Expression and Clinical Significance of the Endoplasmic Reticulum PERK/eIF-2α Signaling Pathway in Cervical Cancer
-
摘要:
目的 探讨内质网应激蛋白激酶R样内质网激酶（stress protein kinase R-like endoplasmic reticulum kinase，PERK）/真核启动因子2α（eukaryotic initiation factor 2α，eIF-2α）信号通路在宫颈上皮内瘤变（cervical intraepithelial neoplasia，CIN）和宫颈癌（cervical carcinoma，CC）中的表达及其对肿瘤进程的影响。 方法 收集29例CC患者活检标本为宫颈癌组，26例宫颈上皮内瘤变（CIN Ⅱ级、CIN Ⅲ级）作为对照组，17例健康子宫患者的宫颈组织为正常组。Western blot法检内质网介导相关蛋白的表达；免疫组织化学法检测PERK/eIF-2α在CC中的染色状况。将HeLa细胞分为对照组、pcDNA3.1-NC组、过表达PERK组、sh-NC组、干扰PERK组，采用CCK-8法检测细胞增殖活性，免疫荧光染色观察细胞内上皮型钙黏蛋白（E-cadherin）表达，应用流式细胞术检测细胞凋亡与周期分布，Transwell实验评估细胞迁移与侵袭能力。 结果 随着CIN分级程度的增加，PERK/eIF-2α表达升高（P < 0.05），且CC组织PERK/eIF-2α表达显著高于CIN组织（P < 0.05）；与pcDNA3.1-NC组比较，过表达PERK组细胞增殖活性升高，细胞内E-cadherin荧光表达明显减弱（P < 0.05），E-cadherin蛋白表达下调（P < 0.05），促进细胞增殖、迁移与侵袭，抑制细胞凋亡（P < 0.05）；与sh-NC组比较，干扰PERK组细胞增殖活性降低，细胞内E-cadherin荧光表达则明显增强（P < 0.05），E-cadherin蛋白表达上调（P < 0.05），抑制细胞增殖、迁移与侵袭，促进细胞凋亡（P < 0.05）。 结论 内质网应激PERK/eIF-2α在子宫内瘤变和CC组织中表达升高，抑制其表达能够降低CC肿瘤细胞增殖，从而减缓肿瘤进程。 -
关键词:
- 宫颈癌 /
- 内质网应激 /
- 应激蛋白激酶R样内质网激酶 /
- 真核启动因子2α
Abstract:Objective : To investigate the expression of the endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase （PERK）/eukaryotic initiation factor 2α （eIF-2α） signaling pathway in cervical intraepithelial neoplasia （CIN） and cervical carcinoma （CC）, and its impact on tumor progression. Methods: Biopsy specimens from 29 CC patients were collected as the CC group, 26 cases of cervical intraepithelial neoplasia （CIN II and CIN III） served as the control group, and cervical tissues from 17 healthy uterine patients constituted the normal group. Western blot was used to detect the expression of endoplasmic reticulum-mediated related proteins; immunohistochemistry was employed to assess the staining status of PERK/eIF-2α in CC tissues. HeLa cells were divided into the following groups: control group, pcDNA3.1-NC group, overexpressed PERK group, sh-NC group, and PERK interference group. Cell proliferation activity was detected using the CCK-8 assay, and the expression of epithelial cadherin （E-cadherin） was observed by immunofluorescence staining. Apoptosis and cell cycle distribution were analyzed by flow cytometry, and cell migration and invasion ability were evaluated by Transwell assay. Results With increasing CIN grade, the expression of PERK/eIF-2α increased （P < 0.05）, and its expression in CC tissues was significantly higher than that in CIN tissues （P < 0.05）. Compared with the pcDNA3.1-NC group, cells in the PERK overexpression group enhanced