灵芝乙醇提取物对宫颈癌细胞生物学行为和JAK1/STAT3信号通路的影响

doi:10.13481/j.1671-587X.20220613

摘要/Abstract

摘要：

目的探讨灵芝乙醇提取物（GLEE）对宫颈癌细胞生物学行为和Janus激酶1（JAK1）/信号传导和转录激活因子3（STAT3）信号通路的影响，并阐明其可能的作用机制。方法将人宫颈癌HeLa细胞随机分为对照组（给予完全培养液）和低、中及高剂量GLEE组（给予25、50、100 mg·L^－1 GLEE）。MTT法检测各组细胞增殖抑制率，平板克隆实验检测各组克隆形成率，Transwell小室实验检测各组迁移细胞数和侵袭细胞数，实时荧光定量PCR（RT-qPCR）法检测各组细胞中JAK1和STAT3 mRNA表达水平，Western blotting法检测各组细胞中JAK1、磷酸化JAK1（p-JAK1）、STAT3、磷酸化STAT3（p-STAT3）、细胞因子信号传导抑制蛋白3（SOCS3）和活化STAT蛋白抑制因子1（PIAS1）蛋白表达水平。结果与对照组比较，低、中和高剂量GLEE组宫颈癌HeLa细胞增殖抑制率升高（P<0.05），细胞克隆形成率、迁移细胞数和侵袭细胞数降低（P<0.05），细胞中JAK1和STAT3 mRNA表达水平降低（P<0.05），细胞中p-JAK1和p-STAT3蛋白表达水平降低（P<0.05），细胞中SOCS3和PIAS1蛋白表达水平升高（P<0.05）。与低剂量GLEE组比较，中和高剂量GLEE组宫颈癌HeLa细胞增殖抑制率升高（P<0.05），细胞克隆形成率、迁移细胞数和侵袭细胞数降低（P<0.05），细胞中JAK1和STAT3 mRNA表达水平降低（P<0.05），细胞中p-JAK1和p-STAT3蛋白表达水平降低（P<0.05），细胞中SOCS3和PIAS1蛋白表达水平升高（P<0.05）。与中剂量GLEE组比较，高剂量GLEE组宫颈癌HeLa细胞增殖抑制率升高（P<0.05），细胞克隆形成率、迁移细胞数和侵袭细胞数降低（P<0.05），细胞中JAK1和STAT3 mRNA表达水平降低（P<0.05），细胞中p-JAK1和p-STAT3蛋白表达水平降低（P<0.05），细胞中SOCS3和PIAS1蛋白表达水平升高（P<0.05）。结论 GLEE对宫颈癌HeLa细胞增殖、克隆形成、迁移和侵袭均有一定的抑制作用，其可能是通过上调细胞中SOCS3及PIAS1表达水平，进而影响JAK1/STAT3信号通路发挥作用。

关键词: 灵芝乙醇提取物, 宫颈肿瘤, Janus激酶1, 信号传导和转录激活因子3, 细胞因子信号传导抑制蛋白3

Abstract:

Objective To investigate the effects of ganoderma lucidum ethanol extract （GLEE） on the biological behavior and Janus kinase 1（JAK1）/signal transduction and transcription activator 3 （STAT3） signaling pathway of the cervical cancer cells， and to clarify its possible mechanisms. Methods The human cervical cancer HeLa cells were randomly divided into control group （given complete culture medium） and low， medium and high doses of GLEE groups（given 25， 50， and 100 mg·L^－1 GLEE）. The inhibitory rates of proliferation of the human cervical cancer HeLa cells in various groups were detected by MTT assay； the clone formation rates of the human cervical cancer HeLa cells in various groups were detected by plate cloning experiment； Transwell chamber assay was used to detect the numbers of migration cells and invasion cells in various groups； the expression levels of JAK1 and STAT3 mRNA in the human cervical cancer HeLa cells in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method； the expression levels of JAK1， phophorylated JAK1（p-JAK1），STAT3， phophorylated STAT3（p-STAT3）， suppressor of cytokine signaling 3（SOCS3）， and protein inhibitor of activated STAT1 （PIAS1） proteins in the human cervical cancer HeLa cells in various groups were detected by Western blotting method. Results Compared with control group， the inhibitory rates of proliferation of the cervical cancer HeLa cells in low， medium and high doses of GLEE groups were increased （P<0.05），the clone formation rates the cervical cancer HeLa cells were decreased （P<0.05），the numbers of migration cells were decreased （P<0.05）， the numbers of invasion cells were decreased （P<0.05），the expression levels of JAK1 and STAT3 mRNA in the cervical cancer HeLa cells were decreased （P<0.05）， and the expression levels of SOCS3 and PIAS1 proteins in the cervical cancer HeLa cells were increased（P<0.05）. Compared with low dose of GLEE group， the inhibitory rates of proliferation of the cervical cancer HeLa cells in medium and high doses of GLEE groups were increased （P<0.05）， the clone formation rates of the cervical cancer HeLa cells were decreased （P<0.05）， the numbers of migration cells were decreased （P<0.05），the numbers of invasion cells were decreased （P<0.05）， the expression levels of JAK1 and STAT3 mRNA in the cervical cancer HeLa cells were decreased （P<0.05），and the expression levels of SOCS3 and PIAS1 proteins in the cervical cancer HeLa cells were increased （P<0.05）. Compared with midium dose of GLEE group， the inhibitory rate of proliferation of the cervical cancer HeLa cells in high dose of GLEE group was increased （P<0.05），the clone formation rate of the cervical cancer HeLa cells was decreased （P<0.05），the number of migration cells was decreased （P<0.05），the number of invasive cells was decreased （P<0.05）， the expression levels of JAK1 and STAT3 mRNA in the cervical cancer HeLa cells were decreased （P<0.05），and the expression levels of SOCS3 and PIAS1 proteins in the cervical cancer HeLa cells were increased （P<0.05）. Conclusion GLEE can inhibit the proliferation， cloning， migration and invasion of the cervical cancer HeLa cells to some extent， which may play a role by up-regulating the expression levels of SOCS3 and PIAS1 in the cells， thereby affecting the JAK1/STAT3 signaling pathway.

