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中华细胞与干细胞杂志(电子版) ›› 2023, Vol. 13 ›› Issue (01) : 1 -9. doi: 10.3877/cma.j.issn.2095-1221.2023.01.001

论著

芒柄花黄素通过调控miR-140-5p/TIGIT轴影响喉鳞状细胞癌细胞增殖和凋亡
武川军1, 彭丽娜1,†(), 韩海平1, 冯志星1   
  1. 1. 056002　邯郸，河北省邯郸市中心医院耳鼻咽喉科
  • 收稿日期:2022-10-31 出版日期:2023-02-01
  • 通信作者: 彭丽娜

Effect of formononetin on proliferation and apoptosis of laryngeal squamous cell carcinoma cells and its possible mechanism

Chuanjun Wu1, Lina Peng1,†(), Haiping Han1, Zhixing Feng1   

  1. 1. Department of Otolaryngology, Handan Central Hospital, Handan 056002, China
  • Received:2022-10-31 Published:2023-02-01
  • Corresponding author: Lina Peng
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武川军, 彭丽娜, 韩海平, 冯志星. 芒柄花黄素通过调控miR-140-5p/TIGIT轴影响喉鳞状细胞癌细胞增殖和凋亡[J/OL]. 中华细胞与干细胞杂志(电子版), 2023, 13(01): 1-9.

Chuanjun Wu, Lina Peng, Haiping Han, Zhixing Feng. Effect of formononetin on proliferation and apoptosis of laryngeal squamous cell carcinoma cells and its possible mechanism[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(01): 1-9.

目的

探讨芒柄花黄素对喉鳞状细胞癌细胞增殖、凋亡的影响及其可能的机制。

方法

体外培养人喉鳞状细胞癌细胞系Hep-2，以MTT法检测0、5、10、20、40、60 μmol/L芒柄花黄素处理24 h后Hep-2细胞活力，根据IC50值筛选出芒柄花黄素合适作用浓度。Hep-2细胞分别采用20、40 μmol/L芒柄花黄素，40 μmol/L芒柄花黄素+阴性对照（转染mirRNA-140-5p抑制剂阴性对照），40 μmol/L芒柄花黄素+miR-140-5p inhibitor干预细胞敲减干预Hep-2细胞。分组转染及药物处理后，采用实时荧光定量PCR （qRT-PCR）实验和免疫印迹检测Hep-2细胞miR-140-5p与T细胞免疫球蛋白和ITIM结构域蛋白（TIGIT）表达；采用MTT法和平板集落形成实验检测Hep-2细胞增殖；采用流式细胞实验检测Hep-2细胞凋亡；采用免疫印迹检测Hep-2细胞增殖（PCNA、Cyclin D1）与凋亡相关蛋白（Bax、cleaved caspase-3）表达。于裸鼠背部皮下注射Hep-2细胞构建裸鼠移植瘤模型，随机分为对照组，25、50 mg/kg芒柄花黄素组，50 mg/kg芒柄花黄素+阴性对照组，50 mg/kg芒柄花黄素+ miR-140-5p敲减组，分组处理后，检测各组裸鼠移植瘤质量与体积；采用双荧光素酶报告实验分析Hep-2细胞miR-140-5p对TIGIT的靶向调控。两组间比较采用t检验，多组间比较采用单因素方差分析，进一步两两比较采用SNK-q检验。

