The effect of adenovirus-mediated SPRY1 overexpression on apoptosis and autophagy in hepatocellular carcinoma

doi:10.11958/20252541

Abstract

Abstract:

Objective To investigate the effect and mechanism of adenovirus-mediated overexpression of Sprouty RTK signaling antagonist 1 (SPRY1) on proliferation, apoptosis and autophagy in human hepatocellular carcinoma (HCC) cells. Methods The recombinant adenovirus vector Ad5-SPRY1 was constructed and used to infect human HCC cell lines Huh7 and Hep3B. Cells were divided into the control group (infected with Ad5-GFP) and the SPRY1 overexpression group (infected with Ad5-SPRY1). SPRY1 mRNA expression was detected by qPCR. Western blot assay was used to analyze the protein levels of SPRY1, c-MYC, BCL2 and autophagy-related proteins LC3Ⅰ/Ⅱ. Cell apoptosis was measured by flow cytometry with Annexin V-PE/7-AAD double staining. Autophagosome formation was observed by transmission electron microscopy (TEM), and autophagic flux was assessed using the Ad-mCherry-GFP-LC3B reporter system. Results After 48 hours of Ad5-SPRY1 infection in Huh7 and Hep3B cells, the expression levels of SPRY1 mRNA and protein were higher than those in the control group and the Ad5-GFP group (P<0.05). Flow cytometry analysis showed that after 24 hours of infection in Huh7 and Hep3B cells, the apoptosis rate in the Ad5-SPRY1 group was lower than that in the control group and the Ad5-GFP group (P<0.05). Western blot results showed that the expression levels of c-MYC and BCL2 proteins in the Ad5-SPRY1 group were higher than those in the control group and the Ad5-GFP group (P<0.05). Transmission electron microscopy observation revealed a large number of double-membrane autophagosome structures in the cytoplasm of the Ad5-SPRY1 group. Western blot results indicated that the expression level of LC3Ⅱ protein in the Ad5-SPRY1 group was higher than that in the control group and the Ad5-GFP group (P<0.05). Ad-mCherry-GFP-LC3B fluorescence assay showed that the number of yellow spots in the Ad5-SPRY1 group was higher than that in the control group and pcDNA3.1 group (P<0.05). Conclusion Adenovirus-mediated SPRY1 overexpression inhibits apoptosis and promotes autophagy in HCC cells, possibly through upregulation of c-MYC and BCL2 and enhancement of LC3-mediated autophagic flux. SPRY1 may represent a novel therapeutic target for hepatocellular carcinoma.

Key words: carcinoma, hepatocellular, apoptosis, autophagy, SPla and RyR domain-containing protein 1, proto-oncogene proteins c-myc, proto-oncogene proteins bcl-2

CLC Number:

