The Mechanism Of Shenqi Fuzheng Injection In The Treatment Of Hepatocellular Carcinoma By STAT1 Synergistic Interferon

Hepatocellular carcinoma(HCC)is one of the most common malignancies in the world and is the third most malignant tumor in the world.It is the third most common cause of global cancer.Due to the high rate of hepatitis B infection,China is a high incidence of liver cancer,at least 300,000 people die each year from liver cancer,accounting for more than half of the world's death of liver cancer,is the second cancer killer.In recent years,the incidence of liver cancer around the world has increased the trend.After several decades of efforts,liver cancer clinical and basic research have made some progress,but the overall prognosis of liver cancer has not been significantly improved,the 5-year survival rate of only 3%-5%.Surgery is still the most effective method for the treatment of liver cancer,but the total recurrence rate of liver cancer after surgery as high as 50%-100%,postoperative high recurrence rate has become a further improvement of long-term efficacy of the bottleneck is the key to capture liver cancer.Therefore,to find clinically useful measures to reduce the recurrence of liver cancer recurrence is particularly necessary.IFN-? has antiviral infection,regulation of immune function,inhibition of cell proliferation,anti-tumor neovascularization and other biological effects,has been widely used in liver cancer and other malignant tumor treatment research.Studies have shown that high doses of long-term use of IFN-? can inhibit the proliferation and invasion of hepatoma cells in nude mice.At present,IFN-? has become a clinical prevention of liver cancer recurrence of one of the conventional treatment program.However,due to some patients with liver cancer can not tolerate a large number of long-term use of IFN-? caused by discomfort and side effects,limiting its wide clinical application,and clinical randomized study found that liver cancer resection after IFN-? treatment can be extended Patients with survival,delayed recurrence,but compared with the control group did not reduce the recurrence rate,some patients in the withdrawal after a period of time after the use of IFN-? ineffective.The reason for this is the long-term use of IFN-? after liver cancer resistance.Therefore,clear the causes of resistance to IFN-? in liver cancer,and accordingly take effective targeted measures will help improve the efficacy of IFN-a,reduce the dosage and side effects of IFN-?,reduce postoperative recurrence and metastasis Rate,for the improvement of the prognosis of patients with liver cancer is of great significance.We found that the agent Qiqi Fuzheng injection can effectively inhibit the growth and metastasis of hepatocellular carcinoma and confirm that it can significantly enhance the expression of STAT1 in hepatocellular carcinoma tissues.In view of the high expression of STAT1,it is the key to reduce the resistance of IFN-a,We speculated that Shenqi Fuzheng injection can effectively reduce the resistance of liver cancer to IFN-a.In this study,MTT,Transwell,ELISA,Western Blot,immunohistochemistry,oligonucleotide microarray and RNA interference were used to establish human hepatocellular carcinoma cell line MHCC97L and lung cancer model with lung metastasis potential.To investigate the effect of Shenqi Fuzheng Injection on the growth and metastasis of hepatocellular carcinoma(HCC),and to explore the effect of Shenqi Fuzheng injection on JAK/STAT signaling pathway,and to elucidate the mechanism of Shenqi Fuzheng injection on reducing the resistance of IFN-a,For the clinical use of Shenqi Fuzheng injection and IFN-a prevention and treatment of liver cancer recurrence and metastasis to provide a theoretical basis.Objective.To observe the effect of Shenqi Fuzheng injection combined with IFN-a on the proliferation and invasion of hepatocellular carcinoma cell line MHCC97L;To construct the MHCC97L tumor cell of STAT1 gene "silence" and to verify the inhibitory effect of STAT1 gene expression;The invasion and proliferation ability of STAT1 gene "silencing"MHCC97L tumor strain were tested to verify the mechanism of Shenqi Fuzheng Injection in synergizing IFN-a to inhibit hepatocarcinoma cells.Methods 1.To observe the effect of Shenqi Fuzheng Injection on the proliferation