FBXO38通过PI3K-Akt信号通路调控眼部黑色素瘤增殖

doi:10.3969/j.issn.1674-8115.2023.12.002

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (12): 1470-1479.doi: 10.3969/j.issn.1674-8115.2023.12.002

• 创新团队成果专栏 • 上一篇

FBXO38通过PI3K-Akt信号通路调控眼部黑色素瘤增殖

吴以加(), 房燕, 沈菲洋, 黄蕊, 沈键锋(), 范先群()

上海交通大学医学院附属第九人民医院眼科，上海市眼眶病眼肿瘤重点实验室，上海 200011

收稿日期:2023-08-13 接受日期:2023-10-25 出版日期:2023-12-28 发布日期:2024-02-01
通讯作者: 沈键锋,范先群 E-mail:wyj_med@sjtu.edu.cn;jfshen@shsmu.edu.cn;fanxq@sjtu.edu.cn
作者简介:吴以加（1997—），女，硕士生；电子信箱：wyj_med@sjtu.edu.cn。
基金资助:
国家自然科学基金(81972667)

FBXO38 regulates ocular melanoma proliferation through the PI3K-Akt signaling pathway

WU Yijia(), FANG Yan, SHEN Feiyang, HUANG Rui, SHEN Jianfeng(), FAN Xianqun()

Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai 200011, China

Received:2023-08-13 Accepted:2023-10-25 Online:2023-12-28 Published:2024-02-01
Contact: SHEN Jianfeng,FAN Xianqun E-mail:wyj_med@sjtu.edu.cn;jfshen@shsmu.edu.cn;fanxq@sjtu.edu.cn
Supported by:
National Natural Science Foundation of China(81972667)

摘要/Abstract

摘要：

目的·研究F-box蛋白38（F-box only protein 38，FBXO38）对眼部黑色素瘤增殖的作用以及潜在的调控通路。方法·使用FBXO38短发夹RNA（short hairpin RNA，shRNA）和FBXO38过表达质粒构建FBXO38敲低以及过表达的人皮肤黑色素瘤A375和葡萄膜黑色素瘤OMM2.3细胞系，并通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）和Western blotting在转录和蛋白水平验证FBXO38的敲低和过表达效率。通过克隆形成实验、BrdU免疫荧光染色和CCK8细胞增殖实验，探究FBXO38对黑色素瘤细胞增殖的影响。使用肿瘤基因组图谱计划数据库（The Cancer Genome Atlas，TCGA），分析FBXO38高表达和低表达组中的差异表达基因，并进行京都基因与基因组数据库（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集，揭示与FBXO38相关的信号通路。进一步通过CCK8细胞增殖实验检测信号通路抑制剂对不同FBXO38表达量细胞的抑制率。同时通过qRT-PCR和Western blotting，验证在敲低FBXO38之后该通路是否激活。结果·qRT-PCR和Western blotting验证A375和OMM2.3细胞系中的FBXO38的mRNA及蛋白质表达水平，发现与对照组相比敲低组的FBXO38表达水平下降，与野生型相比过表达组的FBXO38的表达水平提高（P<0.05）。克隆形成实验、BrdU免疫荧光染色和CCK8细胞增殖实验显示，敲低FBXO38显著增强A375和OMM2.3细胞的增殖能力（P<0.05），反之过表达FBXO38抑制A375和OMM2.3细胞增殖（P<0.05）。KEGG通路富集分析显示，在皮肤黑色素瘤和葡萄膜黑色素瘤中，FBXO38的表达影响磷脂酰肌醇3激酶/蛋白激酶B（phosphoinositide 3-kinase/protein kinase B，PI3K-Akt）通路激活。与对照组相比，PI3K抑制剂LY294002和mTOR1抑制剂Everolimus对FBXO38敲低组的抑制率显著提升（P<0.05），对FBXO38过表达组的抑制率则显著下降（P<0.05）。Western blotting结果显示，敲低FBXO38之后，与PI3K-Akt通路相关的PTEN、P21和P53蛋白水平下降，而MDM2蛋白水平上升。qRT-PCR结果显示在FBXO38敲低细胞中P53转录水平显著下降（P<0.05），而MDM2转录水平显著上升（P<0.05）。结论·FBXO38通过PI3K-Akt信号通路参与调控眼部黑色素瘤细胞的增殖。

