Application Of Plasma RASSF1A And P16 Gene Hypermethylation Measurement In Detection Of Non-small Lung Cancer

PrefaceLung cancer is one kind of malignant tumors with the highest incidence andmortality. The early-stage diagnosis and suitable treatment is important for improvingthe patient's life quality and survival rate, especially the former as the crucial clinicalpractice. Once diagnosed, lung cancer usually has progressed to its middle oradvanced-stages. So identification of the specific biomarkers has become one of thereliable approaches in the early-stage diagnosis. Recent studies have demonstratedthat there was the free-DNA in normal human plasma. The increase in free-DNAcontent were discovered in cancer patients plasma, and these free-DNAs arepresented some biological features including promoter hypermethylation, aberrationand among others. Plasma could be obtained conveniently and with little harm topatients. So the identification of tumor makers in plasma is considered as an idealmethod of early-stage diagnosis for the lung cancer.Ras association domain family 1A gene (RASSF1A) is a candidate lung tumorsuppressor gene and was discovered in 2000, which is localized at 3p21.3 in aminimum deletion sequence. Homozygotic and heterozygotic loss was found in morethen 90% of small cell lung cancers (SCLC) and in 50～80% of non-small cell lungcancers(NSCLC). It has been suggested that RASSFIA may be a special lung cancersuppress gene. Previous studies have shown that the frequent loss of RASSF1A in lung cancer and other tumors, and methylation of the RASSF1A promoter was themain cause of the epigenetic inactivation of it. So promoter methylation of RASSF1Ahas been recognized as the main cause of the gene inactivation and the earliest eventsin the pathogenesis of lung cancer pathogenesis. But less studies in this respect havebeen conducted in China.The p16 tumor-suppressor gene is located at 9p21, which is frequentlyinactivated in lung cancer cells, and play an essential role in the tumorigenesis anddevelopment process. Promoter hypermethylation is the main event causing p16 geneinactivation in lung cancer tissue. And the promoter hypermethylation of p16 genecan be detected in plasma free-DNA of the tumor patients.Tumorigenesis, development and prognosis of lung cancer could be acomplicated process, which could be divided into some different phases andcontrolled by multiple factors, and the promoter hypermethylation of suppressor geneswas recognized as an earlier event in the pathogenesis of tumors. If the promoterhypermethylation is associated with RASSF1A and p16 gene inactivation, it should bedetected in plasma free-DNA of patients with lung cancer in Chinese, if there aresignifficant correlation between both gene methylation levels in plasma DNA andtumor tissues, methylation analysis of RASSF1A and p16 genes in plasma free-DNAmay become a promising new diagnostic approach to screen putative cancer patients.As a tumor suppressor gene, the biological function of RASSF1A remains unclear.By means comparative proteomic analysis, we can observe the variations of effectorproteins and regulatory proteins priceded by inactivation of RASSF1A, andunderstand more biological functions of RASSF1A.Objectives1. To explore the inactivation conditions of RASSF1A and p16 genes in tumor tissuesfrom patients with NSCLC in Chinese, and relationship between the promoterhypermethylation and gene inactivation; To determine the promoterhypermethylation conditions of RASSF1A and p16 genes in plasma and tumortissues DNA from patients with NSCLC, and the correlation of promoter hypermethylation in plasma and tumor tissue DNA; then to analyze and evaluatethe benefits for early-stage diagnosis of NSCLC by combining detection ofpromoter hypermethylation of RASSF1A and p16 genes in plasma DNA.2. To establish the differential two-dimentional gel electrophoresis maps includinglung adenocarcinoma tissue with the expression of RASSF1A and that with loss ofRASSF1A protein. To pick up the differential protein spots and identify them bymass spectrometry (MS). To explore the exact cancer-suppress pathways ofRASSF 1A by analyzing the functions of these differential proteins.Materials and methods1. Samples and GroupsExperimental group: 96 tumor tissues and matched plasma from patients withNSCLC in Chinese.Control group: 96 matched normal lung