| [Background]In the worldwide,hepatocellular carcinoma(HCC)are the fifth most common cancer and the second leading cause of cancer-related deaths.HCC has become one of the most serious diseases which harm people's health.The main treatments of HCC include liver transplantation,surgical operation and interventional therapy and so on.Due to diagnosis delay,the high recurrence and metastasis rate,the 5-year survival rate is less than 30%in HCC patients after surgical resection.The discovery of tumor biomarkers will help to improve the early diagnosis rate,reduce the cancer related mortality,improve the quality of life of patients and prolong the survival in HCC patients.Tumor biomarkers are produced by tumor cells or others cell which response to tumor cells.Tumor biomarkers can screen the high risk groups,identify the character of tumor,reflect the development of tumor,and monitor therapy and recurrence and prognosis in tumor.Traditionally,tumor biomarkers mainly refer to the protein in plasma or tumor tissue.With the advances in biotechnology,the molecules,which are related genomics,proteomics and metabolomics,can be very good to clarify the tumor biological mechanism,and more and more tumor biomarker,which included epigenetics,CNV,fusion gene,mutation,mRNA,miRNA,lncRNA,protein,metabolites and antibody,are discovered.DNA methylation is the most common epigenetic modification in the genomic DNA of the eukaryotic organisms,and can regulate the genes which are closely related to the normal functions,genetic imprinting,gene silencing,the development of embryo and tumor.In cells,the changes of DNA methylation,which are located to the promoter of the tumor suppressor gene or its adjacent region,occur in the early stage of carcinogenesis,directly affect the binding of transcription factors to the promoter,regulate gene transcription,and finally lead to excessive cell growth and tumorigenesis.With the development of its research,DNA methylation will help to reveal the mechanism of the tumorigenesis and tumor progress.As the tumor biomarkers,DNA methylation not only can be used for the molecular pathological type of HCC,clinical staging and the judgment of the curative effect,but also bring great hope for the early diagnosis and the monitoring of tumor.Compared with the reference genome,CNV refers to the increase or decrease of DNA segment which longth are 1kb or some Mb.CNV is an important genetic variation in the human genome,and the increase of CNV can enhance the expression activity of genes which wre located to its covering area or its adjacent region.TM6SF1 gene is an important member of the transmembrane 6 superfamily.TM6SF1 gene is located in 15q24-26,the size of mRNA is 1.4 kb and the molecular weight of protein,which contains 370 amino acids,is 41.6 k Da.Refering to its functions,some scholars have found that hypermethylation of TM6SF1 gene happen in the patients with breast cancer and HBV-related HCC.In addition,we have found that the variations of TM6SF1 gene are frequent in HCC in the previous experiments.These results indicate that TM6SF1 gene may be a tumor suppressor gene in breast cancer and HCC.Part one:Correlation between methylation and CNV of TM6SF1 and the clinical characters and prognosis,and diagnostic value of its methylation in HCC[Objective]To detect the methylation and CNV of TM6SF1,to analyze the correlation between methylation and CNV of TM6SF1 gene and the clinical characters and prognosis,and to detect the diagnostic value of its methylation in HCC.[Materials and methods]1 HCC specimens were collected and the genomic DNA were extracted from HCC and its adjacent tissues.2ThemethylationofTM6SF1weredetectedby Mass ARRAY?Epi TYPER?in HCC.3 CNV of TM6SF1 were detected by q PCR in HCC.4 The software of SPSS19.0 was used for statistical description and analysis.The measurement data were showed according to mean±SD and compared using wilcoxon test between groups.The enumeration data were analyzed using?2 test or Fisher exact test between groups.Overall survival rate and disease-free survival rate were analyzed with Kaplan-Meier method.The methods of logistic regression,kNN,naive Bayes,support vector machine and decision tree were used to detect the diagnostic value of TM6SF1 methylation in HCC.ROC curves and heat map were plotted using pROC and pheatmap software package in R programming environment,Scatter plots and survival curves were plotted using GraphPad Prism 5.0.All statistical tests were two-tailed,and differences with P<0.05 were considered statistically significant.