proliferation activity, markedly reduced intracellular E-cadherin fluorescence expression （P < 0.05）, and downregulated E-cadherin protein expression （P < 0.05）, accompanied by promotion of cell proliferation, migration, and invasion, as well as inhibition of apoptosis （P < 0.05）. In contrast, compared with the sh-NC group, cells in the PERK interference group showed decreased proliferation activity, significantly increased intracellular E-cadherin fluorescence expression （P < 0.05）, upregulated E-cadherin protein expression （P < 0.05）, along with inhibition of cell proliferation, migration, and invasion, and promotion of apoptosis （P < 0.05）. Conclusion The expression of the endoplasmic reticulum stress-related PERK/eIF-2α pathway is upregulated in uterine intraepithelial neoplasia and CC tissues. Inhibition of its expression can reduce the tumor cell proliferation in CC, thereby slowing tumor progression. -
图 1 各组组织中PERK、eIF-2α染色水平（×100）（ni = 3，$\bar x \pm s $）
Figure 1. PERK and eIF-2αstaining levels in tissues of each group （×100）（ni = 3，$\bar x \pm s $）
图 2 Western blot检测各组组织中PERK/eIF-2α通路及内质网相关指标蛋白条带及各组组织中PERK、p-eIF-2α、ATF4、ATF6、CHOP、XBP-1和Caspase12蛋白表达比较分析（ni = 3，$\bar x \pm s $）
注：A：Western blot检测各组组织中PERK/eIF-2α通路及内质网相关指标蛋白条带；B：各组组织中PERK、p-eIF-2α、ATF4、ATF6、CHOP、XBP-1和Caspase12蛋白表达比较分析。与正常组相比，*P < 0.05，**P < 0.01，***P < 0.001；与CINⅡ组相比，#P < 0.05，##P < 0.01，###P < 0.001；与CIN Ⅲ组相比，△P < 0.05，△△P < 0.01，△△△P < 0.001。
Figure 2. Western blot analysis of PERK/eIF-2α pathway proteins and endoplasmic reticulum-related protein bands in tissues of each group，and comparative analysis of PERK，p-eIF-2α，ATF4，ATF6，CHOP，XBP-1，and Caspase12 protein expression （ni = 3，$\bar x \pm s $）
图 3 各组HeLa细胞中PERK、eIF-2α蛋白表达条带及PERK、eIF-2α mRNA和蛋白表达比较（ni = 3，$\bar x \pm s $）
注：A、C：各组HeLa细胞中PERK、eIF-2α蛋白表达条带；B、D：各组细胞中PERK、eIF-2α mRNA和蛋白表达比较。与pcDNA3.1-NC组相比，*P < 0.05；与sh-NC组相比，*P < 0.05。
Figure 3. PERK and eIF-2α protein expression bands in HeLa cells of each group，and comparison of PERK and eIF-2α mRNA and protein expression （ni = 3，$\bar x \pm s $）
图 4 各组细胞吸光度值比较（ni = 6，$\bar x \pm s $）
注：A：pcDNA3.1-NC组和表达PERK组吸光度值比较；B：sh-NC组与干扰PERK组吸光度值比较。与pcDNA3.1-NC组相比，*P < 0.05；B注：与sh-NC组相比，*P < 0.05。
Figure 4. Comparison of absorbance values among cell groups （ni = 6，$\bar x \pm s $）
图 5 各组HeLa细胞转化标志物E-cadherin表达检测（×100）（ni = 3）
Figure 5. Expression of the transformation marker E-cadherin in HeLa cells of each group （×100） （ni = 3）
图 6 Transwell实验检测细胞迁移能力（ni = 3）
Figure 6. Cell migration ability assessed by Transwell assay （ni = 3）
