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

doi:10.3877/cma.j.issn.1674-0793.2021.01.003

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。

关键词: 山药多糖, 肝癌细胞, miR-98-5p, TGFβR1, 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.

Key words: Chinese Yam polysaccharide, Hepatocellular carcinoma cells, miR-98-5p, TGFβR1, Apoptosis

表1 基因及引物列表

名称	上游引物（5'→3'）	下游引物（5'→3'）
miR-98-5p	AGGATTCTGCTCATGCCAG	TGAATATGCCACACACCAGG
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

表1 基因及引物列表

名称	上游引物（5'→3'）	下游引物（5'→3'）
miR-98-5p	AGGATTCTGCTCATGCCAG	TGAATATGCCACACACCAGG
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT

表2 TGFβR1 3'-UTR目的基因片段

名称	序列（5'→3'）
正义链	CACCTCTCAAGAGACCAAGGTACATTTACCTCATTGTGTATATAATGTTTAATATTTGTCAGAGCATTCTCCAGGTTTGCAGTTTTATTTCTATAAAGTATGC
反义链	TCGAGCATACTTTATAGAAATAAAACTGCAAACCTGGAGAATGCTCTGACAAATATTAAACATTATATACACAATGAGGTAAATGTACCTTGGTCTCTTGAGAGGTGAGCT

表2 TGFβR1 3'-UTR目的基因片段

名称	序列（5'→3'）
正义链	CACCTCTCAAGAGACCAAGGTACATTTACCTCATTGTGTATATAATGTTTAATATTTGTCAGAGCATTCTCCAGGTTTGCAGTTTTATTTCTATAAAGTATGC
反义链	TCGAGCATACTTTATAGAAATAAAACTGCAAACCTGGAGAATGCTCTGACAAATATTAAACATTATATACACAATGAGGTAAATGTACCTTGGTCTCTTGAGAGGTGAGCT

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

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