Jianpi Huayu Fang Promote Hepatic Carcinoma Dormancy By MiRNA-570 To The Pathway Of B7-H1/PD-1

hepatocellular carcinoma(HCC) is one of the common malignant tumors in our country, its morbidity and mortality rate is the highest in the world, and each year, the new cancer of liver happened in our country account for about half the global total, Currently the treatment of hepatocellular carcinoma by operation is the first choice and the main treatment methods, postoperative recurrence are still the main causes leading to death in patients, after radical resection of hepatocellular carcinoma, metastatic recurrence rate is as high as 50%-70% above after 5 years, Therefore, after hepatic resection for hepatocellular carcinoma dormancy recently becomes a research hotspot. Traditional Chinese medicine (TCM) in this field has a huge advantage, "prevention of the recrudescence of disease" in TCM theory of liver cancer dormancy is an important guiding role, application of traditional Chinese medicine to promote liver cancer dormancy, prevention of postoperative recurrence of liver cancer patients to improve the quality of life has great significance, The prescription of Jianpi Huayu is our experience prescription for patients with hepatocellular carcinoma after operation, but th mechanism is not clear, According to the related research, this pathway of B7-H1 mediated immune suppression of microenvironment may be an important mechanism for HCC dormancy, and miRNA-570 can significantly inhibit B7-H1 expression. in human gastric cancer cells(SGC-7901) and human breast cancer cells(MDA-MB-435), Therefore, we speculate that the The prescription of Jianpi Huayu may play an important role in regulation of B7-H1/PD-1 pathway through the miRNA-570 in hepatocellular carcinoma after operation。ObjectiveResearch the inhibitory effect of invasion and metastasis on hepatic carcinoma cell in vitro of Jianpi Huayu Fang, by miRNA-570 to the pathway of B7-H1/PD-1 of inhibiting hepatic carcinoma cell growth, proliferation, invasion, and promote its apoptosis, and certifying that Jianpi Huayu Fang promote hepatic carcinoma dormancy in vivo.Methods:First of all, the human hepatoma cell line HepG-2 with the lowest expression of miRNA570 and the highest expression of B7-H1 protein was detected by qRT-PCR and bolt Western detection was used as the experimental object, Lentiviral vector, transfection of miRNA570, real-time PCR validation transfection efficiency, and building of wild and mutant type B7-H1 3’UTR psi-check2 carrier and Western blot to detect B7-H1 protein expression, then expression of luciferase activity was detected miRNA570 in wild and mutant type psi-check2 carrier. MTT detected the HepG-2 cell’s proliferation in 24,48,72,96 hours after the transfection of miRNA570, the migration of HepG-2 cells after transfection of miRNA570 was detected by transwell and the apoptosis was detected by flow cytometry, and the role of B7-H1 protein reverse miRNA570 further validation of the relationship between the two. Then, using CCK-8 method to observe the proliferation in different concentrations of Jianpi Huayu Fang on the HepG-2 cell and obtained IC50 (50% inhibitory concentration)through data, the effect of invasion which at 50% inhibitory concentration of Jianpi Huayu Fang in HepG-2 cells was observed through Transwell, the apoptosis of HepG-2 was observed at the half inhibition concentration of Jianpi Huayu Fang by using flow cytometry. At last, established the model of postoperative dormancy model of the HCC in nude mice, the model mice was randomly divided into normal control group, the low dose group, the middle dose group, the high dose group, stimulating the mice by truncating trauma, and then interventing by giving physiological saline, low dose, medicine and high dose of prescription, observed and recorded the health condition of the mice, the size of the tumors observed by the naked eye and measured by vernier caliper, the shortest path (a) and the longest diameter (b) of tumor tissue in mice were recorded, the volume of tumor were calculated according to the formula v=1/2ab2, we can obtain that the effect of Jianpi Huayu Fang on on the dormancy of HCC after operation.Results(1) Blot Western was detected B7-H1 protein expression in normal liver cell lines L02 and HL-7702 as well as liver cancer cell lines Bel-7404, Huh-7, HepG2, and we discovered the expression of B7-H1 protein in HepG2 was highest (p<0.05)。 Real-time PCR was detected miRNA-570 expression levels in normal liver cell lines L02 and HL-7702 as well as liver cancer cell lines Bel-7404, Huh-7, HepG2, and we discovered the expression of miRNA-570 expression levels in HepG2 was lowest (p<0.05)。The negative feedback regulation mechanism of miRNA-570 and B7-H1/PD-1 pathway:slow virus transfected miRNA-570 in HepG2 cells, real-time PCR detected that the group after transfection of miRNA-570 were significantly higher than the NC group, constructed Psi-check2 vectors with wild and mutant type UTR 3’B7-H1, though the blot Western,the expression of B7-H1 in HepG2 cells was significantly decreased after transfection with miRNA-570; though the real-time PCR, the expression of miRNA-570 in HepG2 cells was not significantly different with the NC group, The expression of miRNA-570 in the mutant-type psi-check2 vector was twice as than that of the wild-type psi-check2 vector (p<0.05). MTT detected in 0,24,48,72,96 h, miRNA-570 high expression of HepG2 cells compared with the NC group, the proliferation was significantly decreased (p<0.05), transwell detected the miRNA-570 high expression of HepG2 cell was migration decreased (p<0.05), the apoptosis of HepG2 cells with high expression of miRNA-570 was significantly increased by flow cytometry (p<0.05). HepG2-miR-570+B7-H1 is the group of B7-H1 overexpression vector, and HepG2-miR-570+Vector as control group, the two group was both transfected the miR-570 and over expressed vector, the expression level of B7-H1 was detected by blot Western, MTT detected the proliferation level of HepG2 cells in the culture of 0,24,48,72,96 h, the apoptosis of HepG2 cells was detected by flow cytometry, and the migration ability of HepG2 cells was detected by Transwell (p<0.05)。(3)The inhibitory effect of Jianpi Huayu Fang on the human hepatocarcinoma cell line HepG-2:CCK8 results showed that the effect of Jianpi Huayu Fang on the human hepatocarcinoma cell line HepG-2 which is inhibiting the proliferation (p<0.05), the IC50 value is 6.8872mg/ml, through analysed the data. (2)Transwell experimental results showed that the 50% inhibitory concentration of Jianpi Huayu Fang effects on HepG-2 cells for 24 hours, the number of invasive was decreased (p<0.05). (3) The results of flow cytometry showed that the 50% inhibitory concentration of Jianpi Huayu Fang could induce apoptosis of HepG-2 cells after 48 hours (p<0.05).(4) the dormancy of tumor tissue:under the repeated trauma stimulation, The dormancy rate of the blank control group was 0, the dormancy rate of low dose group and middle dose group were 80% and 60%, respectively (p<0.05).ConclusionThe prescription of Jianpi Huayu have a significant inhibitory effect on the proliferation and invasion of hepatocellular carcinoma cells(HepG-2), can induce the apoptosis of HepG-2, have recovering effect for hepatocellular carcinoma after operation, all those results may be related to regulation of the negative feedback regulation mechanism of miRNA570 and B7-H1/PD-1 pathway.