麝香酮调节SHH介导的自噬对卵巢癌细胞恶性进展的影响

doi:10.11958/20230541

摘要/Abstract

摘要：

目的探究麝香酮调节超音刺猬蛋白（SHH）介导的自噬对卵巢癌细胞恶性进展的影响。方法检测0、2、4、8、16、24 μmol/L的麝香酮处理后的人卵巢癌细胞SKOV3存活率，筛选出麝香酮最佳细胞作用浓度。体外培养SKOV3细胞并构建其移植瘤小鼠模型，随机均分为对照组，麝香酮组，麝香酮+自噬抑制剂氯喹（CQ）组，麝香酮+空载组，麝香酮+SHH过表达组，按照分组分别以麝香酮、CQ、空载质粒及SHH过表达质粒处理后，检测各组移植瘤小鼠肿瘤体积和质量；分别以EdU染色、TUNEL染色、细胞划痕、Transwell侵袭实验、免疫印迹法、实时荧光定量PCR检测SKOV3细胞增殖、凋亡、迁移、侵袭、SKOV3细胞及其移植瘤小鼠肿瘤组织自噬相关蛋白（LC3Ⅱ/LC3Ⅰ、Beclin-1）与SHH表达。结果与对照组相比，麝香酮组细胞增殖率、迁移率、侵袭数目、肿瘤体积和质量、肿瘤组织SHH mRNA及蛋白表达水平均降低（P<0.05），细胞凋亡率、细胞及肿瘤组织LC3Ⅱ/LC3Ⅰ和Beclin-1蛋白表达水平均升高（P<0.05）。与麝香酮组相比，麝香酮+CQ组和麝香酮+SHH过表达组细胞增殖率、迁移率、侵袭数目、肿瘤体积和质量升高（P<0.05），细胞凋亡率、细胞及肿瘤组织LC3Ⅱ/LC3Ⅰ和Beclin-1蛋白表达水平均降低（P<0.05）；麝香酮+SHH过表达组细胞及肿瘤组织SHH mRNA及蛋白表达水平升高（P<0.05）；麝香酮+空载组细胞各指标变化差异无统计学意义（P>0.05）。结论麝香酮可通过下调SHH而促进卵巢癌细胞自噬，进而抑制其增殖、体内生长、迁移及侵袭，促进其凋亡，最终抑制其恶性进展。

关键词: 卵巢肿瘤, 麝香酮, 超音刺猬蛋白, 自噬, 恶性进展

Abstract:

Objective To investigate the effect of muscone on malignant progression of ovarian cancer cells mediated by regulating sonic hedgehog (SHH) mediated autophagy. Methods Survival rates of human ovarian cancer cell line SKOV3 treated with 0, 2, 4, 8, 16, and 24 μmol/L muscone were detected, and the optimal cell action concentration of muscone was selected. SKOV3 cells were cultured in vitro, and their transplanted tumor mouse models were constructed. Cells were randomly grouped into the control group, the muskone group, the muskone+chloroquine (CQ，an autophagy inhibitor) group, the muskone+empty group and the muskone+SHH overexpression group. After grouping and treatment with musconeand, CQ, empty plasmid and SHH overexpression plasmid, the tumor volume and weight in transplanted tumor mice were detected. EdU staining, TUNEL staining, cell scratch, Transwell invasion assay, immunoblotting and real-time fluorescence quantitative PCR were used to detect proliferation, apoptosis, migration and invasion of SKOV3 cells, the expression of autophagy related proteins (LC3Ⅱ/LC3Ⅰ, Beclin-1) and SHH in SKOV3 cells and transplanted tumor mice in each. Results Compared with the control group, the cell proliferation rate, cell migration rate, number of invasions, tumor volume and weight, and the expression of SHH mRNA and protein in tumor tissue were decreased in the muskone group (P<0.05), and the apoptosis rate, LC3Ⅱ/LC3Ⅰ, Beclin-1 protein in cells and tumor tissue were increased (P<0.05). Compared with the muscone group, the cell proliferation rate, cell migration rate, number of invasion, tumor volume and weight were increased in the muscone+CQ group and the muskone+SHH overexpression group (P<0.05), and the apoptosis rate, LC3Ⅱ/LC3Ⅰ and the expression of Beclin-1 protein in cells and tumor tissue were decreased (P<0.05). There were no significant differences in the above indicators in the muscone+empty group (P>0.05). Conclusion Muscone can promote autophagy of ovarian cancer cells by down-regulating SHH, thereby inhibiting their proliferation, in vivo growth, migration and invasion, promoting their apoptosis, and ultimately inhibiting their malignant progression.

