Ras相关结构域家族成员5对头颈鳞癌细胞迁移和侵袭能力的影响

doi:10.3969/j.issn.1674-8115.2021.06.003

摘要/Abstract

摘要：

目的·探讨Ras相关结构域家族成员5（Ras association domain family 5，RASSF5）对头颈鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）细胞侵袭和迁移能力的影响。方法·通过基因表达谱交互分析（Gene Expression Profiling Interactive Analysis，GEPIA）平台分析癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库中563例HNSCC的RASSF5基因的表达情况及其对患者预后生存的影响。通过CMV-MCS-PGK-Puro载体构建RASSF5过表达慢病毒（LV RASSF5）和对照慢病毒（LV vector）。通过划痕实验、Transwell迁移和侵袭实验分析RASSF5过表达对HNSCC细胞Cal27和HN30迁移和侵袭能力的影响；通过Western blotting分析RASSF5过表达对上皮型钙黏蛋白（epithelial cadherin，E-cadherin）、Snail蛋白、糖原合酶激酶3β（glycogen synthase kinase 3β，GSK-3β）和磷酸化GSK-3β（phosphorylated GSK-3β，p-GSK-3β）的作用。构建BALB/c裸鼠肺转移模型，分别经尾静脉注射转染LV RASSF5（RASSF5过表达组）或LV vector（对照组）的Cal27细胞，每组5只小鼠；通过观察裸鼠肺部转移瘤结节数量分析过表达RASSF5对Cal27细胞转移能力的影响。结果·RASSF5基因在HNSCC中的表达水平低于正常组织（P=0.001），低表达RASSF5的患者总体生存期（P=0.005）和无病生存期（P=0.004）较差。划痕实验显示转染LV RASSF5后，Cal27细胞和HN30细胞在观察终点的划痕面积高于相应的对照组细胞（P=0.003，P=0.015）。Transwell迁移实验显示转染LV RASSF5后，Cal27细胞和HN30细胞穿出小室膜的数量低于相应对照组细胞（P=0.005，P=0.001）。Transwell侵袭实验同样显示转染LV RASSF5的Cal27细胞和HN30细胞穿出预铺基质胶小室膜细胞数量低于对照组（P=0.001，P=0.001）。Western blotting结果显示过表达RASSF5的Cal27和HN30细胞中，E-cadherin表达水平提高，而p-GSK-3β/GSK-3β比值以及Snail蛋白质水平降低。在裸鼠肺转移模型中，RASSF5过表达组的肺部瘤结节数量少于对照组（P=0.049）。结论·RASSF5过表达抑制HNSCC细胞的侵袭和迁移能力，而通过GSK-3β/Snail信号通路抑制上皮间质转化可能在其中起到关键作用。

Abstract:

Objective·To investigate the effect of Ras association domain family 5 (RASSF5) on migration and invasion of head and neck squamous cell carcinoma (HNSCC).

Methods·The expression of RASSF5 gene in 563 cases of HNSCC from the Cancer Genome Atlas (TCGA) database and the effect of RASSF5 expression on prognosis and survival of patients were analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) platform. The lentivirus overexpressing RASSF5 (LV RASSF5) and control lentivirus (LV vector) were constructed by CMV-MCS-PGK-Puro vector. The wound-healing assay and the Transwell assay were performed to test the invasion and metastasis capacity of overexpressed-RASSF5 HNSCC cells. In addition, the expressions of epithelial cadherin (E-cadherin), Snail, glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β (p-GSK-3β) were determined by Western blotting in RASSF5-overexpressed HNSCC cells. The lung metastasis model of HNSCC in 10 BALB/c nude mice were established. Cal27, which were transfected with LV RASSF5 or LV vector, were respectively injected into 5 BALB/c nude mice via tail vein, and the quantity of metastasis nodules was counted to evaluate the effect of RASSF5 overexpression on tumor metastasis ability.

