The MicroRNAs Associated With Recurrence And Metastasis Of Stage Ⅰ Lung Adenocarcinoma The Genes Associated With The Potential Of Liver Metastasis Of Colorectal Cancer

This PhD project consists of two parts of relatively independent research work.Chapter I The microRNAs associated with recurrence and metastasis of stage I lung adenocarcinomaBackground:About 30～35%early stage lung adenocarcinoma(ADC)patients undergone the tumor resection died of tumor recurrence and distant metastasis.Screening prognostic biomarkers associated with metastasis,and taking targeted drug therapy on the basis of the molecular classification could prolong the survival period of patients with lung cancer to a certain extent.The microRNAs(miRNAs)play important roles in the occurrence,development and metastasis of lung cancer as oncogenes or tumor suppressor genes,and their expression are related to the prognosis of patients and sensitivity or resistance of radiation and chemotherapy.This study aims to identify the miRNAs that associate with recurrence and metastasis of stage I lung ADC,and find out biology function and target genes of candidate miRNAs,and evaluate efficiency of candidate miRNAs and the protein products of the target genes as molecular marker for prognosis of stage I lung ADC.Methods:A total 160 cases of stage I lung ADC patients were enrolled,including 80 cases in the "initial cohort",and 80 cases in the "independent cohort".For the patients in the group RM,the tumor recurrence and/or metastasis occurred within 2 years after the surgical resection,including 40 cases in the "initial cohort" and 39 cases in the"independent cohort".For the patients in the group NRM,the tumor recurrence and/or metastasis had not appeared for 5 years after the surgery,including 40 cases in the "initial cohort" and 41 cases in the "independent cohort".The tumor tissues were enriched from formalin-fixed and paraffin-embedded tissue sections,and the total RNAs were then prepared.The Agilent Human microRNA arrays were used to generate miRNA profiles and explore the aberrantly expressed miRNAs in the samples dierived from the"initial cohort".A prognostic evaluation model for patients with stage I lung ADC based on the aberrantly expressed miRNAs between the two groups was constructed by Cox proportional hazards regression method,and validated in the "independent cohort".The interesting differentially expressed miRNAs between the two groups were also validated by Real-time PCR in the "independent cohort".In addition,the target genes and biology function of the candidate miRNAs were investigated through the dual luciferase reporter gene and cell function experiments in vitro.Finally,the protein expression status of target genes of the candidate miRNAs in the tumor tissues both of the "initial cohort" and "independent cohort",and its clinical significance were examined by immunohistochemical analysis.Results:According to the permutation test(p<0.05 and Fold Change>1.5)and survival analysis(log-rank test p<0.05),eight candidate differentially expressed miRNAs(miR-1260b,miR-21-3p,miR-4514,miR-4659a-3p,miR-2467-3p.miR-3621,miR-1471,and miR-92a-3p)were selected from the "initial cohort".In the"independent cohort",miR-21-3p expression was significantly higher in the RM group than that in the NRM group(p=0.02).Data of the prognostic evaluation model for the patients with stage I lung ADC tested in "independent cohort" showed that the sensitivity was 74.4%,the specificity was 68.3%and the curacy was 71.3%,indicating that miR-1260b and miR-92a-3p were risk factor and miR-2467-3p was protect factor for progression-free survival(PFS)of the patients with stage I lung ADC.Presently,there is no research report on the biological function of mir-2467-3p.In this study,the results of cell proliferation experiment in vitro showed that overexpression of miR-2467-3p could inhibit proliferation of lung adenocarcinoma cells A549 and H1975 through blocking the cells in GO/G1 phase indicated by the cell cycle detection experiment.Furthermore,overexpression of miR-2467-3p could significantly inhibits invasion ability of A549 and H1975 cells(p<0.001)in vitro.CHEK1 was one of the target genes of miR-2467-3p,and its protein expression was significantly higher in the cytoplasm of the tumor tissues of patients in the RM group than that in the NRM group,both in the "initial cohort" and "independent cohort".Conclusions:The miR-21-3p was associated with recurrence and metastasis of stage I lung ADC.The prognostic evaluation model for patients with stage I lung ADC could predict and evaluate the prognosis of the patients.The miR-2467-3p was associated with recurrence and metastasis of lung ADC and could inhibit invasion of lung adenocarcinoma cells;therefore,it may be a tumor suppressor.CHEK1 was one of the target genes of miR-2467-3p and was likely to evaluate the