To investigate the effect of formononetin on the proliferation and apoptosis of laryngeal squamous cell carcinoma cells and its possible mechanism.

The human laryngeal squamous cell carcinoma cell line Hep-2 was cultured in vitro, and the cell viability of Hep-2 cells was detected by the MTT method after treatment with 0, 5, 10, 20, 40, 60 μmol/L formononetin for 24 h. The appropriate concentration of mancoxanthin was screened according to the IC₅₀ value. Hep-2 cells cultured were treated with, 20 μmol/L formononetin (20 μmol/L) , 40 μmol/L formononetin (40 μmol/L) , formononetin (40 μmol/L) + negative control (transfected with miR-140-5p inhibitor negative control) , formononetin (40 μmol/L) + miR-140-5p inhibitor (transfected with miR-140-5p inhibitor) . After transfection and drug treatment, the expression of miR-140-5p and T cell immunoglobulin and ITIM domain protein (TIGIT) in Hep-2 cells in each group was detected by real-time fluorescence quantitative PCR (qRT-PCR) experiment and western blot; the proliferation of Hep-2 cells in each group was detected by MTT assay and plate colony formation assay; the apoptosis of Hep-2 cells in each group was detected by flow cytometry; Western blot was used to detect the proliferation (PCNA, Cyclin D1) and apoptosis-related protein (Bax, cleaved caspase-3) expression of Hep-2 cells in each group. Nude mice were subcutaneously injected with Hep-2 cells on the back to establish a transplanted tumor model in nude mice, and they were randomly grouped into a control group, a low-dose formononetin (25 mg/kg) group, a high-dose formononetin (50 mg/kg) group, high-dose formononetin (50 mg/kg) + negative control (transfected with miR-140-5p antagomir negative control) group, high dose of forsythia (50 mg/kg) + miR-140-5p knockdown (transfected with miR-140-5p antagomir) group. After treatment, the quality and volume of transplanted tumors in nude mice of each group were detected; a dual-luciferase reporter assay was used to analyze the targeted regulation of TIGIT by miR-140-5p in Hep-2 cells.

Compared with the control, the expression of miR-140-5p (1.79±0.16, 2.58±0.22 vs 1.04±0.11) , the apoptosis rate [ (36.17±8.14) %、(68.65±14.20) %vs (2.10±0.65) %], the protein expression of Bax and cleaved caspase-3 were increased, in the 20 μmol/L formononetin and the 40 μmol/L formononetin treated cells (P < 0.05) , the expression of TIGIT protein (0.56±0.12, 0.17±0.01 vs 0.98±0.10) and mRNA (0.68±0.10, 0.34±0.03 vs 1.05±0.13) , colony formation rate [ (60.82±12.24) %、(35.36±8.20) %vs (100.0±0.00) %], cell viability (63.12±11.03, 39.75±7.14 vs 100.0±0.00) , protein expression of PCNA and Cyclin D1 were decreased (P < 0.05) ; compared with the 40 μmol/L formononetin, the expression of miR-140-5p (1.11±0.13 vs 2.58± 0.22) , the apoptosis rate [ (5.04±1.51) %vs (68.65±14.20) %], the protein expression of Bax and cleaved caspase-3 were decreased in the high-dose formononetin+miR-140-5p knockdown cells (P < 0.05) , the expression of TIGIT protein (0.92±0.18 vs 0.17±0.01) and mRNA (0.97±0.14 vs 0.34±0.03) , colony formation rate[ (90.76±15.02) %vs (35.36±8.20) %], cell viability (92.01±17.13 vs 39.75±7.14) , protein expression of PCNA and Cyclin D1 were increased (P < 0.05) ; Compared with the control group, the weight [ (609.23±58.14) 、(465.87±41.26) vs (762.14±76.51) mg] and volume [ (532.82±60.28) 、(396.34±48.10) vs (681.70±72.44) mm³] of transplanted tumor were decreased in nude mice in the low-dose formononetin group and the high-dose formononetin group (P < 0.05) ; Compared with the high-dose formononetin group, the weight [ (735.48±80.12) vs (465.87±41.26) mg] and volume [ (654.79±75.85) vs (396.34±48.10) mm³] of transplanted tumor were increased in nude mice in the high-dose formononetin+miR-140-5p knockdown group (P < 0.05) ; MiR-140-5p in Hep-2 cells targeted to down-regulate TIGIT expression.

Formononetin inhibits proliferative activity and promotes apoptosis in laryngeal squamous cell carcinoma cells by regulating miR-140-5p/TIGIT axis expression.