R735.7

Figures/Tables 11

References 22

[1]	SIEGEL R L, KRATZER T B, GIAQUINTO A N, et al. Cancer statistics,2025[J]. CA Cancer J Clin, 2025, 75(1):10-45. doi:10.3322/caac.21871.
[2]	SUN L, HE M, LIU D, et al. Deacetylation of ANXA2 by SIRT2 desensitizes hepatocellular carcinoma cells to donafenib via promoting protective autophagy[J]. Cell Death Differ, 2025, 32(9):1630-1647. doi:10.1038/s41418-025-01499-3.
[3]	ZHANG G P, SONG Z B, CHEN D H, et al. Syntaxin-6 mediated autophagy confers lenvatinib resistance in hepatocellular carcinoma[J]. Oncogene, 2025, 44(25):2025-2039. doi:10.1038/s41388-025-03371-7.
[4]	MONTICO B, GIURATO G, GUERRIERI R, et al. Suppression of Spry1 reduces HIF1α-dependent glycolysis and impairs angiogenesis in BRAF-mutant cutaneous melanoma[J]. J Exp Clin Cancer Res, 2025, 44:53. doi:10.1186/s13046-025-03289-8.
[5]	SHI T, LI X, ZHENG J, et al. Increased SPRY1 expression activates NF-κB signaling and promotes pancreatic cancer progression by recruiting neutrophils and macrophages through CXCL12-CXCR4 axis[J]. Cell Oncol, 2023, 46(4):969-985. doi:10.1007/s13402-023-00791-z.
[6]	LIU Z, ZHANG Y, MA N, et al. Progenitor-like exhausted SPRY1(+)CD8(+) T cells potentiate responsiveness to neoadjuvant PD-1 blockade in esophageal squamous cell carcinoma[J]. Cancer Cell, 2023, 41(11):1852-1870.e9. doi:10.1016/j.ccell.2023.09.011.
[7]	CHAKRABORTY S, NANDI P, MISHRA J, et al. Molecular mechanisms in regulation of autophagy and apoptosis in view of epigenetic regulation of genes and involvement of liquid-liquid phase separation[J]. Cancer Lett, 2024, 587:216779. doi:10.1016/j.canlet.2024.216779.
[8]	YU Y S, KIM I S, BAEK S H. Decoding the dual role of autophagy in cancer through transcriptional and epigenetic regulation[J]. FEBS Lett, 2025, 599(16):2237-2249. doi:10.1002/1873-3468.70060.
[9]	JIA H, WEI J, ZHENG W, et al. The dual role of autophagy in cancer stem cells:implications for tumor progression and therapy resistance[J]. J Transl Med, 2025, 23:583. doi:10.1002/1873-3468.14999.
[10]	DEBNATH J, GAMMOH N, RYAN K M. Autophagy and autophagy-related pathways in cancer[J]. Nat Rev Mol Cell Biol, 2023, 24(8):560-575. doi:10.1038/s41580-023-00585-z.
[11]	HUANG X, ZHANG J, YAO J, et al. Phase separation of p62:roles and regulations in autophagy[J]. Trends Cell Biol, 2025, 35(10):992-1006. doi:10.1016/j.tcb.2025.03.003.
[12]	YANG S, HU C, CHEN X, et al. Crosstalk between metabolism and cell death in tumorigenesis[J]. Mol Cancer, 2024, 23:71. doi:10.1186/s12943-024-02018-5.
[13]	LI J, MA R, WANG X, et al. Sprouty genes regulate activated fibroblasts in mammary epithelial development and breast cancer[J]. Cell Death Dis, 2024, 15(4):256. doi:10.1038/s41419-024-06573-9.
[14]	NIU Z, WANG L, QIN X, et al. Macrophage derived miR-7219-3p-containing exosomes mediate fibroblast trans-differentiation by targeting SPRY1 in silicosis[J]. Toxicology, 2022, 479:153310. doi:10.1016/j.tox.2022.153310.
[15]	ZHAO G, PAN A Y, FENG Y, et al. Sprouty and Spred temporally regulate ERK1/2-signaling to suppress TGFβ-induced lens EMT[J]. Exp Eye Res, 2022, 219:109070. doi:10.1016/j.exer.2022.109070.
[16]	ZHANG M F, WAN S C, CHEN W B, et al. Transcription factor Dmrt1 triggers the SPRY1-NF-κB pathway to maintain testicular immune homeostasis and male fertility[J]. Zool Res, 2023, 44(3):505-521. doi:10.24272/j.issn.2095-8137.2023.202219.
[17]	FANG X, LU X, MA Y, et al. Possible involvement of a MEG3-miR-21-SPRY1-NF-κB feedback loop in spermatogenic cells proliferation,autophagy,and apoptosis[J]. iScience, 2024, 27(10):110904. doi:10.1016/j.isci.2024.110904.
[18]	LIU X, LAN Y, ZHANG D, et al. SPRY1 promotes the degradation of uPAR and inhibits uPAR-mediated cell adhesion and proliferation[J]. Am J Cancer Res, 2014, 4(6):683-697.
[19]	CROCE C M, VAUX D, STRASSER A, et al. The BCL-2 protein family:from discovery to drug development[J]. Cell Death Differ, 2025, 32(8):1369-1381. doi:10.1038/s41418-025-01454-2.
[20]	LU Y, JIANG X, LI Y, et al. NL101 synergizes with the BCL-2 inhibitor venetoclax through PI3K-dependent suppression of c-Myc in acute myeloid leukaemia[J]. J Transl Med, 2024, 22:867. doi:10.1186/s12967-024-05377-3.
[21]	RUIZ-FERNÁNDEZ DE CÓRDOBA B, VALENCIA K, WELCH C, et al. Dual ENPP1/ATM depletion blunts DNA damage repair boosting radioimmune efficacy to abrogate triple-negative breast cancer[J]. Signal Transduct Target Ther, 2025, 10:185. doi:10.1038/s41392-025-02114-0.
[22]	RINNERTHALER G, EGLE D, BARTSCH R, et al. Neoadjuvant atezolizumab in combination with dual HER2 blockade plus epirubicin in women with early HER2-positive breast cancer:the randomized phase 2 ABCSG-52/ATHENE trial[J]. Nat Cancer, 2025, 6(1):41-50. doi:10.1038/s43018-024-00809-7.