Results·In HNSCC, the gene expression level of RASSF5 was lower than that in normal tissues (P=0.001). Patients with lower gene expression of RASSF5 had worse overall survival (P=0.005) and disease-free survival (P=0.004). The relative healing areas of Cal27 and HN30 transfected with LV RASSF5 were higher than those of the control group (transfected with LV vector) at the experimental endpoint of the wound-healing assay (P=0.015, P=0.003). The number of cells that had traversed the cell-permeable membrane was higher in Cal27 and HN30 cells transfected with LV RASSF5 than those in the control group in the Transwell migration assay (P=0.005, P=0.001). And in the Transwell invasion assay, the number of invaded cells that had traversed the matrigel-coated membrane was also higher in Cal27 and HN30 cells transfected with LV RASSF5 than those in the control group (P=0.001, P=0.001). Western blotting showed an increased level of E-cadherin, and decreased level of Snail and p-GSK-3β/GSK-3β ratio in RASSF5 overexpression Cal27 and HN30 cells. And in the metastasis assay of BALB/c nude mice, the quantity of metastases nodes was lower in the RASSF5-overexpressed group than that in the control group (P=0.049).

Conclusion·RASSF5 may inhibit epithelial-mesenchymal transition through GSK-3β/Snail pathway, and then inhibits the invasion and metastasis ability of HNSCC cells.

Key words: head and neck cancer, Ras association domain family 5 (RASSF5), cell migration, invasion, epithelial-mesenchymal transition

中图分类号:

739.91

鲁昱晟, 杨文艺, 马海龙, 胡镜宙. Ras相关结构域家族成员5对头颈鳞癌细胞迁移和侵袭能力的影响[J]. 上海交通大学学报(医学版), 2021, 41(6): 717-723.

Yu-sheng LU, Wen-yi YANG, Hai-long MA, Jing-zhou HU. Impact of Ras association domain family 5 on cell migration and invasion of head and neck squamous cell carcinoma[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(6): 717-723.

图/表 4

图1 在HNSCC中RASSF5基因表达水平以及生存分析Note: A. The gene expression of RASSF5 in tumor (n=519) and normal (n=44) tissues of HNSCC. B. OS and DFS of HNSCC patients with high RASSF5 expression (n=130) and low RASSF5 expression (n=130). TPM—transcripts per kilobase of exon model per million mapped reads. ①P=0.001.

Fig 1 Gene expression of RASSF5 in HNSCC and the related survival analysis

图2 RASSF5对Cal27和HN30迁移和侵袭能力的影响Note: A. Relative expression of RASSF5 mRNA by qPCR. B. Cell migration ability of LV RASSF5 or LV vector-transfected HN30 was determined using wound healing assay (×40). C. Cell migration ability of LV RASSF5 or LV vector-transfected Cal27 was determined using wound healing assay (×40). D. Transwell assays were performed to assess migration ability (×50). E. Transwell assays were performed to assess invasion ability (×50). ①P=0.004, ④P=0.001, compared with 0 h LV vector; ②P=0.001,⑤P=0.016, compared with 0 h LV RASSF5; ③P=0.003, compared with 24 h LV vector, ⑥P=0.015, compared with 36 h LV vector; ⑦P=0.001, ⑧P=0.005, compared with LV vector.

Fig 2 Effect of RASSF5 on the ability of cell migration and invasion

图3 过表达RASSF5对EMT相关蛋白表达的影响Note: A. The correlation of gene expression between RASSF5 and CDH1. B. Protein level of RASSF5, p-GSK-3β, GSK-3β, Snail and E-cadherin in Cal27 and HN30 after transfection with LV RASSF5 or LV vector by Western blotting. C. Relative protein level of RASSF5, p-GSK-3β, GSK-3β, Snail and E-cadherin compared with GAPDH.

Fig 3 Effects of RASSF5 overexpression on protein expression of EMT-related proteins

图4 RASSF5对Cal27细胞肺转移的影响Note: A. Metastatic nodules in lung in the LV RASSF5 group and LV vector group. B. The number of metastatic nodules in the LV RASSF5 group and LV vector group. ①P=0.049, compared with LV vector.