图 7 Transwell实验检测细胞侵袭能力（×100）（ni = 3）
Figure 7. Cell invasion ability assessed by Transwell assay （×100） （ni = 3）
表 1 各组组织中PERK、eIF-2α、ATF4、ATF6、CHOP、XBP-1和Caspase12 mRNA表达比较（ni = 6，$\bar x \pm s $）
Table 1. Comparison of mRNA expression of PERK，eIF-2α，ATF4，ATF6，CHOP，XBP-1，and Caspase12 in tissues of each group（ni = 6，$\bar x \pm s $）
分组 PERK
mRNAeIF-2α
mRNAATF4
mRNAATF6
mRNACHOP
mRNAXBP-1
mRNACaspase12
mRNA正常组 1.01 ± 0.12 1.00 ± 0.12 1.01 ± 0.03 1.00 ± 0.07 1.01 ± 0.04 1.00 ± 0.10 1.00 ± 0.13 CINⅡ组 1.29 ± 0.17∆ 1.28 ± 0.18∆ 1.27 ± 0.06∆∆∆ 1.36 ± 0.35∆ 1.22 ± 0.18∆ 1.25 ± 0.18∆ 1.27 ± 0.19∆ CIN Ⅲ组 1.36 ± 0.22∆∆ 1.31 ± 0.14∆∆ 1.45 ± 0.04∆∆∆ 1.39 ± 0.17∆∆ 1.38 ± 0.22∆∆ 1.33 ± 0.20∆∆ 1.30 ± 0.13∆ 宫颈癌组 1.57 ± 0.12∆∆∆▲# 1.72 ± 0.17∆∆∆▲▲## 1.60 ± 0.05∆∆∆▲▲▲### 1.75 ± 0.13∆∆∆▲# 1.63 ± 0.17∆∆∆▲▲▲## 1.65 ± 0.12△△△▲▲▲### 1.69 ± 0.30∆∆∆▲▲# F 12.29 22.20 170.4 13.04 15.48 17.82 12.38 P <0.01** <0.01** <0.001*** <0.01** <0.01** <0.01** <0.01** MS组内 0.026 0.024 0.384 0.044 0.027 0.024 0.040 注：**P < 0.01，***P < 0.001；与正常组相比，∆P < 0.05，∆∆P < 0.01，∆∆∆P < 0.001；与CINⅡ组相比，▲P < 0.05，▲▲P < 0.01，▲▲▲P < 0.001；与CIN Ⅲ组相比，#P < 0.05，##P < 0.01，###P < 0.001。 
下载: 导出CSV
表 2 流式细胞仪检测细胞凋亡（ni = 3，$\bar x \pm s $）
Table 2. Cell apoptosis measured by flow cytometry（ni = 3，$\bar x \pm s $）
组别 早期凋亡率（%） 晚期凋亡率（%） 总凋亡率（%） Control 组 2.23 ± 0.06 0.15 ± 0.05 2.38 ± 0.10 pcDNA3.1-NC 组 3.50 ± 0.10 0.32 ± 0.03 3.82 ± 0.12 过表达 PERK 组 3.83 ± 0.06 1.67 ± 0.08 5.50 ± 0.09 sh-NC 组 4.77 ± 0.08 0.65 ± 0.05 5.42 ± 0.12 干扰 PERK 组 13.53 ± 0.35 1.31 ± 0.11 14.85 ± 0.41 F 2117.00 118787.00 1674.00 P <0.001* <0.001* <0.001* *P < 0.05。
下载: 导出CSV
-
[1] Buskwofie A, David-West G, Clare C A. A review of cervical cancer: Incidence and disparities[J]. J Natl Med Assoc, 2020, 112(2): 229-232. [2] Bertone F, Serusi E, Liscia D S, et al. Thyroid gland metastasis from endometrial carcinoma causing acute severe dyspnea[J]. Ear Nose Throat J, 2024, 103(3): NP142-NP144. doi: 10.1177/01455613211045564 [3] 徐海花, 孙蓬明. Toll 样受体与输卵管疾病[J]. 中国生育健康杂志, 2020, 31(3): 289-291. [4] 李海英, 蔡竞, 朱虹丽. 宫颈癌的临床治疗研究进展[J]. 宁夏医科大学学报, 2020, 42(4): 419-422. doi: 10.19844/j.cnki.1672-397X.2024.11.020 [5] 李洵, 付晓霞, 樊丽伟, 等. 乔松素抑制人胃癌细胞系 AGS 增殖、迁移和侵袭的研究[J]. 基础医学与临床, 2022, 42(11): 1744-1749. doi: 10.16352/j.issn.1001-6325.2022.11.1744 [6] 李聪聪, 赵鹏, 秦燕勤, 等. 淫羊藿苷的药理活性研究进展[J]. 