结果

与对照比较，20、40 μmol/L芒柄花黄素干预细胞miR-140-5p表达（1.79±0.16、2.58±0.22比1.04±0.11）、凋亡率[（36.17±8.14）%、（68.65±14.20）%比（2.10±0.65）%]、Bax与cleaved caspase-3蛋白表达升高（P < 0.05），TIGIT蛋白（0.56±0.12、0.17±0.01比0.98±0.10）与mRNA表达（0.68±0.10、0.34±0.03比1.05±0.13）、集落生成率[（60.82±12.24）%、（35.36±8.20）%比（100.0±0.00）%]、细胞活力（63.12±11.03、39.75±7.14比100.0±0.00）、PCNA与Cyclin D1蛋白表达降低（P < 0.05）；与40 μmol/L芒柄花黄素比较，芒柄花黄素+miR-140-5p敲减细胞miR-140-5p表达（1.11±0.13比2.58±0.22）、凋亡率[（5.04±1.51）%比（68.65±14.20）%]、Bax与cleaved caspase-3蛋白表达降低（P < 0.05），TIGIT蛋白（0.92±0.18比0.17±0.01）与mRNA表达（0.97±0.14比0.34±0.03）、集落生成率[（90.76±15.02）%比（35.36±8.20）%]、细胞活力（92.01±17.13比39.75±7.14）、PCNA与Cyclin D1蛋白表达升高（P < 0.05）；动物实验中，与对照组比较，25、50 mg/kg芒柄花黄素组裸鼠移植瘤质量[（609.23±58.14）、（465.87±41.26）比（762.14±76.51） mg]降低，体积[（532.82±60.28）、（396.34±48.10）比（681.70±72.44）mm3]减小（P < 0.05）；与50 mg/kg芒柄花黄素组比较，芒柄花黄素+miR-140-5p敲减组裸鼠移植瘤质量[（735.48±80.12）比（465.87±41.26）mg]升高，体积[（654.79±75.85）比（396.34±48.10） mm3]增加（P < 0.05）；Hep-2细胞miR-140-5p可靶向下调TIGIT表达。

结论

芒柄花黄素通过调控miR-140-5p/TIGIT轴表达抑制喉鳞状细胞癌细胞增殖活性，并促进其凋亡。

关键词: 芒柄花黄素, miR-140-5p/TIGIT, 喉鳞状细胞癌细胞, 增殖, 凋亡
Objective

To investigate the effect of formononetin on the proliferation and apoptosis of laryngeal squamous cell carcinoma cells and its possible mechanism.

Methods

The human laryngeal squamous cell carcinoma cell line Hep-2 was cultured in vitro, and the cell viability of Hep-2 cells was detected by the MTT method after treatment with 0, 5, 10, 20, 40, 60 μmol/L formononetin for 24 h. The appropriate concentration of mancoxanthin was screened according to the IC50 value. Hep-2 cells cultured were treated with, 20 μmol/L formononetin (20 μmol/L) , 40 μmol/L formononetin (40 μmol/L) , formononetin (40 μmol/L) + negative control (transfected with miR-140-5p inhibitor negative control) , formononetin (40 μmol/L) + miR-140-5p inhibitor (transfected with miR-140-5p inhibitor) . After transfection and drug treatment, the expression of miR-140-5p and T cell immunoglobulin and ITIM domain protein (TIGIT) in Hep-2 cells in each group was detected by real-time fluorescence quantitative PCR (qRT-PCR) experiment and western blot; the proliferation of Hep-2 cells in each group was detected by MTT assay and plate colony formation assay; the apoptosis of Hep-2 cells in each group was detected by flow cytometry; Western blot was used to detect the proliferation (PCNA, Cyclin D1) and apoptosis-related protein (Bax, cleaved caspase-3) expression of Hep-2 cells in each group. Nude mice were subcutaneously injected with Hep-2 cells on the back to establish a transplanted tumor model in nude mice, and they were randomly grouped into a control group, a low-dose formononetin (25 mg/kg) group, a high-dose formononetin (50 mg/kg) group, high-dose formononetin (50 mg/kg) + negative control (transfected with miR-140-5p antagomir negative control) group, high dose of forsythia (50 mg/kg) + miR-140-5p knockdown (transfected with miR-140-5p antagomir) group. After treatment, the quality and volume of transplanted tumors in nude mice of each group were detected; a dual-luciferase reporter assay was used to analyze the targeted regulation of TIGIT by miR-140-5p in Hep-2 cells.