tissues and those from patients withNSCLC in Chinese. Plasmas from 12 patients with benign lung disease and 20volunteer with no lung diseases were prepared.2. Methods2.1 Determination of inactivation status of RASSF1A and p16 genes in NSCLCtissues2.1.1 Determination of transcription inactivation of RASSF1A and p16 genesTotal RNA was extracted from NSCLC tissues and matched normal lung tissuesfar from tumor areas, and cDNA was synthesized with oligo (dT) n primer byreverse transcription, the fragments of RASSF1A and p16 genes was amplifiedwith PCR, and the transcriptional expression of RASSF1A and p16 genes wasanalyzed. Chi-square test compares the relation of the transcriptional inactivationof RASSF1A and p16 genes and the clinicopathological characteristics ofpatients with NSCLC.2.1.2 Measurement of expression inactivation of RASSF1A The soluble proteinsfrom NSCLC tissues and matched normal lung tissues far from tumor areas wereextracted. And the protein concentrations were measured using Bradford's method. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed,the proteins were transferred to nitrocellulous membranes. Afterantigen-antibody reaction, DAB kit was used to measure the result ofimmnuoreactivity. Finally the expression and inactivation status of RASSF1Agene was evaluated.2.2 Characterization of the promoter hypermethylation of RASSF1A and p16gene in NSCLC tissues and matched plasma DNA2.2.1 DNA extraction and modification Following the protocol provided by themanufacture, Genomic DNA was extracted from tumor tissues, matched normaltissues far from tumor areas, and plasma of patients with NSCLC or benign lungdiseases and health volunteers. Then Genomic DNA was quantitated by UVspectrophotometer. 50μg DNA from placenta tissues was modified by M.Sss I,1.2μg tissues DNA or 45μl plasma DNA from 600μl plasma was modified bysodium bisulfite.2.2.2 Examination of primer specificity In order to test primer's specificity ofRASSF1A and p16 genes, PCR and mythylation-specific PCR (MSP) wasperformed to amplify the DNA from placenta tissues modified and unmodifiedby M.Sss I, with wild-type primers, methylated-specifie primers andunmethylated-specific primers.2.2.3 The investigation of promoter hypermethylation of RASSF1A and p16genes in tissues DNA In order to detect the promoter hypermethylation ofRASSF1A and p16 genes in tissues DNA, PCR and mythylation-specific PCR(MSP) was performed to amplify DNA from NSCLC tissues and matched normallung tissues with wild-type, methylated and unmethylated primers of RASSF1Aand p16 genes. Chi-square test was used to compare the relation between thepromoter hypermethylation of RASSF1A and p16 genes and theclinicopathological characteristics of patients with NSCLC; The correlationbetween the promoter hypermethylation and the transcriptional expression ofRASSF1A and p16 genes was evaluated by Spearman correlation analysis. Theinactivation mechanism and status of the two genes in patients with NSCLC in Chinese was investigated.2.2.4 Measurement of promoter hypermethylation of RASSF1A and p16 genes inplasma DNA In order to detect the promoter hypermethylation of RASSF1Aand p16 genes in plasma DNA of NSCLC patients, the bisulfite-convertedplasma DNA was amplified by PCR with methylated-specific andunmethylated-specific primers of RASSF1A and p16 genes. The correlation forboth gene hypermethylation between plasma and tumor tissues was analyzedby Spearman correlation analysis, to assess the concordance of the promoterhypermethylation of RASSF1A and p16 gene in plasma and tumor tissues DNAfrom patients with NSCLC.2.2.5 Efficiency indices for diagnosis To evaluate the significance of RASSF1A andp16 genes hypermethylation in plasma DNA in early detection of non-smalllung cancer in Sensitivity, specificity, negative predictive value and positivepredictive value.2.3 Identification of differential proteins in two different lung adenocarcinomatissues with expressing or devoid of RASSF1A2.3.1 Extraction of the soluble proteins in tissues The soluble proteins from twokinds of different lung adenocarcinoma tissues with the expression andexpression loss of RASSF1A were extracted with lysis solution of 2-DEingredient. And the protein concentration was measured with Bradford's method.2.3.2 Screening of the differential protein spots The two-dimentional gel maps forthe soluble proteins of