Experimental Research On The Prevention Of Hepatocellular Carcinoma Postoperative Recurrence By Jiedu Fang Regulating Sec62

Background:Hepatocellular carcinoma?HCC?is one of the most common malignancies and is the fourth leading cause of cancer death worldwide.As a large country with liver cancer in the world,new cases of liver cancer account for 55%of the total number of new cases in the world each year,and its mortality rate is the second highest of malignant tumors in China.Currently,surgical resection,liver transplantation or local ablation is recommended for patients with very early liver cancer?Barcelona Clinic Liver Cancer[BCLC]0/A?in clinical practice.However,regardless of therapy methods,liver cancer recurrence is a major problem in the treatment.Relevant studies have found that tumour recurrence commonly occurs within two years of resection?19%to 25%in the first year?and exceeds 70%at 5 years.Metastasis is the major reason for the high recurrence and poor survival of HCC patients.Traditional Chinese medicine?TCM?prescription,"Jiedu Fang"?JDF?,is significantly effective in the treatment of a variety of tumors,especially liver cancer,which can inhibit HCC metastasis and reduce postoperative recurrence,thus then prolong the survival and increase the life quality of HCC postsurgical patients.By microarray analyses and variable survival analysis,our group has found that Sec62 is expected to be a new independent predictor of tumor-free survival after HCC surgery.Therefore,in this study we mainly demonstrate whether JDF can play the pivotal role in preventing HCC postoperative recurrence through Sec62 and further explore its downstream mechanism so as to provide a new gene target for clinical prevention and treatment of HCC postoperative recurrence.Objective:1.Verify that JDF prevents HCC postoperative recurrence and explore its optimal medication time through animal experiments.2.Explore that if JDF prevents HCC postoperative recurrence by down-regulating Sec62 and further study its downstream mechanism.Methods:1.The model animal established by Luc-Huh7 was used for intragastric administration at different times.The effect of JDF in preventing HCC postoperative recurrence was further verified by animal experiments and its optimal medication time was explored.2.The model animal was established by Luc-Huh7.JDF was used for intragastric administration according to the optimal administration time explored above.The expression of Sec62 in recurrent tissues of HCC were analyzed quantitativelyto explore the relationship between JDF and Sec62 in preventing HCC recurrence.3.Different concentrations of JDF were applied to Huh7 and Huh7-Sec62^OE,respectively,and cell scratch assay and transwell assay were used to investigate the relationship between JDF,Sec62 and cell migration and invasion.4.Huh7-Sec62^KD and Huh7-Sec62^OE were used to further investigate the effects of Sec62 on Huh7 migration,invasion and proliferation using cell scratch assay,transwell assay and MTT assay.5.Microarray analyses,IPA and GO were used to further explore the downstream mechanism of JDF in preventing postoperative recurrence of HCC by down-regulating Sec62,and q RT-PCR,Western Blot,scratch assay,transwell assay,and Co-IP were used to further validate the above results.Results:1.The model mice was established by Luc-Huh7 cells.By intragastric administration of JDF and periodic observation of recurrence,preliminary conclusion was drawn that JDF was effective in preventing recurrence of HCC in mice.And It is tentatively proposed that the optimal administration time of JDF for preventing recurrence of HCC in mice is 10 days.2.The intragastric administration of JDF to the model mice for 10 days showed that the Sec62 expression of the recurrent tumors in JDF intragastric administration group was significantly lower than that in saline intragastric administration group?P<0.05?.3.Different concentrations of JDF?0.2 mg/m L,0.5 mg/m L?were applied to Huh7-Sec62^OE and Huh7-NC,respectively.Cell scratch assay and transwell assay showed that JDF inhibited the migration and invasion of Huh7 cells,while when Huh7 overexpressed Sec62,the inhibitory effect of JDF on the migration and invasion of Huh7 cells was significantly attenuated.4.Cell scratch assay,transwell assay and MTT assay using Huh7-Sec62^KDand Huh7-Sec62^OE showed that overexpression of Sec62 promoted the migration and invasion of Huh7,while knockdown of Sec62 significantly inhibited the migration and invasion of Huh7?P<0.01?;knockdown or overexpression of Sec62 had no significant effect on the proliferation of Huh7 cells at 24 h.5.Microarray analyses and IPA of human gene expression in Huh7-Sec62^KD and Huh7-Sec62^OE showed that the change in Integrin signalling in both Huh7-Sec62-sh RNA cells and Huh7-Sec62overexpressing cells is consistent with the expression of Sec62.GO showed that it was most correlated with cell movement.Integrative analysis of Integrin signaling based on IPA database and Huh7-Sec62^KD cell microarray data,and q RT-PCR and Western Blot validation results,confirmed that:Sec62 regulates Integrin pathway including Integrin?2b,Integrin?4,Integrin?5,Integrin?V,CAV1,calpain,MLCK.Further cell validation results showed that overexpression of Integrin?2b,Integrin?4,Integrin?5,and Integrin?V in Huh7-Sec62^KD cells,only ectopic overexpression of Integrin?5 or?V abolished the knockdown of Sec62-induced inhibition of migration and invasion.And Co-IP results of Huh7 cells showed that Sec62 indeed binds to Integrin?V or Integrin?5 to initiate downstream signaling,calpain and MLCK,to promote migration and invasion of HCC.Conclusion:1.JDF is effective in preventing HCC postoperative recurrence in mice and it is tentatively proposed that the optimal administration time of it is 10 days.2.JDF mainly inhibits migration and invasion of tumor cells by down-regulating Sec62/Integrin?/CAV1 signaling,thus preventing HCC postsurgical recurrence.