Identification And Study Of The Target Of Anti-cancer Drug Molecule Erianin In HepG2 Cells

OBJECTIVE: Erianin is a natural small molecule compound extracted from the precious Chinese medicinal materials Dendrobium officinale and Dendrobium chrysotoxum.Current studies have shown that erianin has a certain therapeutic effect on various diseases such as cancer,tumor angiogenesis,microbial infection and diabetic nephropathy,but there are still few reports on the specific mechanism and target of erianin.This article focuses on the identification and study of the target protein of erianin in human hepatoma HepG2 cells.METHODS: Firstly,the active probe and the non-active probe with the erianin analog as the active group were identified by pull down of the HepG2 cell lysate and mass spectrometry to find a protein capable of specifically binding to the active probe.Next we used the molecular docking simulation software Discovery studio 2016 V16.1 for homology modeling of human pyruvate carboxylase(h PC),and docked its simulated structure with erianin and analyzed the results.Then we overexpressed PC protein in HepG2 cells and interfering with the expression of PC protein by using lentiviral expression vector,RNA interference vector and lentivirus packaging strategy to explore the response of the protein in the HepG2 cell to the response to the treatment of erianin.Afterward we purified and expressed PC protein by using the eukaryotic expression system to affinity with the corresponding tag purified gel beads and further investigated its interaction with RH active probes as well as unmodified erianin small molecules in vitro.RESULTS: By analyzing the mass spectrometric identification results of the differential proteins in the pull down results of the active probe and the inactive probe,it was confirmed that the protein interacting with the active probe is pyruvate carboxylase(PC).A homology structure model of human pyruvate carboxylase(h PC)was obtained.Molecular docking simulations show that h PC can bind to erianin by conventional hydrogen bonding,hydrocarbon hydrogen bonding,?-anion interaction,?-disulfide interaction and ?-alkyl interaction.The lentiviral expression vector p LVX-puro-flag-PC and the RNA interference vector p ENTRTM/U6-sh PC1/2 were constructed.After co-transfection with p MD2.G and ps PAX2,a lentivirus liquid with higher titer was obtained.After lentivirus infection and puromycin screening,a HepG2/flag-PC cell line stably expressing flag-PC was obtained.The cell viability assay for HepG2/flag-PC cells and HepG2 cells infected with sh PC2 lentivirus treated with erianin for 48 h showed that the cytotoxicity of erianin was positively correlated with the expression of PC.After subculture of HepG2/flag-PC cells in a large amount,the flagPC protein with very high purity was purified from the cell lysate by using anti-flag affinity beads.The active probe and the inactive probe are separately incubated with the purified flagPC protein and cross-linked to introduce an RH fluorophore.After SDS-PAGE electrophoresis,the fluorescence imaging system revealed that the flag-PC protein could bind to a large number of active probes,while the non-active probe group had only weak fluorescence.When the erianin was directly incubated with the purified flag-PC protein,the PC enzyme activity was significantly decreased.CONCLUSION: Firstly,we have determined that probes based on the synthesis of erianin analogs can specifically capture PC protein in HepG2 cells.Molecular docking results also showed that erianin interacts with h PC.Secondly,the cytotoxicity of erianin was positively correlated with the expression of PC in HepG2 cells.Then,the purified flag-PC protein can also specifically bind to the active probe in vitro;finally,erianin also significantly affects PC enzyme activity.This study basically determined that the target protein of erianin in HepG2 cells is PC protein,which laid a foundation for further study of the anti-cancer mechanism of erianin.