红景天苷调控miRNA-1343-3p/MAP3K6/MMP24信号分子抑制胃癌细胞的增殖和侵袭

Salidroside Regulates the miRNA-1343-3p/MAP3K6/MMP24 Signaling Molecules to Inhibit Proliferation and Invasion of Gastric Cancer Cells

【目的】本研究旨在探讨红景天苷调控miR-1343-3p/MAP3K6(丝裂原激活激酶激酶激酶6)/MMP24(膜型基质金属蛋白酶24)信号分子,在抑制胃癌细胞增殖和迁移中的作用。【方法】将人胃癌细胞(MGC-803)按照红景天苷药物浓度不同分为对照组(0μmol/mL)和低(6μmol/mL)中(12μmol/mL)高(24μmol/mL)红景天苷剂量组;CCK-8实验检测红景天苷药物对人胃癌细胞抗增殖作用;克隆形成实验检测红景天苷药物对人胃癌细胞克隆形成能力的影响;Transwell实验检测红景天苷对人胃癌细胞侵袭能力的影响;细胞划痕实验检测红景天苷对人胃癌细胞迁移能力的影响;高通量测序(RNA-seq)检测了胃癌细胞在红景天苷作用后24 h时对照组和药物组miRNA和mRNA的表达量,将具有明显差异表达的miRNA聚类并预测其靶基因。通过Gene Ontology(GO)和KEGG富集分析,预测差异表达miRNA靶基因的相关功能及相关代谢通路,并建立miRNA与靶基因mRNA间的互作网络。免疫细胞荧光检测相关靶蛋白的表达;q-PCR检测相关候选基因的转录。【结果】CCK-8细胞毒性实验表明,红景天苷抑制了MGC-803细胞的增殖(P<0.01)。细胞克隆形成实验表明,红景天苷降低了MGC-803细胞克隆形成能力(P<0.0001)。细胞侵袭实验表明,红景天苷降低了MGC-803细胞侵袭能力(P<0.0001)。划痕实验表明红景天苷降低了细胞迁移能力(P<0.0001)。高通量测序发现红景天苷作用人胃癌细胞后,有44个miRNA的表达量发生了显著差异性变化(P<0.05),生物信息学分析结果显示差异表达miRNA所对应的靶mRNA有1384个,红景天苷作用后引起抑癌因子miR-1343-3p表达明显上调(P<0.01),而受其调控与增殖和迁移相关的MAP3K6和MMP24基因转录及表达明显下调(P<0.05)。免疫荧光实验证明红景天苷降低了MAP3K6和MMP24基因的蛋白表达水平(P<0.0001)。q-PCR实验证明红景天苷降低了MAP3K6和MMP24基因的mRNA表达水平(P<0.0001),而miR-1343-3p基因的miRNA表达上调(P<0.0001)。【结论】红景天苷调控miRNA-1343-3p/MAP3K6/MMP24信号分子抑制胃癌细胞增殖和侵袭。

【Objective】The aim of this study is to investigate the role of salidroside in regulating the miR-1343-3p/MAP3K6(mitogen-activated protein kinase kinase kinase 6)/MMP24(membrane-type matrix metalloproteinase 24)sig⁃naling pathway to inhibit gastric cancer cell proliferation and migration.【Methods】Human gastric cancer cells(MGC-803)were divided into several groups based on different salidroside concentrations:a control group(0μmol/mL),a low�dose group(6μmol/mL),a medium-dose group(12μmol/mL),and a high-dose group(24μmol/mL).The anti prolifer⁃ative effects of salidroside on human gastric cancer cells were evaluated by CCK-8 assay.Clonogenic assay was used to ex⁃amine the effects of salidroside drugs on the clonogenic ability of human gastric cancer cells.Transwell assay was per⁃formed to detect the effect of salidroside on the invasive ability of human gastric cancer cells.Cell scratch assay was per⁃formed to detect the effect of salidroside on the migration ability of human gastric cancer cells.The miRNA expression pro⁃file was analyzed by using RNA-seq in cancer cells for 24 h after salidroside treatment.The differentially expressed miR⁃NAs were clustered and their target genes were predicted.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Ge⁃nomes(KEGG)were used to analyze and predict the functions of these target genes,and the interaction networks were es⁃tablished.Immunocytofluorescence was used to detect the expression of target proteins,and the transcription of candidate genes was detected by q-PCR.【Results】CCK-8 cytotoxicity experiments showed that salidroside inhibited the prolifera⁃tion of MGC-803 cells(P<0.01).Cell cloning experiments showed that salidroside reduced the clonal formation capacity of MGC-803 cells(P<0.0001).Cell invasion experiments showed that salidroside reduced the MGC-803 cell invasion capacity(P<0.0001).Cell scratch experiments showed that salidroside reduced the cell migration capacity(P<0.0001).RNA-seq findings showed that the expression of 44 miRNAs changed significantly after salidroside treatment in cancer cells(P<0.05).Bioinformatic analysis showed that there were 1384 target mRNAs corresponding to the differentially ex⁃pressed miRNAs,and the expression of the tumor suppressor miR-1343-3p was significantly upregulated after salidroside treatment(P<0.01),and resulted in down-regulated transcription of MAP3K6 and MMP24 genes which are related to the proliferation and migration of cancer cells(P<0.05).Immunofluorescence experiments demonstrated that salidroside re⁃duced protein expression levels in MAP3K6 and MMP24 genes(P<0.0001).q-PCR experiments showed that salidroside reduced the mRNA expression level of MAP3K6 and MMP24 genes(P<0.0001),while miRNA expression in miR-1343-3p gene was upregulated(P<0.0001).【Conclusion】Salidroside regulates the miRNA-1343-3p/MAP3K6/MMP24 signaling molecules to inhibit proliferation and invasion of gastric cancer cells.