基因名称	引物序列（5′→3′）	产物大小/bp
SPRY1	上游：TCCCTGGTCATAGGTCTGAAAG	187
SPRY1	下游：TGCCGGTTACAGGCCAAAC	187
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	197
GAPDH	下游：GGCTGTTGTCATACTTCTCATGG	197

基因名称	引物序列（5′→3′）	产物大小/bp
SPRY1	上游：TCCCTGGTCATAGGTCTGAAAG	187
SPRY1	下游：TGCCGGTTACAGGCCAAAC	187
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	197
GAPDH	下游：GGCTGTTGTCATACTTCTCATGG	197

组别	Huh7细胞		Hep3B细胞
组别	mRNA	蛋白	mRNA	蛋白
对照组	1.00±0.12	1.00±0.08	1.00±0.03	1.00±0.03
Ad5-GFP组	1.00±0.07	1.00±0.07	1.00±0.06	1.00±0.04
Ad5-SPRY1组	81.89±4.07^ab	3.31±0.19^ab	69.19±3.07^ab	2.61±0.11^ab
F	1 180.719^＊	320.415^＊	1 477.800^＊	541.412^＊

组别	Huh7细胞		Hep3B细胞
组别	mRNA	蛋白	mRNA	蛋白
对照组	1.00±0.12	1.00±0.08	1.00±0.03	1.00±0.03
Ad5-GFP组	1.00±0.07	1.00±0.07	1.00±0.06	1.00±0.04
Ad5-SPRY1组	81.89±4.07^ab	3.31±0.19^ab	69.19±3.07^ab	2.61±0.11^ab
F	1 180.719^＊	320.415^＊	1 477.800^＊	541.412^＊

组别	Huh7细胞
组别	细胞凋亡率/%			c-MYC蛋白		BCL2蛋白
对照组	32.14±0.78			1.00±0.03		1.00±0.13
Ad5-GFP组	33.14±1.05			1.00±0.07		1.00±0.04
Ad5-SPRY1组	17.02±1.58^ab			2.30±0.14^ab		1.94±0.11^ab
F	181.832^＊			216.545^＊		85.748^＊
组别		Hep3B细胞
组别		细胞凋亡率/%	c-MYC蛋白		BCL2蛋白
对照组		27.51±2.90	1.00±0.07		1.00±0.02
Ad5-GFP组		28.84±2.35	1.00±0.10		1.00±0.10
Ad5-SPRY1组		3.53±0.63^ab	2.94±0.07^ab		1.74±0.10^ab
F		127.296^＊	638.889^＊		115.649^＊