Key words: ovarian neoplasms, muscone, sonic hedgehog, autophagy, malignant progression

中图分类号:

R737.31

图/表 13

表1 基因引物序列

Tab.1 Gene primer sequences

基因名称	引物序列（5′→3′）	产物大小/bp
SHH	上游：TGTCTGCTGCTAGTCCTCGTCTC 下游：GTGCCTCCTCTTCGAACCC	265
GAPDH	上游：CAGGAGGCATTGCTGATGAT 下游：GAAGGCTGGGGCTCATTT	178

图1 不同浓度麝香酮对SKOV3细胞存活率的影响 F=83.820，n=6，P<0.05；a与0 μmol/L麝香酮比较，P<0.05。

Fig.1 Effects of different concentrations of muskone on survival rates of SKOV3 cells

表2 各组SKOV3移植瘤小鼠肿瘤体积和质量比较（n=10，$\bar{x}±s$）

Tab.2 Comparison of tumor volume and mass of SKOV3 transplanted tumor mice between five groups

组别	肿瘤体积/mm³	肿瘤质量/g
对照组	914.36±78.10	0.81±0.08
麝香酮组	218.75±43.56^a	0.22±0.05^a
麝香酮+CQ组	780.84±92.81^b	0.65±0.09^b
麝香酮+空载组	240.13±51.73	0.29±0.06
麝香酮+SHH过表达组	847.82±85.64^b	0.74±0.07^b
F	219.700^＊	142.100^＊

图2 EdU染色检测各组SKOV3细胞增殖（×200）

Fig.2 EdU staining for detecting SKOV3 cell proliferation in each group (×200)

图3 TUNEL染色检测各组SKOV3细胞凋亡（×200）

Fig.3 TUNEL staining for detecting apoptosis of SKOV3 cells in each group (×200)

表3 各组SKOV3细胞增殖率和凋亡率比较（n=6，%，$\bar{x}±s$）

Tab.3 Comparison of the proliferation rate and apoptosis rate of SKOV3 cells between five groups

组别	增殖率	凋亡率
对照组	42.50±5.14	1.63±0.51
麝香酮组	11.76±3.42^a	62.45±10.26^a
麝香酮+CQ组	32.36±4.80^b	2.14±0.63^b
麝香酮+空载组	12.13±2.94	54.35±11.24
麝香酮+SHH过表达组	38.85±4.87^b	2.48±0.73^b
F	69.090^＊	123.700^＊

图4 细胞划痕实验检测各组SKOV3细胞迁移（×200）

Fig.4 Wounding healing assay for detecting SKOV3 cell migration in each group (×200)

图5 Transwell侵袭实验检测各组SKOV3细胞侵袭（结晶紫染色，×200）

Fig.5 Transwell invasion assay for detecting invasion of SKOV3 cells in each group (crystal violet stain, ×200)

表4 各组SKOV3细胞迁移率与侵袭数目比较（n=6，$\bar{x}±s$）

Tab.4 Comparison of SKOV3 cell migration rate and invasion number between five groups

组别	迁移率/%	侵袭数目/（个/视野）
对照组	84.13±13.12	251.64±36.58
麝香酮组	19.79±3.51^a	85.42±12.23^a
麝香酮+CQ组	70.85±14.83^b	215.14±30.64^b
麝香酮+空载组	20.14±3.74	90.38±15.20
麝香酮+SHH过表达组	76.87±15.63^b	239.87±35.72^b
F	45.620^＊	51.230^＊

图6 免疫印迹检测各组SKOV3细胞及其移植瘤小鼠肿瘤组织自噬相关蛋白表达 A：对照组；B：麝香酮组；C：麝香酮+CQ组；D：麝香酮+空载组；E：麝香酮+SHH过表达组。

Fig.6 Immunoblotting detection of autophagy related protein expression in tumor tissue of SKOV3 cells and transplanted tumor mice in each group

表5 各组SKOV3细胞及其移植瘤小鼠肿瘤组织自噬相关蛋白相对表达水平比较（$\bar{x}±s$）

Tab.5 Comparison of relative expression levels of autophagy related proteins in tumor tissue of SKOV3 cells and transplanted tumor mice between five groups