Key words: Ganoderma lucidum ethanol extract, Cervical neoplasms, Janus kinase 1, Signal transduction and transcription activator 3, Cytokine signaling inhibitor protein 3

中图分类号:

R730.52

任璐,曹芹雪,杨少琴. 灵芝乙醇提取物对宫颈癌细胞生物学行为和JAK1/STAT3信号通路的影响[J]. 吉林大学学报(医学版), 2022, 48(6): 1474-1480.

Lu REN,Qinxue CAO,Shaoqin YANG. Effects of ganoderma lucidum ethanol extract on biological behavior and JAK1/STAT3 signaling pathway of cervical cancer cells[J]. Journal of Jilin University(Medicine Edition), 2022, 48(6): 1474-1480.

图/表 7

表1

表2

图1

表3

表4

图2

表5

参考文献 20

1	HE Y H， SU R J， ZHENG J. Detection of DKK-1 gene methylation in exfoliated cells of cervical squamous cell carcinoma and its relationship with high risk HPV infection［J］.Arch Gynecol Obstet，2021，304（3）： 743-750.
2	BAIK S H， SEO J W， KIM J H， et al. Prognostic value of cervical nodal necrosis observed in preoperative CT and MRI of patients with tongue squamous cell carcinoma and cervical node metastases： a retrospective study［J］.AJR Am J Roentgenol，2019，213（2）： 437-443.
3	JIANG X F， SHI M Q， SUI M， et al. Oleanolic acid inhibits cervical cancer Hela cell proliferation through modulation of the ACSL4 ferroptosis signaling pathway［J］. Biochem Biophys Res Commun， 2021， 545： 81-88.
4	LAI P， CAO X， XU Q， et al. Ganoderma lucidum spore ethanol extract attenuates atherosclerosis by regulating lipid metabolism via upregulation of liver Ⅹreceptor alpha［J］. Pharm Biol， 2020， 58（1）： 760-770.
5	PARK M H， KIM M. Antioxidant and anti-inflammatory activity and cytotoxicity of ethanol extracts from rhynchosia nulubilis cultivated with ganoderma lucidum mycelium［J］.Prev Nutr Food Sci，2018，23（4）： 326-334.
6	LI P， LIU L X， HUANG S， et al. Anti-cancer effects of a neutral triterpene fraction from ganoderma lucidum and its active constituents on SW620 human colorectal cancer cells［J］. Anticancer Agents Med Chem， 2020， 20（2）： 237-244.
7	FAN Y R， SHENG W W， MENG Y， et al. LncRNA PTENP1 inhibits cervical cancer progression by suppressing miR-106b［J］. Artif Cells Nanomed Biotechnol， 2020， 48（1）： 393-407.
8	CAO L P， JIN H L， LIANG Q， et al. A new anti-tumor cytotoxic triterpene from ganoderma lucidum［J］. Nat Prod Res， 2022， 36（16）： 4125-4131.
9	ZHU L P， WU M， LI P， et al. High-pressure supercritical CO₂ extracts of ganoderma lucidum fruiting body and their anti-hepatoma effect associated with the ras/raf/MEK/ERK signaling pathway［J］. Front Pharmacol， 2020， 11： 602702.
