The effect of Biejiajian Pills on regulating the EGFR/MAPK/ERK pathway in MHCC-97H liver cancer cells.

Objective We aimed to investigate the effects of Biejiajian Pills on MHCC-97H hepatoma cells and whether Biejiajian Pills regulate the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway through miR-885-5p. Methods SPF SD rats (n = 10) were randomly divided into the blank group and the Biejiajian Pills (1.1 g/kg) group to prepare blank and Biejiajian Pills-containing serum. MHCC-97H cells in the logarithmic growth phase were divided into the model group, blank serum groups with different concentrations (5%, 10%, 15%, and 20%), the serum containing Biejiajian Pills group, and the blank group without cells. Cell proliferation was detected by the CCK-8 assay, and the optimal intervention time and concentration of drug-containing serum were screened. MHCC-97H cells were divided into the blank control group (no intervention), the Biejiajian Pills-containing serum group (20% Biejiajian Pills-containing serum), the miR-885-5p mimics group (transfected with miR-885-5p mimics), the miR-NC group (transfected with miR-885-5p NC), and the Biejiajian Pills-containing serum + miR-885-5p mimics group (treated with 20% Biejiajian Pills-containing serum and transfected with miR-885-5p mimics). Cells in each group were cultured for 72 hours. A dual luciferase reporter assay was conducted to verify the targeting relationship between miR-885-5p and EGFR. Cell proliferation was detected by the CCK-8 assay, cell migration and invasion abilities were detected by the cell scratch assay and the Transwell invasion assay. Annexin V-APC/PI double staining was performed to detect the apoptosis level, and real-time fluorescence quantitative PCR (RT-qPCR) analysis was conducted to determine the mRNA expression levels of miR-885-5p, EGFR, MEK, and ERK1/2. The expression levels of EGFR, p-EGFR, MEK, p-MEK, ERK1/2, p-ERK1/2, matrix metalloproteinase 1 (MMP1), and CyclinD1 were determined by Western blotting analysis. The subcutaneous tumor model of MHCC-97H hepatoma cells in nude mice was established by subcutaneous injection to observe the inhibitory effect of Biejiajian Pills of different doses(0.55, 1.1, 2.2 g/kg). Results The optimal concentration and intervention time of Biejiajian Pills-containing serum were 20% and 72 hours, respectively. Meanwhile, the dual luciferase reporter assay showed that miR-885-5p could directly target EGFR. No statistical significances between the blank control group and the miR-NC group were observed (P>0.05). Compared with the blank control group, the proliferation rates of MHCC-97H hepatoma cells in the Biejiajian Pills-containing serum group, the miR-885- 5p mimics group, and the Biejiajian Pills-containing serum + miR-885-5p mimics group were significantly decreased (P<0.01), and their migration and invasion abilities were significantly decreased (P<0.05, P<0.01). At the same time, the protein expression levels of CyclinD1 and MMP1, which are closely related to cell proliferation and invasion, were significantly downregulated (P<0.05, P<0.01). The proportions of late apoptotic cells and the proportion of total apoptotic cells were significantly increased (P<0.01). In the Biejiajian Pills- containing serum group, the miR-885-5p mimics group, and the Biejiajian Pills-containing serum + miR-885-5p mimics group, miR-885-5p mRNA was significantly upregulated (P<0.01) and EGFR, MEK, and ERK1/2 were significantly downregulated at the mRNA level (P<0.05, P<0.01). EGFR, MEK, and ERK1/2 phosphorylation was inhibited (P<0.01), and the Biejiajian Pills-containing serum + miR-885-5p mimics group showed the best effect (P<0.05, P<0.01). The subcutaneous liver tumor model in nude mice verified that Biejiajian Pills can inhibit tumor growth in a dose-dependent manner. Conclusion Biejiajian Pills can promote apoptosis of MHCC-97H hepatoma cells and inhibit their proliferation, invasion, and migration. The mechanism may be related to the targeted regulation of the EGFR/MAPK/ERK signaling pathway by miR-885-5p. [ABSTRACT FROM AUTHOR]