槐耳多糖对宫颈癌CaSki细胞增殖、凋亡、迁移侵袭的影响及其作用机制研究

Study on effect of Huai′er polysaccharide on proliferation, apoptosis, migration and invasion of cervical cancer CaSki cells and its mechanism

摘要

目的 探讨槐耳多糖(HP)对宫颈癌CaSki细胞生物学行为的影响及其作用机制。方法 体外培养人正常宫颈上皮细胞HcerEpic及宫颈癌细胞CaSki,根据不同处理方法将CaSki分为Con组、HP-L组、HP-M组、HP-H组、pcDNA组、pcDNA-非受体型蛋白质酪氨酸磷酸酶9(PTPN9)组、HP-H+si-NC组、HP-H+si-PTPN9组.采用实时定量PCR(qRT-PCR)分析PTPN9在HcerEpic细胞及各组Caski细胞中的表达情况;利用甲基噻唑基四唑(MTT)法评估细胞增殖能力;通过克隆形成实验测定细胞克隆生成效率;应用流式细胞术检测细胞凋亡水平;划痕实验用于观察细胞迁移能力,Transwell小室实验则评估细胞的侵袭行为;Western blot技术用于检测细胞PTPN9、活化半胱氨酸天冬氨酸蛋白酶3(cleaved-caspase3)、基质金属蛋白酶-2(MMP-2)、活化半胱氨酸天冬氨酸蛋白酶9(cleaved-caaspase9)、基质金属蛋白酶-9(MMP-9)表达水平。结果 与人正常宫颈上皮细胞HcerEpic相比,PTPN9 mRNA、PTPN9蛋白在宫颈癌CaSki细胞中表达水平显著降低(t=18.665、9.758,P<0.05);与Con组比较,细胞凋亡率、PTPN9 mRNA、PTPN9蛋白、cleaved-caspase3蛋白、cleaved-caspase9蛋白表达水平在HP-L、HP-M、HP-H组显著升高,且HP-H组>HP-M组>HP-L组(F=114.333、104.110、45.244、101.093、88.405,P<0.05),(24h、48h、72h)吸光度(OD)值、细胞克隆形成数、划痕愈合率、侵袭细胞数、MMP-2蛋白、MMP-9蛋白水平在HP-L、HP-M、HP-H组显著降低,且HP-H组<HP-M组<HP-L组(F=34.286、17.209、18.749、40.868、44.577、27.125、110.816、60.721,P<0.05);与Con组、pcDNA组相比,pcDNA-PTPN9组细胞凋亡率、PTPN9 mRNA、PTPN9蛋白、cleaved-caspase3蛋白、cleaved-caspase9蛋白表达水平较高(F=226.034、217.858、95.392、196.636、168.796,P<0.05),(24h、48h、72h)OD值、侵袭细胞数、划痕愈合率、MMP-2蛋白、MMP-9蛋白水平、细胞克隆形成数较低(F=21.828、19.793、15.525、31.634、49.353、109.400、65.314、42.140,P<0.05);与HP-H、HP-H+si-NC组相比,HP-H+si-PTPN9组细胞凋亡率、PTPN9 mRNA、PTPN9蛋白、cleaved-caspase3蛋白、cleaved-caspase9蛋白表达水平较低(F=100.025、74.414、46.500、69.500、63.495,P<0.05),(24h、48h、72h)OD值、侵袭细胞数、划痕愈合率、MMP-9蛋白、MMP-2蛋白水平、细胞克隆形成数较高(F=28.773、35.559、22.211、74.114、75.064、111.636、102.091、56.908,P<0.05)。结论 槐耳多糖可能通过调控PTPN9的表达进而调控宫颈癌CaSki细胞的增殖、凋亡、迁移侵袭.

