from August 2020 to January 2021 were collected from the intelligent Biobank of Peking University Shenzhen Hospital at -80 ℃. Real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) was used to verify the expression of CBX3 in 20 frozen lung adenocarcinoma tissue samples and paired normal lung tissues. A549 cells were transfected with small interfering RNA (siRNA) to silence the expression of CBX3, and A549 cells were divided into silent group (si-CBX3 group) and negative control group (si-NC group) according to the type of siRNA transfection. The effects of CBX3 on proliferation, migration and invasion of A549 cells were investigated by cell counting kit-8 (CCK-8) assay, clone formation assay, scratch assay and transwell assay, respectively. Results: The difference analysis based on TCGA lung adenocarcinoma data and RTFQ-PCR results of lung adenocarcinoma samples showed that CBX3 was significantly overexpressed in lung adenocarcinoma (P<0.05). The infiltration of T helper 2 (Th2) cells was positively correlated with the expression of CBX3 [correlation coefficient (r)=0.437], while mast cells (r=-0.444), eosinophils (r=-0.380) and other immune cells were negatively correlated with the expression of CBX3. The area under ROC curve (AUC) value was 0.912, indicating that CBX3 has a good diagnostic value for lung adenocarcinoma. Kaplan-Meier analysis showed that the survival period of the group with high expression of CBX3 was shorter, suggesting a poor prognosis. After the expression of CBX3 was silenced in A549 cells, significantly decreased proliferation ability of A549 cells was detected by CCK-8 and clone formation assays (P<0.01). The results of scratch assay showed that the mobility of A549 cells was (22.68±3.44)% after silencing, which was significantly lower compared with the control group (P<0.05). Transwell assay results showed that the relative invasion rate of A549 cells was (53.94±5.39)%, and the invasion ability was significantly decreased (P<0.01). Conclusion: CBX3 is significantly overexpressed in lung adenocarcinoma, which predicts poor prognosis of lung adenocarcinoma and can be used as a prognostic marker of lung adenocarcinoma, and the high expression of CBX3 promotes the proliferation, migration and invasion of A549 cells.

Key words: CBX3, Lung adenocarcinoma, Immune infiltration, Prognosis, Cell function experiment

CLC Number:

R734.2

HOU Qinghua, ZHONG Yanfeng, LIU Linzhuang, WU Liusheng, LIU Jixian. Expression, prognostic value of CBX3 in lung adenocarcinoma and its effect on biological behavior of cancer cells[J]. China Oncology, 2022, 32(2): 152-160.

