| Introduction:Liver cancer is one of the most common malignancy in the world,and the incidence and mortality rate is in the forefront among various types of tumors.According to Report of Cancer Incidence and Mortality in China,the morbidity of liver cancer ranks third,and the mortality rate ranks second n males and third in females.More alarming is that the number of new cases and the deaths in China account for about 50%of global data.From now on,there must be more concerns about liver cancer in China.At present,the treatment of liver cancer is given with comprehensive treatment program which is based on surgery.While after radical operation,postoperative tumor recurrence is the main problem to the liver cancer patients with long-term survival and high quality of life.Nowadays,traditional Chinese medicine has been used more and more in the prevention and treatment of tumor recurrence,especially in improving the quality of life and prolong the survival.Due to the clinical experience of Professor Qian Bowen and series of experiments,Jianpi Huayu Decoction shows satisfactory clinical effects in preventing and treating postoperative tumor recurrence.It has the effects of invigorating spleen to tonifyi qi,regulating qi to dissipate dampness,promoting blood and resolve blood stasis to dissolve lump,and clearing heat-toxin,which shows the characteristics of strengthening resistance to eliminating evil,and the combination of tonification and purgation.Therefore,it is of great significance to study the specific anti-tumor mechanism of Jianpi Huayu Decoction.According to the former research,we proved the negative feedback regulation mechanism between miR-570 and B7-H1/PD-1 pathway at the cellular level.Now we further hypothesize that Jianpi Huayu Decoction can up-regulate miR-570,which depress the B7-H1/PD-1 pathway on postoperative recurrence of liver cancer,leading to the tumor dormancy.In this study,on the one hand,we inoculated human hepatoma cell SMMC-7721 and miR-570 mimic SMMC-7721 to build orthotopic transplantation model of human hepatocellular carcinoma in nude mice.Through further resection and serial trauma,nude mice model of postoperative recurrence or dormancy was established.With the use of PET/CT imaging,histopathological examination,qRT-PCR analysis,Western blot analysis and the Flow Cytometry,the in vivo mechanism of miR-570 and B7-H1/PD-1 pathway in postoperative liver cancer have been verified.on the other,after intervened with Jianpi Huayu Decoction or 5-fluorouracil,the nude mice in postoperative recurrence or dormancy model of HCC were also observed by the use of PET/CT imaging,histopathological examination,qRT-PCR analysis,Western blot analysis and the Flow Cytometry.These were of help to proof the preventive and theraputic effect of Jianpi Huayu Decoction on postoperative tumor recurrence and approach the mechanism.Purpose:To clarify the mechanism of miR-570 regulate B7-H1/PD-1 pathway on postoperative recurrence of liver cancer in vivo studies.To discuss the probably mechanism of Jianpi Huayu Decoction that mediate the postoperative recurrences by increasing miR-570 expression to down-regulate B7-H1/PD-1 pathway.Method:First of all,we inoculated human hepatoma cell SMMC-7721 and miR-570 mimic SMMC-7721 in nude mice to build orthotopic transplantation model of human hepatocellular carcinoma.After further resection and serial trauma,nude mice model of postoperative recurrence or dormancy was also established.Nude mice were observed and recorded during the experimentation.Using PET/CT imaging,pathological examination to assess the situation of tumor recurrence.RT-PCR analysis and Western blot analysis is tested to know the expression level of miR-570 and B7-H1.Flow Cytometry is used to detect the levels of CD4+ and CD8+ T lymphocyte and the ratio of CD4+/CD8+.On the other,after intervened with Jianpi Huayu Decoction or 5-fluorouracil,the nude mice in postoperative recurrence or dormancy model of HCC were also observed by the use of PET/CT imaging,histopathological examination.RT-PCR analysis and the Western blot analysis is tested to know the expression level of miR-570 and B7-H1.At last,Flow Cytometry is also used to detect the levels of CD4+ and CD8+ T lymphocyte and the ratio of CD4+/CD8+.Result:1.Inhibitory effect of miR-570 on postoperative recurrence of hepatocellular carcinoma:1)At the end of the experiment,the nude mice all survived,without prominent tumor observed in the abdomen surface.On the twenty-fourth day after operation in NC group,5 mice weight decreased significantly appearing cachectic like hunched