10	LIU Z X， LI Y J， SHE G H， et al. Resveratrol induces cervical cancer HeLa cell apoptosis through the activation and nuclear translocation promotion of FOXO3a［J］. Pharmazie， 2020， 75（6）： 250-254.
11	方艳惠，马彩娟，张纯丽，等. miRNA-496通过mTOR影响宫颈癌HeLa细胞的恶性生物学行为及裸鼠移植瘤的生长［J］. 中国肿瘤生物治疗杂志， 2021， 28（11）： 1068-1074.
12	SOHRETOGLU D， HUANG S L. Ganoderma lucidum polysaccharides as an anti-cancer agent［J］. Anticancer Agents Med Chem， 2018， 18（5）： 667-674.
13	AHMAD M F， AHMAD F A， KHAN M I， et al. Ganoderma lucidum： a potential source to surmount viral infections through β-glucans immunomodulatory and triterpenoids antiviral properties［J］. Int J Biol Macromol， 2021， 187（9）： 769-779.
14	徐凤姣，曾琴，刘宏壁，等. 灵芝乙醇提取物抗肿瘤活性研究［J］. 中药药理与临床， 2016， 32（6）： 127-130.
15	ZENG R M， LU X H， LIN J， et al. FOXM1 activates JAK1/STAT3 pathway in human osteoarthritis cartilage cell inflammatory reaction［J］. Exp Biol Med （Maywood）， 2021， 246（6）： 644-653.
16	TAVERNA J A， HUNG C N， DEARMOND D T，et al.Single-cell proteomic profiling identifies combined AXL and JAK1 inhibition as a novel therapeutic strategy for lung cancer［J］. Cancer Res， 2020， 80（7）： 1551-1563.
17	罗保卫. miR-19a靶向调控SOCS3激活JAK1-STAT3通路对非小细胞肺癌生物学特性的影响［D］. 衡阳：南华大学， 2021.
18	李坤，王静，金春亭，等. lncRNA GAS5通过miR-26a靶向调控JAK1/STAT3信号通路对子宫内膜样癌细胞凋亡及侵袭的影响［J］. 中国优生与遗传杂志， 2022， 30（3）： 357-363.
19	张琰，马娜，王芳，等. 莪术醇抑制JAK1/STAT3信号通路对肺癌细胞增殖、凋亡和侵袭的影响［J］. 临床医学研究与实践， 2021， 6（34）： 10-15.
20	SIMS N A. The JAK1/STAT3/SOCS3 axis in bone development， physiology， and pathology［J］. Exp Mol Med， 2020， 52（8）： 1185-1197.

Group	Inhibitory rate		t	P
Group	(t/h) 24	48	t	P
Control	0	0	－	－
GLEE Low dose	7.83±0.60^*○	15.75±0.75^*○	11.619	<0.001
Medium dose	21.69±0.85^*△○	30.92±1.43^*△○	7.801	<0.001
High dose	38.36±1.25^*△#○	49.53±1.48^*△#○	8.163	<0.001

Group	Clone formation rate
Control	53.42±2.13
GLEE Low dose	38.69±1.13^*
Medium dose	19.77±0.94^*△
High dose	6.04±0.58^*△#

Group	Number of migration cells	Number of invasion cells
Control	112.61±6.31	94.56±4.39
GLEE Low dose	93.29±5.28^*	79.21±3.04^*
Medium dose	66.38±2.57^*△	56.44±2.34^*△
High dose	24.26±1.39^*△#	37.91±1.22^*△#

Group	JAK1 mRNA	STAT3 mRNA
Control	1.04±0.08	0.95±0.07
GLEE Low dose	0.82±0.07^*	0.64±0.04^*
Medium dose	0.66±0.04^*△	0.49±0.03^*△
High dose	0.41±0.02^*△#	0.33±0.02^*△#

Group	JAK1	p-JAK1	STAT3	p-STAT3	SOCS3	PIAS1
Control	0.79±0.05	0.75±0.05	0.84±0.07	0.83±0.07	0.35±0.02	0.27±0.03
GLEE Low dose	0.72±0.05	0.61±0.05^*	0.86±0.06	0.64±0.06^*	0.48±0.04^*	0.44±0.04^*
Medium dose	0.74±0.04	0.43±0.04^*△	0.85±0.04	0.49±0.04^*△	0.64±0.04^*△	0.59±0.05^*△
High dose	0.76±0.03	0.31±0.03^*△#	0.87±0.02	0.27±0.02^*△#	0.81±0.06^*△#	0.76±0.06^*△#