中医学报, 2020, 35: 781-786. doi: 10.16368/j.issn.1674-8999.2020.04.175 [7] Chen H, Chen G M, Liu Y J, et al. Alpha-fetoprotein/endoplasmic reticulum stress signaling mitigates injury in hepatoma cells[J]. Neoplasma, 2021, 68(5): 983-993. doi: 10.4149/neo_2021_210205n180 [8] Izadpanah A, Willingham K, Chandrasekar B, et al. Unfolded protein response and angiogenesis in malignancies[J]. Biochim Biophys Acta BBA Rev Cancer, 2023, 1878(2): 188839. doi: 10.1016/j.bbcan.2022.188839 [9] Kumar A, Kumar N, Pathak Z, et al. Extra cellular matrix remodeling: An adjunctive target for spinal cord injury and intervertebral disc degeneration[J]. Neurospine, 2022, 19(3): 632-645. doi: 10.14245/ns.2244366.183 [10] Oakes S A. Endoplasmic reticulum stress signaling in cancer cells[J]. Am J Pathol, 2020, 190(5): 934-946. doi: 10.1016/j.ajpath.2020.01.010 [11] Lemmer I L, Willemsen N, Hilal N, et al. A guide to understanding endoplasmic reticulum stress in metabolic disorders[J]. Mol Metab, 2021, 47: 101169. doi: 10.1016/j.molmet.2021.101169 [12] 周琦, 吴小华, 刘继红, 等. 宫颈癌诊断与治疗指南(第四版)[J]. 中国实用妇科与产科杂志, 2018, 34(6): 613-622. [13] 中华医学会妇科肿瘤学分会. 中国宫颈癌及癌前病变诊疗指南(2022年版)[J]. 中华妇产科杂志, 2022, 57(4): 256-269. [14] Magaki S, Hojat S A, Wei B, et al. An introduction to the performance of immunohistochemistry[J]. Methods Mol Biol, 2019, 1897: 289-298. [15] Hernandez I, Cohen M. Linking cell-surface GRP78 to cancer: From basic research to clinical value of GRP78 antibodies[J]. Cancer Lett, 2022, 524: 1-14. doi: 10.1016/j.canlet.2021.10.004 [16] Khan M, Sharma A, Hathorn T, et al. The mucosal manifestations of syphilis in the head and neck[J]. Ear Nose Throat J, 2025, 104(12): NP868-NP881. [17] Dhara V, Hoda N, Rajini B C, et al. Significance of cervical node necrosis in preoperative MRI as a prognostic indicator: Retrospective study of patients with SCC of tongue[J]. J Oral Med Oral Surg, 2021, 27(3): 43. doi: 10.1051/mbcb/2021021 [18] 刘红, 王增四, 高文, 等. 黄芪甲苷对 STZ 诱导糖尿病大鼠肾组织内质网应激及 CHOP 信号通路的影响[J]. 中国医院药学杂志, 2021, 41(13): 1318-1323. doi: 10.13286/j.1001-5213.2021.13.08 [19] Yoo Y M, Joo S S. Melatonin can modulate neurodegenerative diseases by regulating endoplasmic reticulum stress[J]. Int J Mol Sci, 2023, 24(3): 2381. doi: 10.3390/ijms24032381 [20] 林善婷, 胡晓, 李来好, 等. 水产蛋白源生物活性肽研究进展[J]. 大连海洋大学学报, 2020, 35: 775-785. doi: 10.16535/j.cnki.dlhyxb.2019-266 [21] 练毅, 宫丽荣, 余剑波, 等. 内质网应激介导的细胞凋亡与急性肺损伤的研究进展[J]. 中国中西医结合外科杂志, 2021, 27(2): 3. doi: 10.3969/j.issn.1007-6948.2021.02.039 [22] 童凯, 罗红兰. 洛铂通过PERK/eIF2α/ATF4/CHOP信号通路调节肺癌细胞A549的作用机制[J]. 世界临床药物, 2023, 44(8): 805. doi: 10.13683/j.wph.2023.08.007 [23] 张超云, 费娜, 郝鹏飞, 等. 基于内质网应激探讨葛根芩连汤对非酒精性脂肪肝大鼠PERK-eIF2α通路的影响[J]. 中国医院药学杂志, 2024, 44(4): 384-390. doi: 10.13286/j.1001-5213.2024.04.04 -
点击查看大图
计量
- 文章访问数: 47
- HTML全文浏览量: 35
- PDF下载量: 14
- 被引次数: 0
