Analysis Of The Treatment And Prognosis Of Hepatocellular Carcinoma And The Mechanism Of TMEM8B With Progression Of Hepatocellular Carcinoma

Objective Hepatocellular carcinoma(HCC)is associated with high recurrence rates and poor survival outcomes after liver resection(LR).Firstly,this study intends to explore the effectiveness of TACE after hepatectomy for patients with intermediate HCC complicated by microvascular infiltration and the beneficiary groups of which.Secondly,the purpose of this study was to investigate the risk predictors of recurrence,recurrence patterns,and survival in patients with large and giant HCC who underwent radical hepatectomy.Thirdly,we intended to explore the effects of TMEM8B on the recurrence,metastasis and overall survival of HCC patients after surgical resection.The molecular mechanism related to the recurrence and metastasis of hepatocellular carcinoma was preliminarily explored.Method In the first place,in this retrospective study,we included patients with intermediate HCC with MVI who received LR treatment from January 2015 to December 2018 and divided them into two groups according to whether they received TACE treatment after surgery.Cox regression analyses were used to determine independent risk factors for recurrence and death.Secondly,the clinicopathologic data of patients diagnosed with large and huge HCC and undergoing radical hepatectomy in our hospital from January 2009 to December 2014 were systematically reviewed.Cox regression analysis were used to analyze the risk predictors of recurrence,postoperative recurrence patterns and survival characteristics.Thirdly,the clinicopathological data of patients with hepatocellular carcinoma who underwent radical surgical resection in Hunan Provincial People’s Hospital from January 2015 to December 2018 were collected,and the paraffin sections of the patients were examined by immunohistochemistry.A stable TMEM8B overexpression cell line was constructed,and the effect of overexpression of TMEM8B was detected and verified by RT-PCR and Western Blot.The effect of TMEM8B on the proliferation of HCC cells in vitro was investigated by cell proliferation experiment and plate clone formation experiment.Transwell experiment and plate scratch experiment were used to study the effect of TMEM8B on the motility of HCC cells.Western Blot was used to detect the expression of epithelial and mesenchymal markers of hepatocellular carcinoma cells overexpressed by TMEM8B.The proliferation of TMEM8B on hepatocellular carcinoma cells in vivo was verified by the subcutaneous tumogenesis model of mice.The mouse tail vein lung metastasis model verified the effect of TMEM8B on the invasion and migration of hepatocellular carcinoma cells in vivo.The functional classification and signaling pathway of potential genes regulated by high-throughput transcriptome sequencing combined with GO functional annotation and KEGG Pathway analysis,and the downstream molecular expression of signaling pathway was detected by Western Blot.Result At the first,among 173 patients with intermediate HCC complicated with MVI,94 patients receiving LR alone were classified as LR group,and 79 patients receiving TACE after LR were classified as LR+TACE group.Estimated 1-,3-,and 5-year RFS and OS rates for the LR+TACE group were higher than LR group.Significant independent predictors of poor OS were LR as treatment,more than 3 tumors,poorly differentiated tumor and cirrhosis,with all differences significant(P<0.05,respectively).Significant independent predictors of poor RFS were LR as treatment,more than 3 tumors,more than 10 centimeter and cirrhosis,with all differences significant(P<0.05,respectively).In the second,among 897 patients with large and huge HCC,significant independent predictors of recurrence were preoperative satellite nodule,preoperative AFP levels above 400 ng/mL,resection margins of 1 cm or less,cirrhosis and microvascular invasion(all P<0.05).All of these except narrow resection margin were also independent risk factors of overall survival.Besides,low tumor differentiation was independent risk factors for death of HCC patients after hepatectomy,P<0.05.At last,The rate of MVI invasion and multiple tumors in the low expression group was significantly higher than that in the high expression group of 146 HCC patients,and the rates of RFS and OS in HCC patients with low TMEM8B expression group were significantly inferior to those in HCC patients with high TMEM8B expression group(all P<0.05).TMEM8B expression level,tumor