the two different lung adenocarcinoma tissues wereobtained with protein standard quantity for analysis gel (17cm pH3-10 IPG strip).After scanning gels with GS-800, the data were analyzed with PDQuest software.The differential protein spots were identified.2.3.3 Identification of the differential protein spots with MS The differentialprotein spots were cut out from preparation gels (17cm pH3-10 IPG strip) andthen digested with typsin. Peptide mass finger printings were obtained bymatrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). The protein database was used to search and identify the protein identities. The function of identified protein could be used to analyze theoncogenicity of RASSF1A.3. Statistical analysisBy means of SPSS program, version 12.0, data analysis was performed using chisquare test and Spearman correlation analysis,αvalue=0.05 was considered thesize of test.Results1. Inactivation of RASSF1A and p16 genes in NSCLC tissues1.1 Transcription inactivation status The rate of transcriptional expression ofRASSF1A and p16 genes was 100% in all normal lung tissues far from cancerareas, but decreased or loss in carcinoma tissues 53.12%(51/96) and36.46%(35/96) respectively. The decrease and loss of RASSF1A transcriptionwas significantly higher in the patients with advanced stages (61.04%) and inthe patients with positive lymph node metastasis (65%) than those in early stage(41.03%, P＜0.05) and those without lymph node metastasis (33.33%, P＜0.05).No significant links of abnormal RASSF1A mRNA expression was identified tosex, age, smoking or histological type, differentiation grade of tumors (allP＞0.05). The rate of transcriptional decrease and transcriptional loss of p16 genewas remarkably higher in female group(52.38%), more then fifty years group(44.93%), adenocarcinoma group (48.15%), stageⅢandⅣtumors (45.61%)and lymph node metastasis group (50.00%) than in male group(24.07), less thanfifty years group(14.81%), squamous cell carcinoma group (21.83%), stageⅠandⅡtumors(23.08%) and non-lymph node metastasis group (13.89%, allP＜0.05). No marked association of abnormal p16 gene mRNA expression wasidentified with smoking or differentiation grade of tumors (all P＞0.05).1.2 Inactivation of gene Expression RASSF1A was expression in all normal lungtissues far from cancer areas. But the expression of RASSF1A was lower or lossin 60.4% (58/96) carcinoma tissues.2. The promoter hypermethylation Status for RASSF1A and p16 genes inNSCLC tissues and matched plasma DNA 2.1 DNA quality and quantity DNA extracted from different tissues and plasma wasmeasured (A260/A280≈1.76～1.80). The content of NSCLC plasma DNA issignificantly higher than that of benign lung diseases and health volunteerplasma DNA.2.2 Characterization of Primers Specificity The three kinds of primers, wild-type,methylated-specific and unmethylated- specifc primers were used to amplifyDNA from placenta tissue, only wild-types primers of RASSF1A and P16genes was amplified. Modified DNA from placenta tissues with M.Sss I afterbisulfite treatment only produced amplification with methylated-specific primers.DNA from placenta tissues after bisulfite treatment only amplified withunmethylated-specific primers. So the primers have a good specificity.2.3 Analysis of RASSF1A and p16 DNA Methylation in NSCLC tissues Thefrequency of promoter hypermethylation detected in NSCLC tissues forRASSF 1A and p16 genes was 48.96%(47/96) and 34.38%(33/96), respectively.But no aberrant promoter methylation was detected in 96 matched normal lungtissues far from lung cancer areas. The promoter hypermethylation of RASSF1Ain NSCLC tissues was only associated with the histological types of the tumor.RASSF1A methylation was more frequently observed in patients with squamouscell carcinomas than in patients with adenocarcinomas (P＜0.05), no significantassociation of the promoter hypermethylation of RASSF1A was detected withsex, age groups, smoking, TNM stages, lymph node metastasis, differentiationgrades of the tumor (all P＞0.05). The frequency of p16 gene aberrantmethylation in NSCLC tissues was remarkably higher in＞50 year age-group,squamous cell carcinoma group and lymph node metastasis group than in＜50group, adenocarcinoma group and non-lymph node metastasis group (allP＜0.05). But hypermethylated p16 gene was not significantly associated with sex,smoking, TNM stage and