Ginsenoside CK Regulates Bclaf1 Mediated HIF-1? Mechanism Of Ubiquitination Inhibiting Hepatoma Cell Proliferation By Glycolysis

Background:Hepatocellular carcinoma is the most important type of primary liver cancer.The incidence rate in my country ranks the fourth among all malignant tumors,and the mortality rate ranks the third.Because the condition of liver cancer is hidden,most patients are already in the middle and advanced stages of liver cancer when they are discovered.In view of the problem of limited treatment drugs for patients with advanced liver cancer and easy development of drug resistance,the development of new chemotherapeutic drugs or targeted therapies is of great significance.Ginsenosides are the main components of ginseng and have various pharmacological effects such as anti-inflammatory,anti-tumor,anti-dementia,and anti-allergic effects.Ginsenoside compound K(ginsenoside CK(20(S)-Ginsenoside CK,ginsenoside CK)is a ginsenoside diol type saponins extracted from ginsenosides Rb1,Rb2 and Rc.As a degradation product of human intestinal bacteria,it has A variety of pharmacological activities,including liver protection,anti-inflammatory and anti-tumor effects.Previous studies have shown that CK regulates STAT3 to induce endoplasmic reticulum stress in human liver cancer cells,promote liver cancer cell apoptosis,and inhibit proliferation.But under hypoxic conditions,ginseng The effect of saponin CK on liver cancer and its related mechanism have not been reported yet.Therefore,in-depth study of the effect and mechanism of ginsenoside CK on liver cancer is of great significance for the further development of anticancer drugs.Bcl-2 associated transcription factor 1(Bcl-2 associated transcription factor 1,Bclaf1)has been reported to be involved in the occurrence and development of liver cancer,and was found to be highly expressed in human hepatocellular carcinoma Huh7 cells.Under the 1% O2 environment,after knocking out the Bclaf1 gene,the expression level of hypoxia inducible factor-1(HIF-1?)in human liver cancer cells decreased,but increased significantly after the overexpression of Bclaf1 gene;In addition,Bclaf1 is a direct transcription target of HIF-1?.HIF-1? is an important transcription factor that cells express in response to hypoxia.It participates in mediating cell responses to hypoxia by promoting glycolysis.At the same time,HIF-1? affects the energy metabolism and proliferation of tumor cells through the degradation of ubiquitin proteasome.Therefore,it is very important to explore the effect of ginsenoside CK on Bclaf1 and HIF-1? and the role of cell glycolysis pathway under hypoxic conditions.In summary,the hypothesis is put forward that under hypoxic conditions,ginsenoside CK can inhibit the proliferation of liver cancer cells through Bclaf1 and promote the degradation of HIF-1? ubiquitination to inhibit the glycolytic pathway,thereby inhibiting the proliferation of liver cancer cells.Objectives:In this study,human liver cancer cells in vitro and primary liver cancer rats in vivo were tested to detect the inhibitory effect of ginsenoside CK on the growth of liver cancer cells under hypoxic conditions.To explore the effect of ginsenoside CK on the glycolysis pathway mediated by Bclaf1 and HIF-1?.At the same time,it was detected that ginsenoside CK regulates Bclaf1 to affect the glycolysis pathway mediated by HIF-1?,which in turn affects the molecular mechanism of liver cancer cell proliferation.The development of the anti-liver cancer effect of ginsenoside CK provides a new theoretical basis.Methods:1.In vitro experiment: The effect of ginsenoside CK on the proliferation of different liver cancer cell lines Bel-7404 and Huh7 under hypoxic conditions was detected by CCK8 method;the colony formation of ginsenoside CK treated liver cancer cells was detected by plate cloning experiment;flow cytometry was used Cytometer to detect cell cycle;immunochemical technology and immunofluorescence technology to detect protein expression and localization of Bclaf1 and HIF-1?;cell energy metabolism instrument to detect glycolytic pressure and real-time ATP production rate of liver cancer cells;use immunoprecipitation method to