number,and the size of the tumor diameter,microvascular invasion and intraoperative blood loss are independent risk factors to affect the postoperative recurrence and overall survival of HCC patients.The in vitro cloning and formation ability,invasion and migration ability and motility of HCC cells with overexpression of TMEM8B were weakened,while the in vitro cloning and formation ability,invasion and migration ability and motility of HCC cells with knockdown of TMEM8B were enhanced.E-cadherin was up-regulated and Vimentin and EMT promoter Snail were down-regulated in Hep 3B cells overexpressing TMEM8B,while Ecadherin was down-regulated and Vimentin and EMT promoter Snail were upregulated in Huh 7 cells downregulated TMEM8B.Compared with the control group,the growth rate of subcutaneous tumors formed by Hep 3B cells overexpressing TMEM8B was significantly slower,the number of tumors was reduced,and the tumor diameter was smaller than that of the control group.However,the growth rate of subcutaneous tumors formed by Huh 7 cells with TMEM8B knockdown was significantly accelerated,and the tumor diameter was significantly larger than that of the control group.Compared with the control group,the number and diameter of lung metastases in Hep 3B cells overexpressing TMEM8B decreased,while the number and diameter of lung metastases in Huh7 cells with TMEM8B knockdown increased.Transcriptome sequencing results showed that the number of down-regulated genes in the Wnt/β-catenin signaling pathway was the largest among the top 20 signaling pathways with the most significant enrichment,and TMEM8B reduced the protein levels of β-catenin and its target genes in Hep 3B cells.Conclusion Firstly,for patients with intermediate-stage HCC and MVI,the use of TACE after LR resulted in significantly better RFS and OS rates than for LR alone.LR with a single adjuvant TACE treatment may be an effective treatment combination for patients with intermediate-stage HCC with MVI.Secondly,patients with large and huge HCC,without hepatic vascular invasion,extrahepatic metastases,or severe chronic liver disease,have acceptable longterm outcomes after liver resection.Risk factors for recurrence in this select patient include satellite nodule,elevated AFP level,resection margins of 1 cm or less,histopathological cirrhosis and microvascular invasion.Patients with postsurgical recurrence that are extrahepatic or that occur within 2 years after surgery have particularly poor prognoses.Knowledge about predictors of recurrence,patterns of recurrence,and survival rates in patients with large and huge HCC may help guide treatment approaches in the event of recurrence.Thirdly,TMEM8B expression level is an independent risk factor affecting the prognosis of HCC patients after radical hepatectomy,and the prognosis of HCC patients with high TMEM8B expression is significantly better than that of patients with low TMEM8B expression.TMEM8B plays an inhibitory role in hepatocellular carcinoma and may inhibit the growth,invasion and metastasis of HCC by inhibiting the Wnt/β-catenin signaling pathway.Part Ⅰ Effectiveness of Transarterial Chemoembolization after Liver Resection in Intermediate-Stage Hepatocellular Carcinoma with Microvascular InvasionObjective Intermediate-stage hepatocellular carcinoma(HCC)with microvascular invasion(MVI)is associated with high recurrence rates and poor survival outcomes after liver resection(LR).Postoperative transarterial chemoembolization(TACE)has been used effectively as an adjuvant therapy for those with HCC considered to be at high risk of recurrence after LR.However,the effectiveness of TACE after hepatectomy for patients with intermediate HCC complicated by MVI has not been reported in relevant literatures.This study intends to explore the effectiveness of TACE after hepatectomy for patients with intermediate HCC complicated by microvascular infiltration and the beneficiary groups of which.Method In this retrospective study,we included patients with intermediate HCC with MVI who received LR treatment from January 2015 to December 2018 and divided them into two groups according to whether they received TACE treatment after surgery.Propensity score matching(PSM)was used to balance the demographic and clinicopathological characteristics of the two groups.Log-rank method was used to compare the relapse-free survival(RFS)and overall survival(OS)between the two groups,and univariate and multivariate regression analyses were used to determine independent risk factors for