Abstract

Objective To investigate effect of Huai′er polysaccharide (HP) on biological behavior of cervical cancer CaSki cells and its mechanism. Methods Human normal cervical epithelial cells HcerEpic and cervical cancer Caski cells were cultured in vitro. The cervical cancer CaSki cells were divided into Con group, HP-L group, HP-M group, HP-H group, pcDNA group, pcDNA-PTPN9 group, HP-H+si-NC group and HP-H+si-PTPN9 group according to different treatment methods. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect expression levels of protein tyrosine phosphatase non-receptor type 9 (PTPN9) in HcerEpic cells and various groups of CaSki cells. Methyl thiazolyl tetrazolium (MTT) assay was used to detect proliferation activity of the cells in each group. Cell clone formation experiment was used to detect number of cell clones formed. Flow cytometry was used to determine apoptosis rate of the cells. The scratch test was used to detect cell migration. The Transwell experiment was used to detect number of cell invasion. Western blot assay was used to detect expression levels of PTPN9, cleaved-caaspase3, matrix metalloproteinase 2 (MMP-2), cleaved-caaspase9 and matrix metalloproteinase 9 (MMP-9) in the cells. Results Compared with normal cervical epithelial cells HcerEpic, the expression levels of PTPN9 mRNA and PTPN9 protein in the cervical cancer CaSki cells were significantly lower (t=18.665 and 9.758 respectively, both P<0.05). Compared with the Con group, the apoptosis rate and the expression levels of PTPN9 mRNA, PTPN9 protein, cleaved-caspase3 protein and cleaved-caspase9 protein in the HP-L group, the HP-M group and the HP-H group were significantly higher, and the data in the HP-H group>those in the HP-M group>those in the HP-L group (F=114.333, 104.110, 45.244, 101.093 and 88.405 respectively, all P<0.05). Optical density (OD) values (at 24h, 48h, 72h), number of cell clones formed, scratch healing rate, number of invasive cells, expression levels of MMP-2 protein and MMP-9 protein in the HP-L group, the HP-M group and the HP-H group were significantly lower, and the data in the HP-H group<those in the HP-M group<those in the HP-L group (F=34.286, 17.209, 18.749, 40.868, 44.577, 27.125, 110.816 and 60.721 respectively, all P<0.05). Compared with the Con group and the pcDNA group, apoptosis rate and expression levels of PTPN9 mRNA, PTPN9 protein, cleaved-caspase3 protein and cleaved-caspase9 protein in the pcDNA-PTPN9 group were higher (F=226.034, 217.858, 95.392, 196.636 and 168.796 respectively, all P<0.05), while OD values (at 24h, 48h, 72h), number of invasive cells, scratch healing rate, expression levels of MMP-2 protein and MMP-9 protein and number of cell clones formed were lower (F=21.828, 19.793, 15.525, 31.634, 49.353, 109.400, 65.314 and 42.140 respectively, all P<0.05). Compared with the HP-H group and the HP-H+si-NC group, apoptosis rate and expression levels of PTPN9 mRNA, PTPN9 protein, cleaved-caspase3 protein and cleaved-caspase9 protein in the HP-H+si-PTPN9 group were lower (F=100.025, 74.414, 46.500, 69.500 and 63.495 respectively, all P<0.05), while OD values (at 24h, 48h, 72h), number of invasive cells, scratch healing rate, expression levels of MMP-9 protein and MMP-2 protein, and number of cell clones formed were higher (F=28.773, 35.559, 22.211, 74.114, 75.064, 111.636, 102.091 and 56.908 respectively, all P<0.05). Conclusion HP may regulate proliferation, apoptosis, migration and invasion of the cervical cancer CaSki cells by regulating expression of PTPN9.

关键词

槐耳多糖 / 非受体型蛋白质酪氨酸磷酸酶9 / 宫颈癌细胞 / 增殖 / 迁移 / 侵袭 / 凋亡

Key words

Huai′er polysaccharide / non-receptor protein tyrosine phosphatase 9 / cervical cancer cell / proliferation / migration / invasion / apoptosis

参考文献

[1] 高飞, 王永存, 尹纪来, 等. miRNA-182水平与宫颈癌患者临床病理特征及预后的关系[J]. 中国妇幼健康研究, 2025, 36(3): 21-29.

[2] 窦丹彬, 张新. IL-6/STAT3在宫颈癌发生发展中作用的研究进展[J]. 现代妇产科进展, 2023, 32(5): 397-398, 400.

[3] 汪军坚, 方静, 赵春. 益气托毒生肌汤联合铜绿假单胞菌治疗宫颈癌淋巴结清扫术后淋巴漏的效果观察[J]. 浙江医学, 2023, 45(15): 1626-1630, 1646.

[4] 雷蕾, 高彦茹, 蔡洲, 等. 槐耳多糖通过抑制AKT/GSK3β/Snail信号通路抑制胰腺癌细胞增殖和上皮间质转化[J]. 现代肿瘤医学, 2023, 31(24): 4536-4541.

[5] 陈嘉丽, 韩梦媛, 王冬明. 槐耳多糖调节YAP/TAZ信号通路对骨肉瘤细胞生物学行为的影响[J]. 西部医学, 2024, 36(3): 344-350.

[6] Hu B, Yan W, Wang M, et al. Huai′er polysaccharide inhibits the stem-like characteristics of ERα-36 high triple negative breast cancer cells via inactivation of the ERα-36 signaling pathway[J]. Int J Biol Sci, 2019, 15(7): 1358-1367.

