The Effect Of Sarcopenia And Inflammatory Cytokine CHI3L1 In Early Warning Of Tumor Recurrence After Liver Transplantation And Its Mechanism

BackgroundHepatocellular carcinoma is one of the main indications for adult liver transplantation in China.How to reduce the rate of tumor recurrence and metastasis in liver transplant recipients is very important.Sarcopenia is a disease associated with age-related decline in skeletal muscle mass and function.Its occurrence is related to various factors such as the body's nutritional status,exercise intensity,and the presence of chronic diseases.Inflammatory cytokines can activate a variety of molecular pathways to participate in skeletal muscle consumption,resulting in an imbalance in muscle protein synthesis and catabolism,leading to sarcopenia.Previous studies have shown that sarcopenia can affect the prognosis of patients with malignant tumors such as liver cancer.Researching the impact of preoperative sarcopenia on the prognosis of recipients of liver transplantation for hepatocellular carcinoma,screening sensitive inflammatory cytokines and conducting in-depth mechanism research to achieve effective early warning of tumor recurrence after liver transplantation for hepatocellular carcinoma are significantly important.AimsThe aim of this research was to study the effect of preoperative sarcopenia on the prognosis of recipients with hepatocellular carcinoma after liver transplantation;To screen and establish inflammatory cytokines related to sarcopenia and tumor recurrence after liver transplantation based on inflammation-related proteomics and other technologies;To study the potential mechanism basing on the key inflammatory cytokines how sarcopenia affecting the prognosis of recipients with hepatocellular carcinoma after liver transplantation.MethodsSection 1:This study retrospectively included 136 patients who underwent liver transplantation for hepatocellular carcinoma at our center from January 1,2012 to December 31,2017.Then we obtained their clinical data and follow-up information,outlined and measured the area of the third lumbar spine skeletal muscle in the CT image of the recipient,and defined skeletal muscle mass(SMI)<43.75 cm~2/m~2 as sarcopenia.Cox regression was used to analyze the effect of sarcopenia and other clinical pathological factors on the prognosis of liver transplant recipients,and a nomogram model was drawn based on the independent risk factors obtained by multivariate analysis.Based on Kaplan-Meier survival analysis,the impact of sarcopenia on overall survival(OS)and recurrence-free survival(RFS)of liver transplant recipients was clarified.At the same time,the clinicopathological factors related to the occurrence of sarcopenia in liver cancer patients were analyzed.Section 2:Bio-Plex Pro?technology was used to detect the levels of 38inflammatory cytokines in the serum of 136 liver transplant recipients before operation,and inflammation-related proteomics data based on the contents of these 38inflammatory cytokines were obtained.Through a comparative analysis of sarcopenia and non-sarcopenia recipients,candidate inflammatory cytokines related to sarcopenia were established,and correlation studies between inflammatory cytokines were carried out.Cox regression analysis was used to determine the correlation between the expression levels of the above inflammatory cytokines and the prognosis of liver transplant recipients.By establishing respective cut-off values,a survival curve was drawn based on Kaplan-Meier survival analysis to determine different levels of inflammatory cytokines on the prognosis of liver transplant recipients.Section 3:The above research found that the inflammatory cytokine CHI3L1 was related to the prognosis of liver transplant recipients.We used plasmids to overexpress chi3l1 in mouse hepato-carcinoma cell line Hep1-6 and human hepatoma cell line Huh7,to explore the effect of CHI3L1 protein on tumor cell proliferation,clonal formation,migration,and invasion,and to detect tumor invasion related proteins by Western blot.The morphological changes of myoblast C2C12 and the expression of CHI3L1 protein were studied during cell differentiation and the changes of C2C12 proliferation and apoptosis after overexpression of chi3l1 were explored.C2C12 overexpressing chi3l1was co-cultured with liver tumor Hep1-6 cells to study the effect of myoblast CHI3L1expression on liver tumor migration and invasion ability.Then,TNF-?and LPS were used as inflammatory stimuli to study the expression change of CHI3L1 and differentiation degree in