back,move slow,and poor endurance.And there were 2 mice observed with suspicious tumor on head or face in NC group.While the mice in miR-570 group were in good condition,and had no suspicious tumor.2)Nude mice in two groups were given serial trauma after operation,and then took PET/CT imaging.It shows high uptake area in the left upper quadrant and epigastrium,while the high uptake area of NC group was significantly increased compared with miR-570 group.3)The pathological examination showed that:13 mice were found tumor recurrence in NC group.Not only that,3 mice were also found with scattered carcinoma nodules in peritoneal cavity,2 with metastatic tumor in lung,and 2 with head facial metastases.While the miR-570 group had 8 mice of tumor recurrence in the liver,but other organs showed no tumor cells.Through pathological examination,the recurrence rate of miR-570 group was lower than the NC group,but it shows no statistical significance(P<0.05).However,there were significant differences in the degree of metastasis between these two groups.Data shows that the number of metastases of miR-570 group was significantly decreased(P<0.01).The results of Flow Cytometry showed that the T lymphocyte subsets of miR-570 group changed remarkably,the level of CD4+ cells and the ratio of CD4+/CD8+ increased significantly,and the level of CD8+ cells decreased(P<0.01).2.Negative regulation mechanism of miR-570 between B7-H1/PD-1 pathway in postoperative hepatocellular carcinoma:RT-PCR analysis showed the expression of miRNA-570 in miR-570 group increased significantly than the NC group(P<0.01),while Western blot analysis showed the expression B7-H1 in the miR-570 group significantly decreased(P<0.01).3.Preventive and therapeutic effect of Jianpi Huayu Decoction on postoperative recurrence of hepatocellular carcinoma:1)At the end of the experiment,the nude mice all survived,without prominent tumor observed in the abdomen surface.On the twenty-fourth day after operation,there were 5 mice in NC group,3 in 5-FU group,and 1 in Jianpi Huayu Decoction group appearing appearing cachectic like weight decrease,hunched back,move slow,and poor endurance.And there were 2 mice observed with suspicious tumor on head or face in NC group.ON the twenty-sixth days after operation,2 mice in 5-FU group were observed with serious cachexia,with aggravated weight loss and poor appetite.2)Nude mice in three groups were given serial trauma after operation,and then took PET/CT imaging.It shows high uptake area in the upper-middle abdomen.3)The pathological examination showed that:13 mice were found tumor recurrence in NC group.Not only that,3 mice were also found with scattered carcinoma nodules in peritoneal cavity,2 with metastatic tumor in lung,and 2 with head facial metastases.5-FU group had 8 mice of tumor recurrence in the liver,of which there were 2 mice found with scattered carcinoma nodules in peritoneal cavity,and 2 with metastatic tumor in lung.While Jianpi Huayu Decoction group had 10 mice of tumor recurrence in the liver,but of which there was only 1 with scattered carcinoma nodules in peritoneal cavity.Through pathological examination,the recurrence rate and the amount of metastasis among these three groups shows no significant difference(P>0.05).But the amount of metastasis between Jianpi Huayu Decoction group and NC group still differ greatly(P<0.05).4.Intervention effect of Jianpi Huayu Decoction on the expression of miRNA-570 and B7-H1:RT-PCR analysis showed the expression of miRNA-570 in 5-FU group and Jianpi Huayu Decoction group increased significantly than the NC group(P<0.01),but there was no statistic difference between 5-FU group and Jianpi Huayu Decoction groups(P>0.05).The expression of B7-H1 in the three groups was detected by Western blot analysis.The expression in the 5-FU group and the Jianpi Huayu Decoction group was significantly lower than that in the NC group(P<0.05),and there was remarkable difference between the 5-FU group and the Jianpi Huayu Decoction group(P<0.05).At last,the results of Flow Cytometry showed that the T lymphocyte subsets of 3 groups changed remarkably.Compared with NC group,the level of CD4+ cells and the ratio of CD4+/CD8+of 5-FU group and the Jianpi Huayu Decoction group increased significantly,while the level of CD8+ cells decreased(P<0.01).Conclusion:miR-570 can negatively control B7-H1/PD-1 pathway in postoperative hepatocellular carcinoma.Jianpi Huayu Decoction can inhibit the B7-H1/PD-1 pathway by up-regulating miR-570,and promote the prognosis of liver cancer. |
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