| Objective: 1.To analyze the differences of intestinal flora and enterotype during hepatocellular carcinoma(HCC)patients’ disease curse using high-throughput sequencing combined with clinical big data The biomarkers of HCC would been screened at microbial level,and thus a unique predictive model based on microbial genomic information could be established to evaluate the prognosis of HCC patients.2.To explore the clinical significance and biological role of fibrinogen γ-chain(FGG),which served as a high weight variable in the established predictive model,in the progression of liver cancer.Methods: 1.The faecal samples of healthy control group(n=20)and HCC patients before operation(n=91),postoperative(n=82)and recurrence(n=15)were collected for 16 S rRNA sequencing and bioinformatics analysis was conducted to determine the intestinal flora and enterotype changes in the progression of hepatocellular carcinoma.Identified species associated with different clinicopathologic features and prognosis were analyzed by statistical method.2.Liver cancer patients enrolled for fecal sample sequencing were included in downstream analysis.The detailed clinical information of liver cancer was collected using clinical big data platform to construct a full-dimensional data set.Then,combined with the binary logistic regression algorithm,the dimensionality reduction of the full-dimensional clinical data set was conducted to construct the dimensionality reduction one,and the dimensionality reduction data set was used to construct model 2 in the same way.Finally,the classification variables of intestinal type were incorporated into the dimensionality reduction data set to generate the comprehensive data set,and the model 3 was constructed using the comprehensive data set in the same way.The prediction accuracy and ROC curve were used to compare the prediction capability of the above models and their high-weight variables were identified.3.The expression of FGG was detected by Western blot /RT-qPCR in 24/35 pairs of HCC tumor tissues and corresponding adjacent tissues,respectively,and Immunohistochemistry was used for another 79 cases of liver cancer.Then the association of FGG expression with clinicopathological factors and prognosis was analyzed.Sk-hep-1 cells with FGG overexpression and knockout were constructed and their migration and invasion abilities were analyzed by cell scratch assay and Transwell assay respectively.Western blot was used to detect the expressions of EMT-related phenotypic markers(E-cadherin 、 N-cadherin)and transcription regulators(Slug,Zeb1).The potential regulatory mechanisms are also verified.Results: 1.Alpha diversity analysis suggested that the species diversity of intestinal flora of normal population was higher than that of HCC patients,while the intestinal flora diversity of postoperative HCC patients was higher than that of preoperative and postoperative recurrence,suggesting that the flora structure improved to be more similar to healthy control after tumor removal.However,intestinal flora diversity decreased again during HCC relapse,showing more similarity with preoperative tumor-bearing status.It indicated that the microbial diversity is closely related to the disease status of HCC patients.The results of the Beta diversity analysis showed that there were great difference in the composition of the flora between the healthy population and the HCC patients,which was consistent with the previous reports.In additional,the phylum,the class,the order,the family,and the genus were analyzed layer by layer.When the preoperative group,the postoperative group,the recurrent group and the control group are compared with each other,the species distribution showed some differences accompanied with the disease progress,which showed potential for further research.Therefore,we compared the flora of the control group and the preoperative group of HCC again,and 10 and 17 species with significant abundance change were found by Lefse and species absolute abundance analysis,respectively.In combination with previous research,strains Verrucomicrobia,Akkermansia,Bacteroides,and Dialister were filtered out from all species with significant abundance change and identified as playing a role in the development of HCC.Using the same analysis strategy,20 and 9 different species were screened by comparison of HCC preoperative group and the postoperative group using Lefse and species absolute abundance analysis,respectively.The abundance of beneficial species tends to increase,while the harmful species tends to decrease,and this trend was consistent with the change of patient disease status,which indicated that the intestinal flora in the postoperative group tends to change toward that in healthy people.According to the data of 15 pairs of paired samples all including sample collected before,after surgical operation and before the recurrence of liver cancer,page-pair analysis was conducted based on the absolute abundance of species,and a total of 5 species with significant difference among different gourps were found.The abundance of Blautia and Bilophila showed a significant negative correlation with the tumor formation,resection and recurrence,which suggested that it may be a beneficial bacterium,which was consistent with the findings in other digestive system tumors.2.Intestinal analysis can directly reflect the structure and composition of intestinal flora,and samples could be generally divided into three types of: enterotype 1(E1),enterotype 2(E2),enterotype 3(E3).In this study,the enterotype analysis of the preand post-operative liver samples showed that the enterotype of the patients was E1 and E2,while the E3 was specifically abesent.Clinical pathological analysis showed that preoperative enterotypes was only significantly correlated with postoperative recurrence,while postoperative enterotype was not correlated with any clinicopathological factors.HCC patients with preoperative enterotype E2 have a shorter disease-free survival than that of patients with preoperative enterotype E1.3.The prediction accuracy of model 1,model 2 and model 3,each constructed with full-dimensional data set,dimensional-reduced data set and comprehensive data set respectively,for postoperative recurrence of liver cancer was 72.22%,85.00% and 89.48%,and the AUC value of ROC curve was 0.8312,0.9471 and 0.9819,respectively.Model 3,which was incorporated with enterotype classification variables,had the highest prediction accuracy.Model 3 variables ranked by weight-valued from highest to lowest were α-fucosidase(AFU),fibrinogen,preoperative and postoperative intestinal type combination,intraoperative blood loss,tumor size,age,preoperative enterotype,etc.Among them,AFU and fibrinogen were the most important factors in the prediction model,which suggested that it may be closely related to the occurrence and deterioration of HCC.4.FGG expression in primary liver cancer tissues was significantly higher than that in paracancerous non-tumor tissues.The clinicopathological analysis showed that the high expression of FGG in cells was significantly correlated with satellite lesion,macrovascular invasion,microvascular invasion and higher TNM vascular staging.HCC patients with high FGG expression had a higher recurrence rate and a shorter overall survival time.High expression of FGG was also an independent risk factor for postoperative recurrence of HCC.Cell experiments in vitro showed that FGG overexpression significantly enhanced the migration and invasion ability of SK-HEP-1 cells in vitro.On the contrary,FGG expression silencing significantly inhibited the migration and invasion ability of SK-HEP-1 cells.Mechanism studies have shown that FGG induces EMT process of HCC cells by regulating the expression of slug and ZEB1,thus promoting the migration and invasion of HCC cells.Conclusions: 1.The species diversity of intestinal flora is negatively correlated with the status of liver cancer.Blautia may be involved in the progression of HCC as a beneficial bacterium,and it may serve as a microbiological indicator to assist in the determination of malignant progression and the prediction of recurrence.Preoperative intestinal type may be a potential biomarker for accessing postoperative recurrence of liver cancer.2.The prediction model of postoperative recurrence and metastasis risk of liver cancer was successfully constructed using XGBoost algorithm,and the enterotype classification variable was added to optimize the prediction model,achieving higher prediction accuracy;Furthermore,as the second most weighted clinical variable in the model,fibrinogen has good clinical value and deserves further study.3.FGG is highly expressed in liver cancer tissues,and its expression level is closely related to postoperative recurrence and poor prognosis of liver cancer patients.FGG can activate EMT through intracellular pathways,thus promoting the migration and invasion of HCC cells,which make it a potential biomarker for HCC prognosis. |
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