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中华普通外科学文献(电子版) ›› 2021, Vol. 15 ›› Issue (01) : 11 -17. doi: 10.3877/cma.j.issn.1674-0793.2021.01.003

所属专题: 文献

论著

山药多糖通过miR-98-5p/TGFβR1分子轴调控肝癌细胞凋亡的机制研究
郑华山1, 陈成辉1, 蔡亲平1, 林献科1, 李合2,()   
  1. 1. 570100 海南医学院第二附属医院东湖外科
    2. 570102 海口,海南医学院第一附属医院普通外科
  • 收稿日期:2020-07-28 出版日期:2021-02-01
  • 通信作者: 李合
  • 基金资助:
    海南省自然科学基金项目(812201)

Chinese Yam polysaccharide regulating apoptosis of hepatocellular carcinoma cells throughmiR-98-5p/TGFβR1 axis

Huashan Zheng1, Chenghui Chen1, Qinping Cai1, Xianke Lin1, He Li2,()   

  1. 1. Department of Surgery, Donghu District of the Second Affiliated Hospital of Hainan Medical College, Haikou 570100, China
    2. Department of General Surgery, the First Affiliated Hospital of Hainan Medical College, Haikou 570102, China
  • Received:2020-07-28 Published:2021-02-01
  • Corresponding author: He Li
引用本文:

郑华山, 陈成辉, 蔡亲平, 林献科, 李合. 山药多糖通过miR-98-5p/TGFβR1分子轴调控肝癌细胞凋亡的机制研究[J/OL]. 中华普通外科学文献(电子版), 2021, 15(01): 11-17.

Huashan Zheng, Chenghui Chen, Qinping Cai, Xianke Lin, He Li. Chinese Yam polysaccharide regulating apoptosis of hepatocellular carcinoma cells throughmiR-98-5p/TGFβR1 axis[J/OL]. Chinese Archives of General Surgery(Electronic Edition), 2021, 15(01): 11-17.

目的

探究山药多糖(CYPS)通过miR-98-5p/TGFβR1分子轴调控肝癌(HCC)细胞凋亡的分子机制。

方法

qPCR检测miR-98-5p在不同HCC细胞系中的表达情况,使用不同浓度的CYPS处理Huh-7细胞,CCK-8检测细胞增殖活力,Annexin V-FITC/PI流式细胞术检测细胞凋亡,Western blotting检测TGFβR1和细胞凋亡相关蛋白Caspase-3、Caspase-8、Bcl-2、Bax的表达,双荧光素酶报告基因系统验证miR-98-5p与TGFβR1的靶向关系。

结果

与人正常肝细胞HL-7702相比,miR-98-5p在HCC细胞系中表达升高(均P<0.05),且在Huh-7细胞中的表达高于其他HCC细胞(P<0.05);CYPS处理可明显抑制Huh-7细胞的增殖活力并诱导细胞凋亡(均P<0.05),且10-3 kg/L CYPS对细胞增殖活力的抑制作用比其他浓度明显。双荧光素酶报告基因实验证实,miR-98-5p靶向调控TGFβ1。与10-3 kg/L CYPS组相比,miR-98-5p mimics可下调10-3 kg/L CYPS对细胞增殖活力(P<0.05)的抑制作用和对细胞凋亡(P<0.01)的促进作用,CYPS+miR-98-5p mimics+pcDNA-TGFβR1组中细胞增殖活力与CYPS+miR-98-5p mimics组相比明显降低(P<0.05),细胞凋亡水平明显升高(P<0.01)。

结论

CYPS可下调miR-98-5p,促进TGFβR1的表达,抑制Huh-7细胞增殖并诱导细胞凋亡。

Objective

To explore the mechanism of Chinese Yam polysaccharide (CYPS) regulating hepatocellular carcinoma (HCC) cells apoptosis through the molecular axis of miR-98-5p/TGFβR1.

Methods

The expression of miR-98-5p in different HCC cell lines was detected by qPCR. Huh-7 cell was treated with CYPS at different concentrations, cell proliferation activity was detected by CCK-8,apoptosis rate was detected by Annexin V-FITC/PI flow cytometry, and the expressions of TGFβR1 and apoptosis-related proteins Caspase-3, Caspase-8, Bcl-2 and Bax were detected by Western blotting. The dual luciferase reporter gene system was used to verify the targeted relationship between miR-98-5p and TGFβR1.

Results

Compared with human normal hepatocytes HL-7702, the expression of miR-98-5pwas increased in HCC cell lines (all P<0.05), and the expression in Huh-7 cell was higher than other cells (P<0.05). CYPS treatment could significantly inhibit the proliferation of Huh-7 cell (all P<0.05) and induce cell apoptosis (all P<0.05), and the inhibitory effect of 10-3 kg/L CYPS on cell proliferation was more obvious than other concentrations. The dual luciferase reporter gene experiment confirmed that miR-98-5p targeted and regulated TGFβR1. Compared with the 10-3 kg/L CYPS group, miR-98-5p mimics could down-regulate the inhibitory effect of 10-3 kg/L CYPS on cell proliferation (P<0.05) and the promotion of cell apoptosis (P<0.01). The cell proliferation activity in the 10-3 kg/L CYPS+miR-98-5pmimics+pcDNA-TGFβR1 group was significantly lower than that in the 10-3 kg/L CYPS+miR-98-5p mimics group (P<0.05), while the level of cell apoptosis was significantly increased (P<0.01).

Conclusion

CYPS can inhibit the proliferation of Huh-7 cell, induce apoptosis, down-regulate miR-98-5p and promote the expression of TGFβR1.

表1 基因及引物列表
表2 TGFβR1 3'-UTR目的基因片段
图1 miR-98-5p在HCC细胞系中表达上调 与HL-7702组相比,*P<0.05,**P<0.01;与Huh-7细胞相比,#P<0.05
图2 CYPS抑制Huh-7细胞增殖活力并诱导其凋亡 A为CCK-8检测细胞增殖;B为Western blotting检测不同浓度CYPS处理后,Huh-7细胞中凋亡相关蛋白;C为Annexin V-FITC/PI检测不同浓度CYPS处理后的Huh-7细胞的凋亡情况 与0 g/L组相比,*P<0.05,**P<0.01
图3 CYPS通过下调miR-98-5p促进TGFβR1表达 A为qPCR检测不同浓度CYPS处理后miR-98-5p的表达水平;B为Western blotting检测不同浓度CYPS处理后TGFβR1的表达;C为starBase数据库预测miR-98-5p和TGFβR1的靶向结合位点;D为双荧光素酶报告基因验证miR-98-5p与TGFβR1的靶向结合关系;E为Western blotting检测TGFβR1的表达 与0 g/L组或miR-NC组相比,*P<0.05,**P<0.01
图4 miR-98-5p通过靶向结合TGFβR1抑制人肝细胞肝癌细胞增殖并促进凋亡 A为Western blotting检测TGFβR1表达水平;B为CCK-8检测各组Huh-7细胞的增殖活力;C为Annexin V-FITC/PI流式细胞术检测Huh-7细胞的凋亡情况;D为Western blotting检测细胞凋亡相关蛋白的表达与NC组相比,**P<0.01,***P<0.001;与CYPS组相比,P<0.05,△△P<0.01;与CYPS+miR-98-5p mimics组相比,#P<0.05,##P<0.01
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