recurrence and death.Result Among 173 patients with intermediate HCC complicated with MVI,94 patients receiving LR alone were classified as LR group,and 79 patients receiving TACE after LR were classified as LR+TACE group.Mean follow-up was 38.3(range,3.0 to 78)months.After PSM was applied,estimated 1-,3-,and 5-year OS rates for the TACE+LR group were 87.6%,48.4%and 32.9%,respectively,and for the LR group were 63.3%,and 35.2%and 25.8%,respectively,with all differences significant(P<0.05,respectively).Estimated 1-,3-,and 5-year RFS rates for the LR+TACE group were 41.3%,24.7%and 19.0%,and for the LR group were 28.5%,11.6%,and 9.9%,respectively,with all differences significant(P<0.05,respectively).Significant independent predictors of poor OS were LR as treatment(HR=1.64,95%CI 1.12-2.39),more than 3 tumors(HR=2.23,95%CI 1.45-3.42),poorly differentiated tumor(HR=1.55,95%CI 1.05-2.3,),and cirrhosis(HR=2.16,95%CI 1.47-3.19),with all differences significant(P<0.05,respectively).Conclusion For patients with intermediate-stage HCC and MVI,the use of TACE after LR resulted in significantly better RFS and OS rates than for LR alone.LR with a single adjuvant TACE treatment may be an effective treatment combination for patients with intermediate-stage HCC with MVI.Part Ⅱ Recurrence Pattern,Risk Factors and Prognosis of Large and Huge Hepatocellular Carcinoma after HepatectomyObjective Some scholars do not recommend hepatectomy as first-line treatment for hepatocellular carcinoma(HCC)beyond the Milan criteria.In fact,many hepatobiliary surgery centers now perform hepatectomy for HCC beyond the Milan criteria.The purpose of this study was to investigate the risk predictors of recurrence,recurrence patterns,and survival in patients with large and giant HCC who underwent radical hepatectomy.Method The data of patients diagnosed with large and huge HCC and undergoing radical hepatectomy in our hospital from January 2009 to December 2014 were systematically reviewed.Information of each patient was collected,including gender,age,body mass index(BMI),BCLC stage,tumor diameter,tumor satellite nodules,past medical history,portal hypertension,preoperative alpha-fetoprotein,blood routine and biochemistry,type of hepatectomy,liver resection margin,cirrhosis,tumor differentiation and microvascular infiltration.Univariate and multivariate Cox regression analysis were used to analyze the risk predictors of recurrence,postoperative recurrence patterns and survival characteristics.Result Among 897 patients,the median follow-up was 48(range,5-140)months.The 1-,3-,and 5-year for RFS rates were 51.6%,36.1%,and 30.1%,respectively,and for OS were 80.2%,55.4%,and 47.7%,respectively.Significant independent predictors of recurrence were preoperative satellite nodule(HR=2.25,95%CI 1.17-4.31),preoperative AFP levels above 400 ng/mL(HR=1.23,95%CI 1.04-1.45),resection margins of 1 cm or less(HR=1.21,95%CI,1.00-1.46),cirrhosis(HR=2.64,95%CI,2.13-3.28),and microvascular invasion(HR=1.71,95%CI 1.45-2.20)(all P<0.05).All of these except narrow resection margin were also independent risk factors of overall survival.Multivariate analysis of OS revealed satellite nodular tumor(HR 2.19,95%CI 1.13-1.93),preoperative AFP level higher than 400 ng/mL(HR 1.31,95%CI 1.09 to 1.58),cirrhosis(HR 2.26,95%CI 1.75-2.92),low tumor differentiation(HR 1.27,95%CI 1.06-1.53)and microvascular invasion(HR 1.60,95%CI 1.33-1.93)were independent risk factors for death of HCC patients after hepatectomy,P<0.05.Conclusion Patients with large and huge HCC,without hepatic vascular invasion,extrahepatic metastases,or severe chronic liver disease,have acceptable long-term outcomes after liver resection.Risk factors for recurrence in this select patient include satellite nodule,elevated AFP level,resection margins of 1 cm or less,histopathological cirrhosis and microvascular invasion.Patients with post-surgical recurrence that are extrahepatic or that occur within 2 years after surgery have particularly poor prognoses.Knowledge about predictors of recurrence,patterns of recurrence,and survival rates in patients with large and huge HCC may help guide treatment approaches in the event of recurrence.PartⅢ Study on the Mechanism of TMEM8B with Progression of Hepatocellular CarcinomaObjective HCC has great genetic molecular heterogeneity,and TMEM8B expression is decreased in a variety of malignant tumors such as hepatocellular carcinoma,but the mechanism of TMEM8B in HCC is still unclear.We intended to explore the effects of TMEM8B on the