Fig 4 Effect of RASSF5 on lung metastasis of Cal27 cells

参考文献 20

1	Chow LQM. Head and neck cancer[J]. N Engl J Med, 2020, 382(1): 60-72.
2	Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020[J]. CA A Cancer J Clin, 2020, 70(1): 7-30.
3	Cohen EEW, Soulières D, Le Tourneau C, et al. Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study[J]. The Lancet, 2019, 393(10167): 156-167.
4	Barnoud T, Schmidt ML, Donninger H, et al. The role of the NORE1A tumor suppressor in oncogene-induced senescence[J]. Cancer Lett, 2017, 400: 30-36.
5	Donninger H, Schmidt ML, Mezzanotte J, et al. Ras signaling through RASSF proteins[J]. Semin Cell Dev Biol, 2016, 58: 86-95.
6	Zhou XH, Yang CQ, Zhang CL, et al. RASSF5 inhibits growth and invasion and induces apoptosis in osteosarcoma cells through activation of MST1/LATS1 signaling[J]. Oncol Rep, 2014, 32(4): 1505-1512.
7	Liu LL, Zhang MF, Pan YH, et al. NORE1A sensitises cancer cells to sorafenib-induced apoptosis and indicates hepatocellular carcinoma prognosis[J]. Tumor Biol, 2014, 35(3): 1763-1774.
8	Steinmann K, Sandner A, Schagdarsurengin U, et al. Frequent promoter hypermethylation of tumor-related genes in head and neck squamous cell carcinoma[J]. Oncol Rep, 2009, 22(6): 1519-1526.
9	Han L, Dong Z, Wang C, et al. Decreased expression and aberrant methylation of RASSF5A correlates with malignant progression of gastric cardia adenocarcinoma[J]. Mol Carcinog, 2015, 54(12): 1722-1733.
10	Liu G, Liu B, Zheng S, et al. Aberrant RASSF5 gene transcribed region hypermethylation in pediatric hepatoblastomas[J]. Int J Clin Exp Pathol, 2018, 11(7): 3612-3617.
11	Guo W, Wang C, Guo YL, et al. RASSF5A, a candidate tumor suppressor, is epigenetically inactivated in esophageal squamous cell carcinoma[J]. Clin Exp Metastasis, 2015, 32(1): 83-98.
12	Zhou BP, Deng J, Xia WY, et al. Dual regulation of Snail by GSK-3β-mediated phosphorylation in control of epithelial-mesenchymal transition[J]. Nat Cell Biol, 2004, 6(10): 931-940.
13	Sun J, Meng D, Yu T, et al. N-terminal truncated carboxypeptidase E represses E-cadherin expression in lung cancer by stabilizing the Snail-HDAC complex[J]. Am J Cancer Res, 2020, 10(3): 925-938.
14	An P, Chen F, Li Z, et al. HDAC8 promotes the dissemination of breast cancer cells via AKT/GSK-3β/Snail signals[J]. Oncogene, 2020, 39(26): 4956-4969.
15	Domoto T, Pyko IV, Furuta T, et al. Glycogen synthase kinase-3β is a pivotal mediator of cancer invasion and resistance to therapy[J]. Cancer Sci, 2016, 107(10): 1363-1372.
16	Jin F, Wu Z, Hu X, et al. The PI3K/Akt/GSK-3β/ROS/eIF2B pathway promotes breast cancer growth and metastasis via suppression of NK cell cytotoxicity and tumor cell susceptibility[J]. Cancer Biol Med, 2019, 16(1): 38-54.
17	Liao TJ, Tsai CJ, Jang H, et al. RASSF5: an MST activator and tumor suppressor in vivo but opposite in vitro[J]. Curr Opin Struct Biol, 2016, 41: 217-224.
18	Donninger H, Calvisi DF, Barnoud T, et al. NORE1A is a Ras senescence effector that controls the apoptotic/senescent balance of p53 via HIPK2[J]. J Cell Biol, 2015, 208(6): 777-789.
19	Li BT, Yu C, Xu Y, et al. TET1 inhibits cell proliferation by inducing RASSF5 expression[J]. Oncotarget, 2017, 8(49): 86395-86409.
20	Sun HY, Yang D, Mi J, et al. Histone demethylase Jmjd3 modulates osteoblast apoptosis induced by tumor necrosis factor-α through directly targeting RASSF5[J]. Connect Tissue Res, 2020, 61(6): 517-525.

