本研究旨在通过调节10号染色体缺失的microRNA-1297(miR-1297)/磷酸酶和张力蛋白同源物(PTEN)信号轴,探讨柴胡-白芍含药血浆对HepG2肝癌细胞的作用及其机制。实时定量PCR(RT-qPCR)检测miR-1297和PTEN在不同肝癌细胞系中的mRNA水平。采用双荧光素酶报告基因测定来验证miR-1297和PTEN之间的靶向相互作用。细胞计数试剂盒-8(CCK-8)用于检测细胞增殖,确定含药血浆的最佳浓度和干预时间。通过Transwell测定和伤口愈合测定检查细胞侵袭和迁移。通过PI染色检测细胞周期分布,AnnexinV-FITC/PI双染色检测细胞凋亡。miR-1297、PTEN、蛋白激酶B(Akt),通过RT-qPCR测定磷脂酰肌醇3-激酶(PI3K)。蛋白质印迹用于确定PTEN的蛋白质水平,Akt,p-Akt,caspase-3,caspase-9,B细胞淋巴瘤-2(Bcl-2),和Bcl-2相关X蛋白(Bax)。结果表明,HepG2细胞是后续实验的最佳细胞系。双荧光素酶报告基因测定证实miR-1297可以结合PTENmRNA中的3'-非翻译区(3'UTR)。含药血浆抑制HepG2细胞增殖,最佳干预浓度和时间分别为20%和72h。与空白血浆相比,柴胡-白芍含药血浆,miR-1297抑制剂,miR-1297抑制剂+含药血浆均抑制增殖,入侵,和HepG2细胞的迁移,增加G_0/G_1期细胞的比例,降低了S期细胞的比例,并增加细胞凋亡率。含药血浆下调miR-1297、PI3K、和Akt并上调PTEN的mRNA水平。此外,它上调了PTEN的蛋白质水平,Bax,caspase-3和caspsae-9下调p-Akt的蛋白水平,p-PI3K,Bcl-2总之,柴胡-白芍含药血浆可抑制HepG2肝癌细胞miR-1297的表达,促进PTEN的表达,并负向调节PI3K/Akt信号通路,从而抑制HepG2细胞的增殖并诱导其凋亡。
The present study aimed to investigate the effect and mechanism of Bupleuri Radix-Paeoniae Radix Alba medicated plasma on HepG2
hepatoma cells by regulating the microRNA-1297(miR-1297)/phosphatase and tensin homologue deleted on chromosome 10(PTEN) signaling axis. Real-time quantitative PCR(RT-qPCR) was carried out to determine the mRNA levels of miR-1297 and PTEN in different
hepatoma cell lines. The dual luciferase reporter assay was employed to verify the targeted interaction between miR-1297 and PTEN. The cell counting kit-8(CCK-8) was used to detect cell proliferation, and the optimal concentration and intervention time of the medicated plasma were determined. The cell invasion and migration were examined by Transwell assay and wound healing assay. Cell cycle distribution was detected by PI staining, and the apoptosis of cells was detected by Annexin V-FITC/PI double staining. The mRNA levels of miR-1297, PTEN, protein kinase B(Akt), and phosphatidylinositol 3-kinase(PI3K) were determined by RT-qPCR. Western blot was employed to determine the protein levels of PTEN, Akt, p-Akt, caspase-3, caspase-9, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). The results showed that HepG2 cells were the best cell line for subsequent experiments. The dual luciferase reporter assay confirmed that miR-1297 could bind to the 3\'-untranslated region(3\'UTR) in the mRNA of PTEN. The medicated plasma inhibited the proliferation of HepG2 cells, and the optimal intervention concentration and time were 20% and 72 h. Compared with the blank plasma, the Bupleuri Radix-Paeoniae Radix Alba medicated plasma, miR-1297 inhibitor, miR-1297 inhibitor + medicated plasma all inhibited the proliferation, invasion, and migration of HepG2 cells, increased the proportion of cells in the G_0/G_1 phase, decreased the proportion of cells in the S phase, and increased the apoptosis rate. The medicated plasma down-regulated the mRNA levels of miR-1297, PI3K, and Akt and up-regulated the mRNA level of PTEN. In addition, it up-regulated the protein levels of PTEN, Bax, caspase-3, and caspsae-9 and down-regulated the protein levels of p-Akt, p-PI3K, and Bcl-2. In conclusion, Bupleuri Radix-Paeoniae Radix Alba medicated plasma can inhibit the expression of miR-1297 in HepG2
hepatoma cells, promote the expression of PTEN, and negatively regulate PI3K/Akt signaling pathway, thereby inhibiting the proliferation and inducing the apoptosis of HepG2 cells.