recurrence,metastasis and overall survival of HCC patients after surgical resection.The molecular mechanism related to the recurrence and metastasis of hepatocellular carcinoma was preliminarily explored.Method The clinicopathological data of patients with hepatocellular carcinoma who underwent radical surgical resection in Hunan Provincial People’s Hospital from January 2015 to December 2018 were collected,and the paraffin sections of the patients were examined by immunohistochemistry.A stable TMEM8B overexpression cell line was constructed,and the effect of overexpression of TMEM8B was detected and verified by RT-PCR and Western Blot.The effect of TMEM8B on the proliferation of HCC cells in vitro was investigated by cell proliferation experiment and plate clone formation experiment.Transwell experiment and plate scratch experiment were used to study the effect of TMEM8B on the motility of HCC cells.Western Blot was used to detect the expression of epithelial and mesenchymal markers of hepatocellular carcinoma cells overexpressed by TMEM8B.The proliferation of TMEM8B on hepatocellular carcinoma cells in vivo was verified by the subcutaneous tumogenesis model of mice.The mouse tail vein lung metastasis model verified the effect of TMEM8B on the invasion and migration of hepatocellular carcinoma cells in vivo.The functional classification and signaling pathway of potential genes regulated by high-throughput transcriptome sequencing combined with GO functional annotation and KEGG Pathway analysis,and the downstream molecular expression of signaling pathway was detected by Western Blot.Result TMEM8B expression was localized in the cell membrane and cytoplasm of hepatocellular carcinoma,presenting light yellow to yellow-brown staining.146 HCC patients were divided into low expression group(n=81)and high expression group(n=65).The rate of MVI invasion and multiple tumors in the low expression group was significantly higher than that in the high expression group(28.4%vs 15.4%,P=0.035;34.6%vs 18.5%,P=0.030),RFS and OS in HCC patients with high TMEM8B expression group were significantly better than those in HCC patients with low TMEM8B expression group(all P<0.05).TMEM8B expression level,tumor number,and the size of the tumor diameter,microvascular invasion and intraoperative blood loss are independent risk factors to affect the postoperative recurrence of HCC patients,and TMEM8B expression level,number of tumor,tumor diameter size,microvascular invasion,and intraoperative blood loss is postoperatively in patients with hepatocellular carcinoma(HCC)independent risk factors for overall survival.The in vitro cloning and formation ability,invasion and migration ability and motility of HCC cells with overexpression of TMEM8B were weakened,while the in vitro cloning and formation ability,invasion and migration ability and motility of HCC cells with knockdown of TMEM8B were enhanced.E-cadherin was up-regulated and Vimentin and EMT promoter Snail were down-regulated in Hep 3B cells overexpressing TMEM8B,while Ecadherin was down-regulated and Vimentin and EMT promoter Snail were upregulated in Huh 7 cells downregulated TMEM8B.Compared with the control group,the growth rate of subcutaneous tumors formed by Hep 3B cells overexpressing TMEM8B was significantly slower,the number of tumors was reduced,and the tumor diameter was smaller than that of the control group.However,the growth rate of subcutaneous tumors formed by Huh 7 cells with TMEM8B knockdown was significantly accelerated,and the tumor diameter was significantly larger than that of the control group.Compared with the control group,the number and diameter of lung metastases in Hep 3B cells overexpressing TMEM8B decreased,while the number and diameter of lung metastases in Huh7 cells with TMEM8B knockdown increased.Transcriptome sequencing results showed that the number of down-regulated genes in the Wnt/β-catenin signaling pathway was the largest among the top 20 signaling pathways with the most significant enrichment,and TMEM8B reduced the protein levels of β-catenin and its target genes in Hep 3B cells.Conclusion TMEM8B expression level is an independent risk factor affecting the prognosis of HCC patients after radical hepatectomy,and the prognosis of HCC patients with high TMEM8B expression is significantly better than that of patients with low TMEM8B expression.TMEM8B plays an inhibitory role in hepatocellular carcinoma and may inhibit the growth,invasion and metastasis of HCC by inhibiting the Wnt/β-catenin signaling pathway.