[7] 李月华, 曹爽. 过表达PTPN9基因抑制宫颈癌细胞恶性表型的机制研究[J]. 中国性科学, 2020, 29(10): 30-34.

[8] 张泽敏, 庞岚. 槐耳多糖抑制AEG-1表达对子宫内膜癌细胞生物学行为的影响[J]. 川北医学院学报, 2023, 38(10): 1299-1305.

[9] Zhao L, Liu J, Li K, et al. PTPN9 dephosphorylates FGFR2 pY656/657 through interaction with ACAP1 and ameliorates pemigatinib effect in cholangiocarcinoma[J]. Hepatology, 2024, 79(4): 798-812.

[10] Wang D, Cheng Z, Zhao M, et al. PTPN9 induces cell apoptosis by mitigating the activation of Stat3 and acts as a tumor suppressor in colorectal cancer[J]. Cancer Manag Res, 2019, 8(11): 1309-1319.

[11] Chen J, Ding C, Yang X, et al. BMSCs-Derived Exosomal MiR-126-3p Inhibits the Viability of NSCLC Cells by Targeting PTPN9[J]. J BUON, 2021, 26(5): 1832-1841.

[12] Zhu L, Guo T, Chen W, et al. CircMMD_007 promotes oncogenic effects in the progression of lung adenocarcinoma through microRNA-197-3p/protein tyrosine phosphatase non-receptor type 9 axis[J]. Bioengineered, 2022, 13(3): 4991-5004.

[13] Ma X, Shi W, Peng L, et al. MiR-96 enhances cellular proliferation and tumorigenicity of human cervical carcinoma cell through PTPN9[J]. Saudi J Biol Sci, 2018, 25(5): 863-867.

[14] Tian Y, Wu J, Zeng L, Zhou L, et al. Huaier polysaccharides suppress triple-negative breast cancer metastasis and epithelial-mesenchymal transition by inducing autophagic degradation of Snail[J]. Cell Biosci, 2021, 11(1): 170-182.

[15] Bao H, Liu P, Jiang K, et al. Huaier polysaccharide induces apoptosis in hepatocellular carcinoma cells through p38 MAPK[J]. Oncol Lett, 2016, 12(2): 1058-1066.

[16] Ma Y, Wang C, Zhang Q, et al. The effects of polysaccharides from Auricularia auricula (Huaier) in adjuvant anti-gastrointestinal cancer therapy: A systematic review and network meta-analysis[J]. Pharmacol Res, 2018, 132(2): 80-89.

[17] Li W T, Wang B L, Yang C S, et al. MiR-613 promotes cell proliferation and invasion in cervical cancer via targeting PTPN9[J]. Eur Rev Med Pharmacol Sci, 2018, 22(13): 4107-4114.

[18] Zhang X, Sun K, Wang X, et al. Chlorpyrifos induces apoptosis and necroptosis via the activation of CYP450s pathway mediated by nuclear receptors in LMH cells[J]. Environ Sci Pollut Res Int, 2023, 30(1): 1060-1071.

[19] Wang H, Liu Y, Guo Z, et al. Aconitine induces cell apoptosis via mitochondria and death receptor signaling pathways in hippocampus cell line[J]. Res Vet Sci, 2022, 143(1): 124-133.

[20] Kazazi I, Ashrafi F, Malekloo M. Synthesis of Gingerol-loaded Uio-66 nanoparticles and its anti-cancer effect against gastric cancer cell line (AGS)[J]. Mol Biol Rep, 2023, 50(4): 3503-3513.

[21] Zhang Y, Lin L, Wang Y, et al. Saikosaponin B4 Suppression Cancer Progression by Inhibiting SW480 and SW620 Cells Proliferation via the PI3K/AKT/mTOR Pathway in Colon Cancer[J]. Curr Cancer Drug Targets, 2022, 22(11): 889-903.

[22] 陈良凤, 吕小慧, 王建, 等. 基于LDHA基因和Wnt/β-catenin信号通路探讨自噬在卵巢癌细胞侵袭和迁移及糖酵解中的作用[J]. 中国妇幼健康研究, 2025, 36(2): 37-46.

[23] Muscella A, Vetrugno C, Cossa L G, et al. TGF-β1 activates RSC96 Schwann cells migration and invasion through MMP-2 and MMP-9 activities[J]. J Neurochem, 2020, 153(4): 525-538.

[24] 李侠, 肖维. 多层螺旋CT联合血清MMP-2、MMP-9在宫颈癌淋巴结转移诊断中的价值分析[J]. 中国CT和MRI杂志, 2021, 19(9): 108-110.

基金资助

国家自然科学基金(81172458)

陕西省自然科学基础研究计划重点项目(2024JCGZDXMG52)