组别	细胞（n=6）		肿瘤组织（n=10）
组别	Beclin-1	LC3Ⅱ/ LC3Ⅰ	Beclin-1	LC3Ⅱ/ LC3Ⅰ
对照组	0.51±0.08	0.33±0.04	0.37±0.04	0.41±0.08
麝香酮组	1.08±0.16^a	0.94±0.12^a	0.95±0.17^a	1.01±0.15^a
麝香酮+CQ组	0.58±0.07^b	0.36±0.06^b	0.43±0.06^b	0.46±0.07^b
麝香酮+空载组	1.10±0.13	0.92±0.15	0.96±0.13	1.02±0.14
麝香酮+SHH 过表达组	0.56±0.09^b	0.35±0.04^b	0.41±0.10^b	0.43±0.08^b
F	42.740^＊	70.190^＊	75.230^＊	85.140^＊

图7 免疫印迹检测各组SKOV3细胞及其移植瘤小鼠肿瘤组织SHH蛋白表达 A：对照组；B：麝香酮组；C：麝香酮+CQ组；D：麝香酮+空载组；E：麝香酮+SHH过表达组。

Fig.7 Immunoblotting detection of SHH protein expression in tumor tissue of SKOV3 cells and transplanted tumor mice in each group

表6 各组SKOV3细胞及其移植瘤小鼠肿瘤组织SHH mRNA、蛋白相对表达水平比较（$\bar{x}±s$）

Tab.6 Comparison of relative expression levels of SHH mRNA and protein in tumor tissue of SKOV3 cells and transplanted tumor mice between five groups

组别	细胞（n=6）		肿瘤组织（n=10）
组别	SHH mRNA	SHH蛋白	SHH mRNA	SHH蛋白
对照组	0.99±0.17	0.67±0.05	0.97±0.14	0.71±0.08
麝香酮组	0.38±0.06^a	0.14±0.03^a	0.32±0.07^a	0.20±0.05^a
麝香酮+CQ组	0.37±0.04	0.13±0.02	0.33±0.06	0.19±0.04
麝香酮+空载组	0.39±0.05	0.15±0.04	0.34±0.08	0.21±0.06
麝香酮+SHH 过表达组	0.94±0.13^b	0.62±0.08^b	0.91±0.19^b	0.65±0.11^b
F	45.090^＊	123.800^＊	71.100^＊	83.400^＊