Figures/Tables 10

Tab. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

References 28

[1]	SUNG H,, FERLAY J,, SIEGEL R L, et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249.
[2]	HUTCHINSON B D,, SHROFF G S,, TRUONG M T, et al.Spectrum of lung adenocarcinoma[J]. Semin Ultrasound CT MR, 2019, 40(3): 255-264.
[3]	ETTINGER D S,, WOOD D E,, AISNER D L, et al.Non-small cell lung cancer, version 5.2017, NCCN clinical practice guidelines in oncology[J]. J Natl Compr Canc Netw, 2017, 15(4): 504-535.
[4]	OSMANI L,, ASKIN F,, GABRIELSON E, et al.Current WHO guidelines and the critical role of immunohistochemical markers in the subclassification of non-small cell lung carcinoma (NSCLC): moving from targeted therapy to immunotherapy[J]. Semin Cancer Biol, 2018, 52(Pt 1): 103-109.
[5]	XING P Y,, WANG S Z,, WANG Q, et al.Efficacy of crizotinib for advanced ALK-rearranged non-small cell lung cancer patients with brain metastasis: a multicenter, retrospective study in China[J]. Target Oncol, 2019, 14(3): 325-333.
[6]	王晓莉,, 葛晓松,, 刘彦魁, 等. EGFR/ALK/ROS1三联检测在非小细胞肺癌中的临床意义[J]. 临床与实验病理学杂志, 2018, 34(10): 1135-1137.
	WANG X L,, GE X S,, LIU Y K, et al.Clinical significance of EGFR/ALK/ros1 triple detection in non-small cell lung cancer[J]. Chin J Clin Exp Pathol, 2018, 34(10): 1135-1137.
[7]	PARK J Y,, JANG S H.Epidemiology of lung cancer in Korea: recent trends[J]. Tuberc Respir Dis (Seoul), 2016, 79(2): 58-69.
[8]	TESTA U,, CASTELLI G,, PELOSI E.Lung cancers: molecular characterization, clonal heterogeneity and evolution, and cancer stem cells[J]. Cancers (Basel), 2018, 10(8): E248.
[9]	CHANG S C,, LAI Y C,, CHEN Y C, et al.CBX3/heterochromatin protein 1 gamma is significantly upregulated in patients with non-small cell lung cancer[J]. Asia Pac J Clin Oncol, 2018, 14(5): e283-e288.
[10]	CANZIO D,, LARSON A,, NARLIKAR G J.Mechanisms of functional promiscuity by HP1 proteins[J]. Trends Cell Biol, 2014, 24(6): 377-386.
[11]	LOVE M I,, HUBER W,, ANDERS S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biol, 2014, 15(12): 550.
[12]	HÄNZELMANN S,, CASTELO R,, GUINNEY J. GSVA: gene set variation analysis for microarray and RNA-seq data[J]. BMC Bioinformatics, 2013, 14: 7.
[13]	BINDEA G,, MLECNIK B,, TOSOLINI M, et al.Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer[J]. Immunity, 2013, 39(4): 782-795.
[14]	CHALELA R,, CURULL V,, ENRÍQUEZ C, et al. Lung adenocarcinoma: from molecular basis to genome-guided therapy and immunotherapy[J]. J Thorac Dis, 2017, 9(7): 2142-2158.
[15]	IMAI H,, KAIRA K,, MINATO K.Clinical significance of post-progression survival in lung cancer[J]. Thorac Cancer, 2017, 8(5): 379-386.
[16]	GREWAL S I,, JIA S.Heterochromatin revisited[J]. Nat Rev Genet, 2007, 8(1): 35-46.
[17]	VAKOC C R,, MANDAT S A,, OLENCHOCK B A, et al.Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin[J]. Mol Cell, 2005, 19(3): 381-391.
[18]	MATEESCU B,, BOURACHOT B,, RACHEZ C, et al.Regulation of an inducible promoter by an HP1beta-HP1gamma switch[J]. EMBO Rep, 2008, 9(3): 267-272.
[19]	KWON S H,, WORKMAN J L.HP1c casts light on dark matter[J]. Cell Cycle, 2011, 10(4): 625-630.
[20]	NONAKA N,, KITAJIMA T,, YOKOBAYASHI S, et al.Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast[J]. Nat Cell Biol, 2002, 4(1): 89-93.
[21]	YAMAGISHI Y,, SAKUNO T,, SHIMURA M, et al.Author correction: heterochromatin links to centromeric protection by recruiting shugoshin[J]. Nature, 2018, 563(7730): E21.
[22]	HAYAKAWA T,, HARAGUCHI T,, MASUMOTO H, et al.Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase[J]. J Cell Sci, 2003, 116(Pt 16): 3327-3338.
[23]	EGUCHI T,, CALDERWOOD S K,, TAKIGAWA M, et al.Intracellular MMP3 promotes HSP gene expression in collaboration with chromobox proteins[J]. J Cell Biochem, 2017, 118(1): 43-51.
[24]	FAN Y,, LI H P,, LIANG X L, et al.CBX3 promotes colon cancer cell proliferation by CDK6 kinase-independent function during cell cycle[J]. Oncotarget, 2017, 8(12): 19934-19946.
[25]	WANG S Q,, LIU F,, WANG Y H, et al.Integrated analysis of 34 microarray datasets reveals CBX3 as a diagnostic and prognostic biomarker in glioblastoma[J]. J Transl Med, 2019, 17(1): 179.
[26]	HAN S S,, KIM W J,, HONG Y, et al.RNA sequencing identifies novel markers of non-small cell lung cancer[J]. Lung Cancer, 2014, 84(3): 229-235.
[27]	LIU J,, ZHANG Y,, ZHAO J, et al.Mast cell: insight into remodeling a tumor microenvironment[J]. Cancer Metastasis Rev, 2011, 30(2): 177-184.
[28]	HUANG B,, LEI Z,, ZHANG G M, et al.SCF-mediated mast cell infiltration and activation exacerbate the inflammation and immunosuppression in tumor microenvironment[J]. Blood, 2008, 112(4): 1269-1279.

Characteristic^*	Low expression of CBX3	High expression of CBX3	P value
T stage n (%)			0.031
T₁	97 (19.0)	71 (13.9)
T₂	126 (24.7)	150 (29.4)
T₃	25 (4.9)	22 (4.3)
T₄	6 (1.2)	13 (2.5)
N stage n (%)			0.005
N₀	179 (35.7)	151 (30.1)
N₁	42 (8.4)	53 (10.6)
N₂	26 (5.2)	48 (9.6)
N₃	0 (0.0)	2 (0.4)
M stage n (%)			0.342
M₀	165 (44.7)	179 (48.5)
M₁	9 (2.4)	16 (4.3)
Pathologic stage n (%)			0.102
Ⅰ	148 (29.3)	126 (25.0)
Ⅱ	60 (11.9)	61 (12.1)
Ⅲ	34 (6.7)	50 (9.9)
Ⅳ	10 (2.0)	16 (3.2)
Gender n (%)			0.504
Female	142 (27.7)	134 (26.1)
Male	114 (22.2)	123 (24.0)
Age/year n (%)			0.024
≤65	105 (21.3)	133 (26.9)
>65	140 (28.3)	116 (23.5)
Smoker n (%)			0.368
No	41 (8.2)	33 (6.6)
Yes	208 (41.7)	217 (43.5)