C2C12 cells under the inflammation stimulation.Finally,using small interfering RNA to knock down chi3l1 in C2C12 cells,we studied the changes of liver tumor cell migration and invasion ability after co-cultivating tumor cell Hep1-6with C2C12,ResultsSection 1:29 of the 136 recipients who underwent liver transplantation included were preoperatively combined with sarcopenia,accounting for 21.3%.In the single factor Cox regression analysis,AFP>250 ng/ml(HR=1.708,p=0.042),exceeds the Hangzhou Criterion(HR=2.066,p=0.006),exceeds the Milan Criterion(HR=2.057,p=0.015),PVTT(HR=2.206,p=0.003),and sarcopenia(HR=2.153,p=0.007)were all related to OS in recipients with hepato-carcinoma after liver transplantation.And the AFP level(p=0.011),the presence of PVTT(HR=2.014,95%CI=[1.182,3.433],p=0.010),combined with sarcopenia(HR=1.982,95%CI=[1.129,3.479],p=0.017)were independent risk factors for poor postoperative prognosis of recipients with hepato-carcinoma after liver transplantation.The nomogram was used to construct a prediction model of overall survival after liver transplantation.Kaplan-Meier curve analysis found that preoperative sarcopenia was the influencing factor of overall survival and tumor-free survival of liver transplant recipients(p=0.007 and 0.041,respectively).Based on whether the patients meet the Hangzhou Criteria of liver transplantation,we conducted a subgroup analysis of sarcopenia.The OS and RFS of liver cancer recipients who exceeded the Hangzhou Criteria and had sarcopenia were significantly inferior to the other three groups.Finally,we analyzed the clinical risk factors affecting the incidence of preoperative sarcopenia in liver transplant recipients and found that only the preoperative BMI level of the recipient was related to the incidence of sarcopenia(21.2±2.0 vs.23.7±2.7,p<0.001),the lower BMI,the higher the risk of sarcopenia.Section 2:Using data from inflammation-related proteomics,we found that the levels of TNFSF13B,CHI3L1,and IL-19 were three inflammatory factors related to the occurrence of sarcopenia.Their contents in the sarcopenia group and the non-sarcopenia group were 154013.92±75095.85 pg/ml and 127301.49±62482.11 pg/ml(p=0.026),13271.64±3897.01 pg/ml and 11400.77±3833.07 pg/ml(p=0.026),97.03±33.57 pg/ml and 81.10±38.95 pg/ml(p=0.033),respectively.And there was a weak correlation between the content of CHI3L1 and TNFSF13B,the correlation coefficient r=0.37.Using 16500 pg/ml as the cut-off value of TNFSF13B content,it was found that the OS(p<0.001)and RFS(p<0.001)of the high TNFSF13B group were inferior to the low TNFSF13B group.Using 13800 pg/ml as the cut-off value of CHI3L1 content,the recipients with high CHI3L1 had shorter OS(p=0.024)and RFS(p=0.020).Section 3:Overexpression of chi3l1 in mouse hepatocellular carcinoma cell line Hep1-6 and human hepatoma cell line Huh7 could upregulate tumor invasion-related proteins and promote tumor's proliferation,clonal formation,migration,and invasion ability.During the differentiation of myoblast C2C12,the expression level of CHI3L1gradually increased.Overexpression of chi3l1 could increase the proliferation ability of myoblast C2C12 and reduce the apoptosis of C2C12 under LPS stimulation.Myoblasts overexpressing chi3l1 could promote liver tumor cells to up-regulate invasion-related proteins,and promote their migration and invasion ability.Inflammatory stimuli caused by TNF-?and LPS could up-regulate CHI3L1 in C2C12cells and promote the expression of myogenic proteins.TNF-?-pretreated C2C12 could promote the migration and invasion ability of tumor cells Hep1-6.After TNF-?pretreatment but knocked down chi3l1,C2C12's ability to promote tumor migration and invasion was obviously weakened.ConclusionPreoperative sarcopenia will increase the recurrence rate of liver cancer after liver transplantation for HCC and shorten the overall survival of the recipient.It is an independent risk factor of poor prognosis for recipients with hepato-carcinoma after liver transplantation.Inflammation-related proteomics studies have found that the inflammatory cytokines CHI3L1,TNFSF13B,and IL-19 can be used as biomarkers for sarcopenia.High levels of CHI3L1 and TNFSF13B in the preoperative serum of the recipient are associated with poor prognosis after liver transplantation for HCC.These two inflammatory cytokines are expected to be early warning molecules for poor prognosis of recipients.Under the stimulation of inflammatory factors,myoblast will upregulate CHI3L1,which will protect myoblast and promote the proliferation and invasion ability of liver cancer cells.