[Results]1 The judgment of hypermethylation sites of TM6SF1 gene in HCC3 pairs of primers,which cover the promoter region of TM6SF1 gene,were designed,and 47 Cp G islands were detected by MassARRAY?EpiTYPER?.The Cp G islands are considered as the hypermethylation sites when the absolute change(T-N)of the average methylation level is greater than 0.1,the relative change(T/N)is greater than 2 and the P value of Wilcoxon test is less than 0.05in the same Cp G islands between the HCC tissues(T)and its adjacent tissues(N).Intheresearch,TM6SF112CpG2,TM6SF112CpG6,TM6SF112CpG13.14.15,TM6SF112CpG35,TM6SF112CpG41,TM6SF112CpG42.43,TM6SF113CpG23.24.25.26and TM6SF113CpG27.28.29.30.31areregardedasthesignificant hypermethylation site.2 The judgment of HCC patients with TM6SF1hypermathylationIn the research,a total of 78 individuals with 5 or more hypermathylation sites,which the relative change(T/N)of hypermethylation sites is greater than 2and the absolute change(T-N)is greater than 0.1,are considered as the HCC patient with TM6SF1 hypermathylation in 146 HCC patients.3 The judgment of HCC patients with TM6SF1 CNV deletionSATB1,LTBP1,the previous and latter of TM6SF1 were detected using qPCR in HCC tissue and its adjacent tissue,and the formula of 2-??Ct??Ct were used.When the ratio of formula is less than 0.75,HCC patients are considered to have TM6SF1 CNV deletion;beyond that,the patients are not considered to have TM6SF1 CNV deletion.Through the analysis,we found that a total of 42 cases have TM6SF1 CNV deletion in 154 cases of HCC.4 Correlation between methylation and CNV of TM6SF1 and the clinical characters in HCC4.1 Correlation between methylation of TM6SF1 and the clinical characters in HCCThe results showed that the incidence of portal vein tumor thrombus were53.23%(33/62)and 29.17%(14/48)respectively in the group with TM6SF1hypermethylation and the group without TM6SF1 hypermethylation in HCC patients.The incidence of portal vein tumor thrombus was significantly different between the group with TM6SF1 hypermethylation and the group with TM6SF1hypomethylation in HCC patients(?2=6.400,P=0.011).4.2 Correlation between CNV of TM6SF1 and the clinical characters in HCCThe results showed that the proportion of HCC patients older than 60 years were 35.71%(15/42)and 16.96%(19/112)respectively in the group with TM6SF1 CNV deletion and the group without TM6SF1 CNV deletion in HCC patients.There was a significant difference in the proportion of HCC patients older than 60 years between the group with TM6SF1 CNV deletion and the group without TM6SF1 CNV deletion in HCC patients(?2=6.242,P=0.012).4.3 Correlation between the concurrence of TM6SF1 hypermethylation and TM6SF1 CNV deletion and the clinical characters in HCCThe results showed that there were 21 cases with the concurrence of TM6SF1 hypermethylation and TM6SF1 CNV deletion among 127 HCC patients.There was not a significant difference in the constisuent ratio of the clinical characters between the group with the concurrence of TM6SF1hypermethylation and TM6SF1 CNV deletion and the group without the concurrence of TM6SF1 hypermethylation and TM6SF1 CNV deletion.5 Correlation between methylation and CNV of TM6SF1 and the prognosis in HCC5.1 Correlation between methylation of TM6SF1 and the prognosis in HCCThe results showed that the disease-free survival rate were 60.26%(47/78)and 58.82%(40/68)respectively,and overall survival rate were 70.51%(55/78)and 64.71%(44/68)respectively in the group with TM6SF1 hypermethylation and the group without TM6SF1 hypermethylation in HCC patients.There are not statistically significant in the disease-free survival rate and overall survival rate between two groups.5.2 Correlation between CNV of TM6SF1 and the prognosis in HCCThe results showed that the disease-free survival rate were 57.14%(24/42)and 62.50%(70/112)respectively,overall survival rate were 61.90%(26/42)and71.43%(80/112)respectively in the group with TM6SF1 CNV deletion and the group without TM6SF1 CNV deletion in HCC patients.There were not statistically significant in the disease-free survival rate and overall survival rate between two groups.5.3 Correlation between the concurrence of TM6SF1 hypermethylation and TM6SF1 CNV deletion and the prognosis in HCCThe results showed that the disease-free survival rate were 66.67%(14/21)and 60.38%(64/106)respectively,overall survival rate were 76.19%(16/21)and66.98%(71/106)respectively in the group with the concurrence of TM6SF1hypermethylation and TM6SF1 CNV deletion and the groups without the concurrence of TM6SF1 hypermethylation and TM6SF1 CNV deletion in HCC patients.There are not statistically significant in the disease-free survival rate and overall survival rate between two groups.6 The diagnostic value of TM6SF1 methylation in HCCHCC patients were randomly divided into A queue and B queue after their methylation data of TM6SF1 being filled with the mean.The methylation data of adjacent tissues from A queue and HCC tissues from B queue were selected to establish training cohort,the methylation data of HCC tissues from A queue and adjacent tissues from B queue were selected to establish validating cohort.The methods of logistic regression,kNN,naive Bayes,support vector machine and decision tree were used to discriminate the HCC and adjacent tissues.Logistic regression reveal that the sensitivity and specificity of the diagnostic model of TM6SF1 methylation are 65.75%(48/73)and 93.15%(68/73)respectively in the training cohorts,the sensitivity and specificity are65.75%(48/73)and 91.78%(67/73)respectively in the validating cohorts,and the AUC are 0.7945 and 0.7877 respectively in the training cohorts and validating