[1]	刘琳, 王慧, 刘宁宁. 侵袭性念珠菌感染在免疫力低下人群中的流行病学和治疗进展[J]. 上海交通大学学报(医学版), 2021, 41(4): 525-529.
[2]	程龙1，徐五琴2，张鹏1，黄建军1，李小宁1. 下调miR-27a表达对三阴乳腺癌细胞增殖、迁移和侵袭的影响[J]. 上海交通大学学报（医学版）, 2020, 40(2): 188-.
[3]	严宇青，沈超琴，陈豪燕，洪洁#，王震华#. lnc-MTBP-5在结直肠癌中的表达及其对细胞侵袭能力的影响[J]. 上海交通大学学报(医学版), 2020, 40(09): 1193-1201.
[4]	张靖. 川楝素通过下调环状RNA circDLST对胃癌细胞BGC-823的抑制作用[J]. 上海交通大学学报(医学版), 2020, 40(09): 1202-1206.
[5]	赵乙汜1，余应喜1，林时辉2，徐昉1, 2. T细胞免疫在脓毒症继发侵袭性真菌感染中作用的研究进展[J]. 上海交通大学学报（医学版）, 2019, 39(11): 1325-.
[6]	张义朋 1，黄华艳 1，仰昳婕 1，徐懂懂 1，张可人 1,2，朱亮 1, 2, 3. 受体酪氨酸激酶AXL在肿瘤耐药中的作用研究进展[J]. 上海交通大学学报（医学版）, 2018, 38(7): 819-.
[7]	徐华丽，刘文雪，沈方倩，席晓薇. UBE3C对卵巢癌SKOV3细胞增殖和侵袭能力的影响[J]. 上海交通大学学报（医学版）, 2018, 38(6): 610-.
[8]	潘玉龙1，齐隽2，周亮1，张彤彤1，李强1. 核糖体蛋白16高表达于前列腺癌并促进肿瘤进展[J]. 上海交通大学学报（医学版）, 2018, 38(4): 394-.
[9]	龚莹靓，董瑜，李玉峰，朱亚菊，金晶，卫敏江. 糖蛋白 130 小分子抑制剂 SC144 对单侧输尿管梗阻小鼠肾间质纤维化的影响[J]. 上海交通大学学报（医学版）, 2018, 38(3): 265-.
[10]	刘霞，余维，余丹，陈娟，骆小华，李兵. 腺病毒介导的 siRNA 沉默PNUTS 基因对喉癌 Hep-2 细胞增殖、侵袭的影响[J]. 上海交通大学学报（医学版）, 2018, 38(2): 161-.
[11]	蔡留芸，罗小东，梁皓，胡建国. 下调 C2CD3通过抑制 Hedgehog通路调控上皮性卵巢癌增殖、侵袭和迁移[J]. 上海交通大学学报（医学版）, 2018, 38(12): 1414-.
[12]	唐中园 1, 2，张宁 2, 3，狄文 2, 3，李卫平 2, 3. 3-甲基腺嘌呤对低氧状态下上皮性卵巢癌细胞自噬、迁移和侵袭的影响[J]. 上海交通大学学报（医学版）, 2018, 38(10): 1152-.
[13]	张钰涵，陈雪华，张浩. 下调极光激酶 A活性抑制 Hep2细胞的促血管生成作用和迁移、侵袭能力[J]. 上海交通大学学报（医学版）, 2018, 38(10): 1181-.
[14]	李泊宁，赖东梅 . 自噬在肿瘤上皮间质转化中的作用#br#[J]. 上海交通大学学报（医学版）, 2017, 37(9): 1271-.
[15]	李栋，胡媛，吴安玥，邱兴堤，邱丽华. TWEAK调控巨噬细胞外泌体中miRNA-7的表达抑制卵巢癌细胞侵袭和迁移的研究[J]. 上海交通大学学报（医学版）, 2017, 37(6): 726-.