detect The binding of Bclaf1 and HIF-1? in liver cancer cells;Western Blot was used to detect the expression of Bclaf1,HIF-1?,glycolysis-related proteins and ubiquitination-related proteins in liver cancer cells;In addition,the use of CRISPR/Cas9 technology to knock out Bclaf1 and HIF-1? HIF-1? gene to further verify that ginsenoside CK inhibits glycolysis of liver cancer cells through Bclaf1,thereby inhibiting proliferation.2.In vivo experiment: A rat model of primary liver cancer was established by diethylnitrosamine(DEN)induction and divided into model group,ginsenoside CK 2.5,5mg/kg administration group.After 2 weeks of continuous administration,PET/CT was used to detect the changes in liver tumors before and after the administration and the SUV value was calculated;immunohistochemistry was used to detect the protein expression and localization of Bclaf1 and HIF-1?;Western Blot was used to detect Bclaf1 in liver cancer cells,HIF-1?,glycolysis-related protein and ubiquitination-related protein expression.Results:1.In vitro experiments: CCK8 results show that under hypoxic conditions,ginsenoside CK can inhibit the proliferation of Bel-7404 and Huh7 cells in a dose-and time-dependent manner.The plate cloning experiment showed that the colony forming ability of the cells was significantly reduced after the ginsenoside CK was used,and the cell cycle results showed cells are blocked in the G0/G1 phase;the results of the cell energy metabolism meter show that ginsenoside CK inhibits glycolytic pressure and the real-time production rate of ATP;the results of immunocytochemistry and immunofluorescence technology show that under hypoxic conditions,ginsenoside CK can be Inhibit the expression of Bclaf1 and HIF-1?,and Bclaf1 is located in the nucleus and cytoplasm,and HIF-1? is located in most of the cytoplasm;immunoprecipitation results show that ginsenoside CK inhibits the binding of Bclaf1 and HIF-1?;Western Blot results Shows that ginsenoside CK can inhibit the expression of glycolysis-related proteins HK2,Glut1,LDHA,PDK1,and activate the ubiquitinated protein of HIF-1?(down-regulate the expression of HSP70,HSP90 and p VHL protein,and up-regulate the expression of PHD and RACK1 protein under hypoxic conditions.expression).In addition,the Bclaf1 gene knockout and HIF-1?gene knockout Bel-7404 and Huh7 cells were constructed.Compared with the ginsenoside CK treatment group,the ginsenoside CK+ Bclaf1 gene knockout group had glycolytic stress and real-time production of ATP.The rate and related proteins were further suppressed,and the ubiquitination of HIF-1? was further promoted.Compared with the Vehicle group,the expression of HIF-1? in the Bclaf1 knockout group was reduced;in the ginsenoside CK+HIF-1? knockout group,Glycolysis pressure,ATP real-time production rate and related proteins were further suppressed.Compared with the Vehicle group,Bclaf1 in the HIF-1? gene knockout group did not change significantly.In vivo experiment: Ginsenoside CK can improve liver nodules and bleeding points in rats with primary liver cancer;PET/CT results show that the tumor area is reduced after administration,and the SUV value is also reduced;immunohistochemistry technology The results showed that ginsenoside CK reduced the expression of Bclaf1 and HIF-1?;Western Blot results showed that ginsenoside CK could inhibit the expression of glycolysis-related proteins and activate the ubiquitinated protein of HIF-1?.Conclusions:1.Under hypoxic conditions,ginsenoside CK can inhibit the proliferation of human liver cancer cells.2.Under hypoxic conditions,ginsenoside CK can inhibit the glycolysis pathway in human liver cancer cells.3.Under hypoxic conditions,ginsenoside CK can inhibit the expression of Bclaf1 and the binding of Bclaf1 and HIF-1? protein in human liver cancer cells.4.Under hypoxic conditions,ginsenoside CK can inhibit the expression of HIF-1? in human liver cancer cells and promote the ubiquitination and degradation of HIF-1?.5.Ginsenoside CK can inhibit tumor growth in rats with primary liver cancer.Inhibit the expression of Bclaf1,promote the ubiquitination of HIF-1?,and inhibit the glycolysis pathway mediated by HIF-1? to inhibit cell proliferation.