参考文献 20

[1]	PORTER R, MATULONIS U A. Immunotherapy for ovarian cancer[J]. Clin Adv Hematol Oncol, 2022, 20(4):240-253.
[2]	KHANLARKHANI N, AZIZI E, AMIDI F, et al. Metabolic risk factors of ovarian cancer:a review[J]. JBRA Assist Reprod, 2022, 26(2):335-347. doi:10.5935/1518-0557.20210067.
[3]	FENG C, YUAN X X. Role of autophagy and its regulation by noncoding RNAs in ovarian cancer[J]. Exp Biol Med, 2023:15353702231151958. doi:10.1177/15353702231151958.
[4]	VIDONI C, FERRARESI A, VALLINO L, et al. Glycolysis inhibition of autophagy drives malignancy in ovarian cancer:exacerbation by IL-6 and attenuation by resveratrol[J]. Int J Mol Sci, 2023, 24(2):1723. doi:10.3390/ijms24021723.
[5]	齐娜, 段文娟, 李雅婧, 等. 麝香酮药理作用的研究进展[J]. 世界科学技术-中医药现代化, 2020, 22(8):3042-3047.
	QI N, DUAN W J, LI Y J, et al. Research Progress on the Pharmacological Action of Muscone[J]. Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology, 2020, 22(8):3042-3047. doi:10.11842/wst.20181223004.
[6]	ZHAO Y R, TAO S X, WANG Q, et al. A network-based pharmacological study on the mechanism of action of muscone in breast cancer[J]. Transl Cancer Res, 2022, 11(5):1195-1206. doi:10.21037/tcr-22-667.
[7]	卢鹏, 樊晶晶, 罗旭, 等. 麝香酮对肺癌细胞的顺铂耐药和小鼠体内的肿瘤生长的作用[J]. 广西医科大学学报, 2020, 37(11):1948-1953.
	LU P, FAN J J, LUO X, et al. The effect of Muscone on lung cancer cells resistance to cisplatin and tumor growth in mice[J]. J Guangxi Med Univ, 2020, 37(11):1948-1953. doi:10.16190/j.cnki.45-1211/r.2020.11.003.
[8]	ZHANG M, TAO Z Y, GAO L J, et al. Toosendanin inhibits colorectal cancer cell growth through the Hedgehog pathway by targeting Shh[J]. Drug Dev Res, 2022, 83(5):1201-1211. doi:10.1002/ddr.21951.
[9]	LONDERO A P, ORSARIA M, VIOLA L, et al. Survivin,sonic hedgehog,krüppel-like factors,and p53 pathway in serous ovarian cancer:an immunohistochemical study[J]. Hum Pathol, 2022, 127:92-101. doi:10.1016/j.humpath.2022.06.023.
[10]	赵飞, 于新平, 赵涵, 等. GLI1及Shh在卵巢型子宫内膜异位症恶变过程中的表达及其意义[J]. 中华妇产科杂志, 2022, 57(2):125-132.
	ZHAO F, YU X P, ZHAO H, et al. Expression and significance of GLI1 and Shh in the malignant transformation of ovarian endometriosis[J]. Chin J Obstet Gynecol, 2022, 57(2):125-132. doi:10.3760/cma.j.cn112141-20211219-00736.
[11]	ZHANG X Y, LIU Q B, ZHANG T T, et al. Bone-targeted nanoplatform enables efficient modulation of bone tumor microenvironment for prostate cancer bone metastasis treatment[J]. Drug Deliv, 2022, 29(1):889-905. doi:10.1080/10717544.2022.2050845.
[12]	PAN Y B, ZHOU J N, ZHANG W D, et al. The Sonic Hedgehog signaling pathway regulates autophagy and migration in ovarian cancer[J]. Cancer Med, 2021, 10(13):4510-4521. doi:10.1002/cam4.4018.
[13]	NAKAI H, MATSUMURA N. The roles and limitations of bevacizumab in the treatment of ovarian cancer[J]. Int J Clin Oncol, 2022, 27(7):1120-1126. doi:10.1007/s10147-022-02169-x.
[14]	BOSE S, SAHA P, CHATTERJEE B, et al. Chemokines driven ovarian cancer progression,metastasis and chemoresistance:potential pharmacological targets for cancer therapy[J]. Semin Cancer Biol, 2022, 86(Pt 2):568-579. doi:10.1016/j.semcancer.2022.03.028.
[15]	王志, 谢建絮, 李婉斯, 等. 麝香及西黄方中麝香酮在正常、乳腺癌癌前病变大鼠体内药动学的比较[J]. 中成药, 2020, 42(10):2545-2550.
	WANG Z, XIE J X, LI W S, et al. Comparison of in vivo pharmacokinetics of muscone from Moschus and Xihuang Decoction in normal rats and rats with precancerous lesions of breast cancer[J]. Chin Tradit Pat Med, 2020, 42(10):2545-2550. doi:10.3969/j.issn.1001-1528.2020.10.001.
[16]	任瑶, 江一鸣, 项蓉蓉, 等. 西黄丸组分中药调节肿瘤微环境中Treg细胞PI3K/AKT通路的抗肿瘤作用机制研究[J]. 药物评价研究, 2019, 42(3):437-443.
	REN Y, JIANG Y M, XIANG R R, et al. Antitumor mechanism of Xihuang Pills component-based Chinese medicine by regulating Treg cells PI3K/AKT pathway in tumor microenvironment[J]. Drug Eval Res, 2019, 42(3):437-443. doi:CNKI:SUN:YWPJ.0.2019-03-010.
[17]	CHEN S, GAO Y, ZHU P, et al. Anti-cancer drug anlotinib promotes autophagy and apoptosis in breast cancer[J]. Front Biosci (Landmark Ed), 2022, 27(4):125. doi:10.31083/j.fbl2704125.
[18]	ZHANG M J, YUE H D, HUANG X, et al. Novel platinum nanoclusters activate PI3K/AKT/mTOR signaling pathway-mediated autophagy for cisplatin-resistant ovarian cancer therapy[J]. ACS Appl Mater Interfaces, 2022, 14(43):48502-48514. doi:10.1021/acsami.2c15143.
[19]	SNEHA S, NAGARE R P, SIDHANTH C, et al. The hedgehog pathway regulates cancer stem cells in serous adenocarcinoma of the ovary[J]. Cell Oncol, 2020, 43(4):601-616. doi:10.1007/s13402-020-00504-w.
[20]	CHEN L P, LI Y, SONG Z H, et al. O-GlcNAcylation promotes cerebellum development and medulloblastoma oncogenesis via SHH signaling[J]. Proc Natl Acad Sci USA, 2022, 119(34):e2202821119. doi:10.1073/pnas.2202821119.