关键词: F-box蛋白38（FBXO38）, 眼部黑色素瘤, PI3K-Akt信号通路, 肿瘤增殖

Abstract:

Objective ·To investigate the effect of F-box only protein 38 (FBXO38) on the ocular melanoma proliferation and the potential regulatory pathway. Methods ·Human skin cutaneous melanoma A375 and human uveal melanoma OMM2.3 cell lines with FBXO38 knockdown and overexpression were constructed by FBXO38 short hairpin RNA (shRNA) and FBXO38 overexpression plasmids respectively. Knockdown and overexpression efficiency of FBXO38 at transcription and protein levels were verified by using quantitative real-time PCR (qRT-PCR) and Western blotting. The effects of FBXO38 on melanoma cell proliferation were detected through clonal formation assay, BrdU immunofluorescence staining and CCK8 cell proliferation assay. By using The Cancer Genome Atlas (TCGA) database, differentially expressed genes were analyzed in the high and low expression groups of FBXO38. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed to reveal the signaling pathways associated with FBXO38. CCK8 cell proliferation assay was used to detect the inhibition rates of the signaling pathway inhibitors on cells with different FBXO38 expression levels. qRT-PCR and Western blotting were used to detect whether the signaling pathway was activated after knocking down FBXO38. Results ·qRT-PCR and Western blotting verified that mRNA and protein expression levels of FBXO38 in FBXO38 knockdown A375 and OMM2.3 cell lines decreased compared with the control group, while the expression levels of FBXO38 in the overexpression cell lines increased compared with wild type group (P<0.05). Clonal formation assay, BrdU immunofluorescence staining and CCK8 cell proliferation assay showed that FBXO38 knockdown significantly enhanced the proliferation of A375 and OMM2.3 cells (P<0.05), while overexpression of FBXO38 inhibited melanoma cell proliferation (P<0.05). Enrichment analysis showed that in skin cutaneous melanoma and uveal melanoma, FBXO38 expression influenced the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) pathway activation. Compared with those in the control group, the inhibition rates of PI3K inhibitor LY294002 and mTOR1 inhibitor Everolimus in the FBXO38 knockdown group significantly improved (P<0.05), while their inhibition rates of the overexpression group significantly decreased compared with those of control cells (P<0.05). Western blotting results showed that after knocking down FBXO38, expression levels of PTEN, P21 and P53 proteins decreased, while expression level of MDM2 protein increased. The qRT-PCR results showed a significant decrease in P53 transcription level (P<0.05) and a significant increase in MDM2 transcription level in FBXO38 knockdown cells (P<0.05). Conclusion ·FBXO38 plays a role in regulating the proliferation of ocular melanoma, and this regulatory effect is related to the PI3K-Akt signaling pathway.

Key words: F-box only protein 38 (FBXO38), ocular melanoma, PI3K-Akt signaling pathway, tumor proliferation

中图分类号:

R739.7

吴以加, 房燕, 沈菲洋, 黄蕊, 沈键锋, 范先群. FBXO38通过PI3K-Akt信号通路调控眼部黑色素瘤增殖[J]. 上海交通大学学报（医学版）, 2023, 43(12): 1470-1479.

WU Yijia, FANG Yan, SHEN Feiyang, HUANG Rui, SHEN Jianfeng, FAN Xianqun. FBXO38 regulates ocular melanoma proliferation through the PI3K-Akt signaling pathway[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(12): 1470-1479.