differentiation of the tumors. The negative correlationbetween the transcription and the promoter hypermethylation of RASSF1A andp16 genes in NSCLC tissues was determined (RASSF1A: r_s=-0.586, P＜ 0.0001, p16: r_s=-0.363, P＜0.0001). The significant negative correlationbetween the expression and the promoter hypermethylation of RASSF1A inNSCLC tissues was observed. So the promoter hypermethylation could beconsidered as the main cause of inactivation of RASSF1A and p16 genes inpatients with NSCLC in Chinese.2.4 DNA Methylation Analysis of RASSF1A and p16 genes in Plasma frompatients with NSCLC Plasma DNA from NSCLC patients after bisulfitetreatment was amplified with semi-MSP, the frequency of aberrant promotermethylation of RASSF 1A and p16 genes was 43.75% (42/96) and 31.25%(30/96),respectively. But no aberrant promoter methylation of RASSF1A and p16 geneswas detected in plasma DNA from 12 benign lung diseases and 20 healthvolunteers. The positive correlation of the promoter hypermethylation of bothgenes was identified in both plasma and tumor tissues from NSCLC patients (r_s=0.932, P＜0.0001). The results display identical epigenetic changes of RASSF1Aand p16 genes in NSCLC tissues and in the paired plasma.2.5 Application of RASSF1A and p16 gene hypermethylation in plasma DNA inearly detection of non-small lung cancer Sensitivity, specificity, negativepredictive value and positive predictive value of RASSF1A and p16 genehypermethylation in plasma DNA for diagnosis of NSCLC were 43.75% and31.25%, 100%, 41.30% and 36.54%, and 100%, respectively. Sensitivity,negative predictive value of hypermethylation in at least one gene for diagnosisof NSCLC was 59.38% and 49.35%.3. Identification of differential proteins in two different lung adenocarcinomatissues with or without RASSF1A expression3.1 The screening results of the differential protein spots The analytical maps of2-DE gel were obtained after being scanned with GS-800. Nine differentialprotein spots on gels for the soluble proteins from two lung adenocarcinomatissues with the expression and expression loss of RASSF1A gene were acquiredafter being examined with PDQuest software.3.2 Identification of the differential protein spots with MS The differential protein spots were cut out from the preparation gels and were identified withMALDI-TOF-MS. Comparing with the protein database, five candidate proteinswere identified with MS. They were Cytochrome b5, 60S acidic ribosomalprotein P2, Carbonic anhydrase 1, Pyrroline-5-carboxylate reductase 1,Apolipoprotein A-I precursor. These Proteins remain unknown relationship withRASSFtA gene. Though the good peptide mass fingerprints(PMF) wereobtained for another 4 protein sports, no matched protein was found in themodern protein database. They will be identified by tandem MS or amino acidsequence analysis.Conclusions1. The frequent transcriptional inactivation of RASSF1A and p16 genes wasobserved in NSCLC tissues in Chinese. And the expression inactivation ofRASSF1A gene was also observed. But the transcriptional and posttranscriptionalexpression of both genes were normal in all matched normal lung tissues far fromcancer areas.2. The frequency of aberrant promoter methylation of RASSF1A and p16 genes wasdetermined in NSCLC tissues in Chinese. The significant positive correlationbetween the expressing inactivation and the promoter hypermethylation of bothgenes in NSCLC tissues was identified. The major reason for silencing ofRASSF1A and p16 genes could be recognized as the promoter hypermethylationof the two genes; The frequency of aberrant promoter methylation of RASSF1Aand p16 genes was also detected in plasma DNA from patients with NSCLC inChinese. The positive correlation of the promoter hypermethylation of RASSF1Aand p16 gene between in plasma and in tumor tissues from NSCLC patients wasidentified. Methylation analysis of RASSF1A and p16 genes in plasma DNA maybe a promising new diagnostic approach to screen putative cancer patients.3. The two-dimentional gel maps for soluble proteins of these two different lungadenocarcinoma tissues with or without RASSF1A expression were obtainedsuccessfully. Nine differential proteins were found. Five candidate proteins were identified with MS: Cytochrome b5, 60S acidic ribosomal protein P2, Carbonicanhydrase 1, Pyrroline-5-carboxylate reductase 1, Apolipoprotein A-I precursor.