Results

Compared with the control, the expression of miR-140-5p (1.79±0.16, 2.58±0.22 vs 1.04±0.11) , the apoptosis rate [ (36.17±8.14) %、(68.65±14.20) %vs (2.10±0.65) %], the protein expression of Bax and cleaved caspase-3 were increased, in the 20 μmol/L formononetin and the 40 μmol/L formononetin treated cells (P < 0.05) , the expression of TIGIT protein (0.56±0.12, 0.17±0.01 vs 0.98±0.10) and mRNA (0.68±0.10, 0.34±0.03 vs 1.05±0.13) , colony formation rate [ (60.82±12.24) %、(35.36±8.20) %vs (100.0±0.00) %], cell viability (63.12±11.03, 39.75±7.14 vs 100.0±0.00) , protein expression of PCNA and Cyclin D1 were decreased (P < 0.05) ; compared with the 40 μmol/L formononetin, the expression of miR-140-5p (1.11±0.13 vs 2.58± 0.22) , the apoptosis rate [ (5.04±1.51) %vs (68.65±14.20) %], the protein expression of Bax and cleaved caspase-3 were decreased in the high-dose formononetin+miR-140-5p knockdown cells (P < 0.05) , the expression of TIGIT protein (0.92±0.18 vs 0.17±0.01) and mRNA (0.97±0.14 vs 0.34±0.03) , colony formation rate[ (90.76±15.02) %vs (35.36±8.20) %], cell viability (92.01±17.13 vs 39.75±7.14) , protein expression of PCNA and Cyclin D1 were increased (P < 0.05) ; Compared with the control group, the weight [ (609.23±58.14) 、(465.87±41.26) vs (762.14±76.51) mg] and volume [ (532.82±60.28) 、(396.34±48.10) vs (681.70±72.44) mm3] of transplanted tumor were decreased in nude mice in the low-dose formononetin group and the high-dose formononetin group (P < 0.05) ; Compared with the high-dose formononetin group, the weight [ (735.48±80.12) vs (465.87±41.26) mg] and volume [ (654.79±75.85) vs (396.34±48.10) mm3] of transplanted tumor were increased in nude mice in the high-dose formononetin+miR-140-5p knockdown group (P < 0.05) ; MiR-140-5p in Hep-2 cells targeted to down-regulate TIGIT expression.

Conclusion

Formononetin inhibits proliferative activity and promotes apoptosis in laryngeal squamous cell carcinoma cells by regulating miR-140-5p/TIGIT axis expression.