图/表 5

表1 用于qRT-PCR检测的引物序列

Tab 1 Primer sequences for qRT-PCR

Primer	Forward (5′→3′)	Reverse (5′→3′)
FBXO38	AACGGTACTCGGCGTTACTC	TGTTTTGGCTACTTCTGACAATTC
ACTIN	CTCCATCCTGGCCTCGCTGT	GCTGTCACCTTCACCGTTCC
MDM2	AGGAGATTTGTTTGGCGTGC	TGAGTCCGATGATTCCTGCTG
TP53	AAGTCTAGAGCCACCGTCCA	CAGTCTGGCTGCCAATCCA

图1 FBXO38 敲低和过表达细胞系构建Note： A/B. Expression of FBXO38 in control and FBXO38-knockdown A375 cell line (A) and OMM2.3 cell line (B) detected by Western blotting. C/D. Transcription level of FBXO38 in FBXO38-knockdown groups compared with control A375 cell line (C) and OMM2.3 cell line (D) detected by qRT-PCR.E/F. Expression of FBXO38 in wild type and FBXO38 overexpression A375 cell line (E) and OMM2.3 cell line (F) detected by Western blotting. G/H. Transcription level of FBXO38 in FBXO38 overexpression compared with wild type A375 cell line (G) and OMM2.3 cell line (H) detected by qRT-PCR. ①P=0.013, ②P=0.012, ③P=0.001, ④P =0.008, ⑤P =0.016.

Fig 1 Construction of FBXO38 knockdown and overexpression cell lines

图2 FBXO38 敲低或者过表达对黑色素瘤细胞增殖的影响Note： A. Representative images of colony formation in FBXO38 knockdown and overexpression A375 cells. B. Colony formation efficiency of A375 cell lines after 7-d incubation. C. Representative images of colony formation on FBXO38 knockdown and overexpression OMM2.3 cells. D. Colony formation efficiency of OMM2.3 cell lines after 7-d incubation. E. Immunofluorescence staining of BrdU in FBXO38 knockdown and overexpression A375 cells. Scale bars, 50 μm. F. The percentage of BrdU positive A375 cells. G/H. Cell viability of FBXO38 knockdown and overexpression A375 cells (G) and OMM2.3 cells (H) detected by CCK8 assay. ①P=0.046, ②P=0.048, ③P=0.039, ④P=0.001, ⑤P=0.003, ⑥P=0.010, ⑦P=0.012, ⑧P=0.036, ⑨P=0.024, ⑩P=0.033, ?P=0.005, ?P=0.016, ?P=0.017.

Fig 2 Effect of FBXO38 knockdown or overexpression on the proliferation of melanoma cells

图3 与 FBXO38 相关的差异表达基因富集分析Note：A/B. Volcano plot of differentially expressed genes between different FBXO38 expression groups in TCGA SKCM (A) and UM (B). C/D. KEGG analysis of differentially expressed genes between different FBXO38 expression groups in SKCM (C) and UM (D) in TCGA datasets.

Fig 3 Enrichment analysis ofdifferentially expressed genes associated with FBXO38

图4 FBXO38 对PI3K-Akt信号通路和下游分子的调节Note： A/B. Detection of inhibition rate of different concentration of LY294002 (A) or Everolimus (B) on A375 cells after 24 h of treatment by using CCK8 assay. C/D. Detection of inhibition rate of different concentration of LY294002 (C) or Everolimus (D) on OMM2.3 cells after 24 h of treatment by using CCK8 assay. E/F. Expression of p-PI3K, p-Akt, p-mTOR, PTEN, MDM2, P53 and P21 in FBXO38 knockdown and control A375 cells (E) and OMM2.3 cells (F) detected by Western blotting. G/H. Transcription level of TP53 and MDM2 in FBXO38 knockdown and control A375 cells (G) and OMM2.3 cells (H) detected by qRT-PCR. ①P=0.003, ②P=0.030, ③P=0.044, ④P=0.006, ⑤P=0.013, ⑥P=0.001, ⑦P=0.041, ⑧P=0.017, ⑨P=0.012, ⑩P=0.004, ?P=0.011, ?P=0.027, ?P=0.015, ?P=0.006, ?P=0.014, ?P=0.033, 17P=0.040, 18P=0.048, 19P=0.005, 20P=0.020, 21P=0.042, 22P=0.024, 23P=0.008, 24P=0.023, 25P=0.010, 26P=0.002.

Fig 4 Regulation of FBXO38 on the PI3K-Akt signaling pathway and downstream molecular

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FBXO38通过PI3K-Akt信号通路调控眼部黑色素瘤增殖

FBXO38 regulates ocular melanoma proliferation through the PI3K-Akt signaling pathway

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