cohorts.k NN method reveal that the sensitivity and specificity of the diagnostic model of TM6SF1 methylation are 68.49%and 97.22%respectively in the validating cohorts,and the AUC of the validating cohorts are 0.8219.Naive Bayes reveal that the sensitivity and specificity of the diagnostic model of TM6SF1 methylation are 63.01%(46/73)and 94.52%(69/73)respectively in the training cohorts,the sensitivity and specificity are 68.49%(50/73)and94.52%(69/73)respectively in the validating cohorts,and the AUC are 0.7877and 0.8151 respectively in the training cohorts and validating cohorts.Support vector machine reveal that the sensitivity and specificity of the diagnostic model of TM6SF1 methylation are 83.56%(61/73)and 98.63%(72/73)respectively in the training cohorts,the sensitivity and specificity are 76.71%and 75.34%respectively in the validating cohorts,and the AUC are 0.9110 and 0.7603respectively in the training cohorts and validating cohorts.Decision tree reveal that the sensitivity and specificity of the diagnostic model of TM6SF1methylation are 83.56%(61/73)and 98.63%(72/73)respectively in the training cohorts,the sensitivity and specificity are 80.82%(59/73)and 84.93%(62/73)respectively in the validating cohorts,and the AUC are 0.9110 and 0.8288respectively in the training cohorts and validating cohorts.The decision tree is considered as the best diagnostic model of TM6SF1 methylation in HCC by comparing their sensitivity,specificity and AUC.[Conclusion]1 compared with its adjacent tissue,TM6SF1 hypermethylation is widespread in the in HCC tissue.HCC patients with TM6SF1 hypermethylation are universal also.2 The incidence of portal vein tumor thrombus in the group with TM6SF1hypermethylation is significantly higher than the group with TM6SF1hypermethylation.The mechanism,which TM6SF1 hypermethylation promote the occurrence of portal vein tumor thrombus,may be related that TM6SF1hypermethylation inhibite the expression of TM6SF1 and promote EMT process.3 The percent of TM6SF1 CNV deletion is 27.27%(42/154)in HCC patients.There is a significant difference in the proportion of HCC patients older than 60 years between the group with TM6SF1 CNV deletion and the group without TM6SF1 CNV deletion in HCC patients.However,its biological function and mechanism need to be studied furtherly.4 The percent of the concurrence of TM6SF1 hypermethylation and TM6SF1 CNV deletion is 16.54%(21/127)in HCC patients.5 The decision tree is considered as the best diagnostic model of TM6SF1methylation in HCC by comparing their sensitivity,specificity and AUC.The diagnostic model of TM6SF1 methylation bulided by the method of decision tree can be used to the diagnosis of HCC.TM6SF1 methylation is tumor biomarker for the diagnosis of HCC.Part two: Correlation between the transcription of TM6SF1 and the clinical characters and prognosis in HCC[Objective]To analyze the correlation between the transcription of TM6SF1 and the clinical characters and prognosis in HCC.[Materials and methods]1 The data of GSE14520-GPL3921 were downloaded and dealed with.2 Data quality of GSE14520-GPL3921 were tested by the software of bioconductor.The software of SPSS19.0 was used for statistical description and analysis.The enumeration data were analyzed using ?2 test or Fisher exact test between groups.Overall survival rate and disease-free survival rate were analyzed with Kaplan-Meier method,survival curves were plotted using the software of Graph Pad Prism5.0.All statistical tests were two-tailed,and differences with P < 0.05 were considered statistically significant.[Result]1 GSE14520-GPL3921 is of better quality.2 The results show that the constituent ratio of TNM stage I-II are71.26%(62/87)and 85.12%(103/121)respectively,and the constituent ratio of CLIP stage 0-1 are 70.11%(61/87)and 84.30%(102/121)respectively in the group without the high transcription of TM6SF1 and the group with the high transcription of TM6SF1 in HCC patients.The constituent ratio of TNM stage and CLIP stage are significantly different between the group without the high transcription of TM6SF1 and the group with the high transcription of TM6SF1 in HCC patients(?2=5.928,P=0.015;?2=6.005,P=0.014).3 The disease-free survival rate are 45.45%(55/121)and 44.94%(40/89)respectively,and overall survival rate are 68.60%(83/121)and 52.81%(47/89)respectively in the group with the high transcription of TM6SF1 and the group without the high transcription of TM6SF1 in HCC patients.There are statistically significant in overall survival rate between two groups.[Conclusion]1 The constituent ratio of TNM stage I-II and CLIP stage 0-1 in the group with the high transcription of TM6SF1 are significantly higher than the group without the high transcription of TM6SF1.The transcription of TM6SF1 can inhibit the progress of TNM stage and CLIP stage.2 Overall survival rate of the group with the high transcription of TM6SF1 are significantly higher than the group without the high transcription of TM6SF1.The transcription of TM6SF1 can improve overall survival in HCC patients. |
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