Key words: Formononetin, miR-140-5p/TIGIT, Laryngeal squamous cell carcinoma cell, Proliferation, Apoptosis
表1 各因引物序列
引物名称 序列（5'-3'） 预期扩增产物长度（bp）
miR-140-5p 上游ACACTCCAGCTGGGCAGTGGTTTTACCCTA 102
  下游TGGTGTCGTGGAGTCG  
U6 上游GCTTCGGCAGCACATATACTAAAAT 91
  下游CGCTTCACGAATTTGCGTGTCAT  
TIGIT 上游TATCCTTCCACCATCTCTCTCA 110
  下游CACAACCTTGCTTCACATTCTC  
β-actin 上游GCTCCCCGGGCTGTATTC 76
  下游CCTTCTGACCCATTCCCACC  
图1 不同浓度芒柄花黄素对Hep-2细胞活力的影响注：与0 μmol/L芒柄花黄素比较，aP < 0.05
图2 免疫印迹检测芒柄花黄素对Hep-2细胞TIGIT蛋白表达注：1为对照；2为20 μmol/L芒柄花黄素；3为40 μmol/L芒柄花黄素；4为芒柄花黄素+阴性对照；5为芒柄花黄素+miR-140-5p inhibitor
表2 芒柄花黄素对Hep-2细胞miR-140-5p与TIGIT的表达影响（ ± s）
分组 实验次数 RNA 蛋白
miR-140-5p/U6 TIGIT/β-actin TIGIT/β-actin
对照 3 1.04±0.11 1.05±0.13 0.98±0.10
20μmol/L芒柄花黄素 3 1.79±0.16a 0.68±0.10a 0.56±0.12a
40μmol/L芒柄花黄素 3 2.58±0.22ab 0.34±0.03ab 0.17±0.01ab
芒柄花黄素+阴性对照 3 2.60±0.19 0.35±0.06 0.16±0.05
芒柄花黄素+miR-140-5p敲减 3 1.11±0.13c 0.97±0.14c 0.92±0.18c
F值   123.975 65.791 78.040
P值   < 0.001 < 0.001 < 0.001
图3 显微镜下观察芒柄花黄素对Hep-2细胞集落生成（结晶紫染色）注：a为对照；b为20 μmol/L芒柄花黄素；c为40 μmol/L芒柄花黄素；d为芒柄花黄素+阴性对照；e为芒柄花黄素+miR-140-5p敲减
图4 流式细胞术检测芒柄花黄素对Hep-2细胞凋亡情况注：a为对照；b为20 μmol/L芒柄花黄素；c为40 μmol/L芒柄花黄素；d为芒柄花黄素+阴性对照；e为芒柄花黄素+miR-140-5p敲减
表3 芒柄花黄素对Hep-2细胞的凋亡率、集落生成率、细胞活力的影响（ ± s）
分组 实验次数 细胞凋亡率（%） 集落生成率（%） 细胞活力（%）
对照 3 2.10±0.65 100.0±0.00 100.0±0.00
20 μmol/L芒柄花黄素 3 36.17±8.14a 60.82±12.24a 63.12±11.03a
40 μmol/L芒柄花黄素 3 68.65±14.20ab 35.36±8.20ab 39.75±7.14ab
芒柄花黄素+阴性对照 3 67.13±15.14 32.40±5.03 40.14±8.01
芒柄花黄素+miR-140-5p敲减 3 5.04±1.51c 90.76±15.02c 92.01±17.13c
F值   62.163 61.559 45.202
P值   < 0.001 < 0.001 < 0.001
图5 免疫印迹检测芒柄花黄素对Hep-2细胞增殖与凋亡相关蛋白的表达注：1为对照；2为20 μmol/L芒柄花黄素；3为40 μmol/L芒柄花黄素；4为芒柄花黄素+阴性对照；5为芒柄花黄素+miR-140-5p敲减
表4 芒柄花黄素对Hep-2细胞增殖与凋亡相关蛋白表达的影响（ ± s）
分组 实验次数 Cyclin D1/β-actin PCNA/β-actin Bax/β-actin cleaved caspase-3/β-actin
对照 3 1.13±0.20 0.94±0.14 0.22±0.03 0.30±0.04
20 μmol/L芒柄花黄素 3 0.64±0.11a 0.50±0.09a 0.64±0.08a 0.71±0.10a
40 μmol/L芒柄花黄素 3 0.17±0.02ab 0.08±0.01ab 1.06±0.16ab 1.13±0.18ab
芒柄花黄素+阴性对照 3 0.15±0.04 0.09±0.01 1.04±0.18 1.14±0.20
芒柄花黄素+miR-140-5p敲减 3 1.07±0.15c 0.88±0.12c 0.26±0.04c 0.34±0.06c
F值   86.718 121.007 73.668 56.962
P值   < 0.001 < 0.001 < 0.001 < 0.001
图6 芒柄花黄素对Hep-2细胞裸鼠移植瘤的影响注：给药后第15天解剖取出的移植瘤大小，n = 10，n为动物数
表5 芒柄花黄素对Hep-2细胞裸鼠移植瘤质量与体积的影响（ ± s，n = 10）
分组 移植瘤质量（mg） 移植瘤体积（mm3）
对照组 762.14±76.51 681.70±72.44
25 mg/kg芒柄花黄素组 609.23±58.14a 532.82±60.28a
50 mg/kg芒柄花黄素组 465.87±41.26ab 396.34±48.10ab
芒柄花黄素+阴性对照组 478.30±44.60 412.06±50.32
芒柄花黄素+miR-140-5p敲减组 735.48±80.12c 654.79±75.85c
F值 29.857 27.033
P值 < 0.001 < 0.001
图7 miR-140-5p与TIGIT之间存在结合位点
表6 miR-140-5p对野生TIGIT和突变TIGIT荧光素酶活性的影响（ ± s，n = 3）
分组 野生TIGIT 突变TIGIT
miR-140-5p mimics阴性对照 1.04±0.17 1.01±0.19
miR-140-5p mimics 0.32±0.03 0.96±0.22
t值 10.216 0.421
P值 < 0.001 0.682
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