prognosis of patients with stage I lung ADC,as a molecular marker.Chapter II The genes associated with the potential of liver metastasis of colorectal cancerBackground:Distant metastasis accounted for 90%of deaths from colorectal cancer(CRC)and complications of metastasis are the leading cause of death in CRC.Liver metastasis is the most common in CRC.Studies have shown that genes in liver metastases of CRC are closely related to the tendency and preference of liver metastasis in CRC,but for now,the genetic basis of the tendency and preference of tumor cell metastasis in CRC is not very clear.The aims of this study is targeted to compare and analyze genome and transcriptome changes both in the primary tumors(PTs)and the liver metastases(CLMs)CRC,looking for the subpopulations with the potential of liver metastasis in the PTs,and the genes associated with the potential of liver metastasis of CRC,for exploring the mechanism of liver metastasis in colorectal cancer further.Methods:Six CRC patients with CLMs were recruited,and underwent the first-stage resection of the primary tumors in colorectum and the metastasis lesion in liver.Multipoint collection of the tissue samples was performed on the PTs and the associated CLMs of the patients.With the next generation sequencing technique,whole-exome sequencing(as controlled by patient’s own peripheral blood mononuclear cells)and transcriptome sequencing(as controlled by the corresponding normal tissues adjacent to the tumors)were carried out,to characterize the gene mutation profiles and gene expression profiles of the the tissue samples investigated.Through cluster analysis,principal component analysis(PCA)and evolutionary tree analysis,the tumor heterogeneity in the PTs and between PTs and the associated CLMs,were applied to explore the potential sources of CLMs.To screen genes associated with the potential liver metastasis of CRC,variant-allele frequencies(VAFs)of mutations,and the number of significantly changed genes introduced by mutant genes in CRC liver metastasis process,and the location of genes in the gene co-expression network through the clonal evolution analysis,were obtained;and Weighted Correlation Network Analysis(WGCNA)and the correlation analysis between gene mutations and gene expression were also used.Results:The sequencing data through cluster analysis and evolutionary tree analysis suggested that the tumor cell subpopulations with the potential of liver metastasis were preliminary identified in 19 samples from 7 PTs of 6 patients.For example,DC1-T3 in case C1,the upper tumor(DC2-Tu1、DC2-Tu2、DC2-Tu3)in case C2,DC3-T1 and DC3-T2 in case C3,DC4-T1 in case C4,DC5-T3 in case C5,DC6-T2 and DC6-T3 in case C6.The WGCNA data showed that gene co-expression networks and the degree of the same gene were different between PTs and CLMs,which implied the same gene had different importance between PTs and CLMs.According to clonal evolution analysis,the average VAFs of 52 subclones in CLMs were higher than that in the PTs,suggesting that the cell populations with these mutant genes were expanding in liver metastasis and these genes were closely related to CRC liver metastasis.The correlation analysis between gene mutations and gene expression showed that although the mutant gene number in CLMs was only 55.3%of that in the PTs,but the number of significant differentially expressed genes caused by mutation were significantly more than that in the PTs,and the number of significant differentially expressed genes caused by the same mutant gene in CLMs were not completely consistent with that in the PTs,which indicate that the importance of the same mutant gene was different between the PTs and CLMs,and that some genes gradually occupied the important position in the process of CRC liver metastasis.Based on the results of above analysis,37 genes associated with the potential of CRC liver metastasis were screened out.These genes have mutated both in PTs and CLMs,and VAFs of mutations and the number of significantly differentially expressed genes introduced by mutant genes in the CLMs were higher and increasing compared with the PTs.Furthermore,the network connection degree of these genes were greater than 2 in the gene co-expression networks of CLMs.Therefore,these genes were thought to be associated with the potential of the liver metastasis of CRC.Validation work for these genes is in progress.Conclusions:The tumor cell subpopulations with the potential of liver metastasis have been indentified from the primary tumors of the colorectal cancer patients investigated in this study.These cell subpopulations mediated tumor metastasis,because of carrying the mutant genes with metastasis potential among them.The molecular mechanism needs further research after validating the 37 genes associated with the potential of CRC liver metastasis screened from this study.