and invasion of hepatocellular carcinoma cell line MHCC97L in combination with IFN-a.1.1 To study the effect of Shenqi Fuzheng injection on the proliferation of MHCC97L tumor strain by MTT assay.The effect of small dose Shenqi Fuzheng injection and IFN-a alone or in combination on the proliferation of MHCC97L cells was detected by cell proliferation assay(MTT).1.2 The effect of Shenqi Fuzheng injection and IFN-Effect of Fuzheng Injection Combined with IFN-? on Invasion of MHCC97L Tumor The effect of Shenqi Fuzheng Injection and IFN-? on invasion of MHCC97L cells was investigated by in vitro invasion assay(Transwell).Western Blot/RT-PCR To investigate the effect of Shenqi Fuzheng injection and IFN-? alone or in combination on the expression of STAT1 and VEGF in MHCC97L cells.2.Construction of STAT1 gene "silencing" of MHCC97L tumor cells and to verify the inhibitory effect of STAT1 gene expression:To collect the tumor cells obtained from the previous experiment(high dose IFN-? alone treatment group and Shenqi Fuzheng injection combined with IFN-? Treatment group hepatocarcinoma cells);the Superovray JAK/STAT signaling oligonucleotide microarray was used to screen for differentially expressed genes in the above-described hepatocellular carcinoma tissues(which contained all known JAK/STAT family members,activated JAK/STAT signaling receptors,STAT protein-related nuclear cofactor and co-activating factor,STAT-induced protein and negative feedback regulatory protein);Western Blot and fluorescence quantitative PCR technique to identify differentially expressed genes from oligonucleotide microarray from protein and mRNA levels.3.The effect of Shenqi Fuzheng injection on the proliferation of MHCC97L tumor strain was studied by MTT assay:the expression of STAT1 gene "silencing"MHCC97L was induced by the STAT1 gene "silence" MHCC97L tumor strain.(MTT)was used to detect the effect of small dose of Shenqi Fuzheng injection and IFN-? alone or in combination on the proliferation of MHCC97L cells with STAT1 gene silencing.3.2 Experimental study of Shenqi Fuzheng injection by in vitro invasion experiment TNF-? was used to detect the expression of MHCC97L cells in the STAT1 gene by in vitro invasion assay(Transwell)Invasive influence.Results Compared with IFN-? alone,Shenqi Fuzheng injection combined with IFN-? could significantly enhance the inhibitory effect on MHCC97-L in human hepatocellular carcinoma.Transwell experiment showed that Shenqi Fuzheng injection combined with low dose IFN(17.67±1.53)and the number of cells in the IFN-? group(27.67±3.06)were significantly lower than those in the control group(P<0.05).The results of RT-PCR showed that MHCC97L cells in Shenqi Fuzheng injection group STAT1 mRNA expression was significantly higher than that of NaCl negative control group,indicating that 0.5g/L Shenqi Fuzheng injection could effectively up-regulate the STAT1 mRNA expression of MHCC97L in hepatocellular carcinoma cells.There was no significant difference between IFN-? group and negative control group.Western blot showed that STAT1 protein expression in MHCC97L cells of Shenqi Fuzheng injection group was significantly higher than that of NaCl negative control group,indicating 0.5g/L Shenqi Fuzheng Injection can effectively upregulate the expression of STAT1 protein in MHCC97L of hepatoma cells.There was no significant difference between IFN-a group and negative control group.The expression of STAT1 gene of MHCC97L was successfully achieved by RNAi,and the STAT1 gene RNAi vector was successfully constructed.After transfection of human hepatoma cell line MHCC97L by lentiviral vector STAT1 gene was significantly inhibited at mRNA level and protein level.The inhibitory effect of Shenqi Fuzheng injection combined with IFN-? on STATCC gene silencing MHCC97L cell line was significantly correlated with negative control group(P>0.05)and IFN-?(P<0.05).The results showed that the number of MHCC97L cells in the small dose of IFN-? group was significantly higher than that in the control group(28.00±2.00)after the STAT1 gene was "silenced" There was no significant difference compared with low dose IFN-a group(28.33±4.04).Conclusion Shenqi Fuzheng injection can improve the expression of STAT1 gene in the treatment of hepatocellular carcinoma,and achieve synergistic effect on IFN-?.