转录产物MALA1的高表达：与早期非小细胞型肺癌的转移有关

　　　　　　 作者：王文兵，周密，严丽荣， 乌慧玲

【摘要】 　　目的： 研究转录产物MALA1的表达水平与早期非小细胞型肺癌(NSCLC)转移之间的关系。方法： 用实时定量PCR分析了MALA1基因在70例NSCLC患者中的表达情况。 结果： 在对Ⅰ期和Ⅱ期NSCLC患者超过5年的随访期中发现，MALA1高表达的患者死亡率超过40 %(12/28)，而MALA1低表达的患者死亡率仅有9 %(2/22) （P=0.01)。说明高表达量的MALA1很可能是早期NSCLC 患者预后不良的一个指标。此外，也发现了MALA1基因与肿瘤组织的转移有关。结论： MALA1的转录水平不仅可以作为预测发生肿瘤转移的高危人群的生物学标记，并有望在今后用于早期NSCLC患者的诊断或治疗中。

【关键词】 转移； 非小细胞型肺癌； MALA1； 表达； 扣除杂交

　　［Abstract］Objective: To investigate the relationship of the expression level of the transcript MALA1 and the metastasis of early stage nonsmall cell lung cancer(NSCLC).Methods： Expression level of MALA1 was analyzed with real time quantitative PCR in 70 patients of NSCLC.Results： In the study of an over fiveyear followup for stage Ⅰand Ⅱ NSCLC patients.It was found that the death rate was more than 40% in patients with high MALA1 expression， whereas only 9%(2/22) in patients with low MALA1 expression died(P=0.01)， suggesting high expression of MALA1 was potentially predictive for a poor prognosis in early NSCLC.Additionally， it was found that the association of MALA1 gene with metastasis depended on the tumor′s histology.Conclusion： These results demonstrated that MALA1 transcript could be used as a biological marker， which may predict high risk for the development of distant metastasis and could be further helpful in improvement of treatment for earlystage NSCLC patients.

　　［Key words］ metastasis; nonsmall cell lung carcinoma; MALA1; expression; subtractive hybridization

　　Bronchogenic carcinoma continues to be the leading cause of cancer death in both men and women ［1］.Nonsmall cell lung carcinoma(NSCLC) accounts for approximately 80% of all bronchogenic carcinomas， with SCLC(small cell lung carcinoma) accounting for the remainder［1］.Survival following treatment of NSCLC is stagerelated， and the patients with earlystage disease have the best chance for curative treatment.Although the patients with stage Ⅰ and Ⅱ disease undergo surgical resection of primary carcinomas， a considerable number of them will eventually die of metastatic neoplasm， implying a need for reliable indicator not only to predict survival of the patients with early stage NSCLC， but to provide information for the high risk patients who might benefit from additional therapy［2］.

　　Up to date， while there are some reports about DNA ploidy and DNA deletions， SPF(Sphase fraction)， PCNA(proliferating cell nuclear antigen)， the gene family of erb B， p53 mutation， EGFR and Ki67 antigen and so on as biological indicators of prognosis in most solid tumors， still have been conflicting results in lung cancer studies［3-9］.Recently， applying a subtractive hybridization procedure， we identified a few new genes whose expression levels were significant differences between NSCLC tumors that were cured by surgical resection and the tumors that subsequently metastasized.An approach to find the genes related to metastasis has been made by a cDNA subtractive hybridization［10］.In the present study， we investigated expression level of the identified genes in the subtracted cDNAs by realtime quantitative RTPCR.The results demonstrated that MALA1(Metastasis associated in lung Adenocarcinoma)， the most frequently appeared gene in identification of subtractive cDNA library， was associated with metastasis in the patients with early stage NSCLC.Therefore it is suggested that expression level of MALA1 could be one valuable molecular biological marker to predict survival for patients in early stage NSCLC.
　　
　　1 Materials and methods

　　1.1 Tumor specimens and survival data

　　Tumor samples were obtained from 70 patients with stage Ⅰ to Ⅱ IA NSCLC at the time of initial surgical resection at Muester university hospital in Germany.Pathohistory examination showed no evidence for remaining tumor.RNA isolation and consequently cDNA preparation， characterization of the cohort of NSCLC patients were described previously ［11］.Parts of the surgically resected tumors were immediately shockfrozen and stored at -70℃ until analysis.All specimens were confirmed to contain a high percentage of tumor cells(&>70%).Lung tissue without histological evidence of tumor infiltration was isolated from 12 inpiduals to serve as a control.

　　1.2 cDNA subtractive hybridization

　　To exclude differences in gene expression independent on the metastatic potential， all the patients in this study were male with more than five years of followup， all the tumors were at stage I and were completely resected by surgery(R0 resection). Histological classification were confirmed to be adenocarcinomas as described ［11］.The cDNA hybridization procedure was described as the method［11］.In brief， cDNA was prepared and amplified using realtime RTPCR in the ABI Prism 7 700 sequence detector(PE Biosystems， Foster City， CA).One gram of total RNA was reverse transcribed using random primers and Moloney murine leukemia virus reverse transcriptase(Promega) following the manufacturer′s protocol.cDNA samples were diluted to 100 μl， and 2.5 μl of cDNA were used for each PCR.PCR amplification of the housekeeping gene glyceraldehyde3phosphate dehydrogenase was used to confirm the quality of cDNA and standardize the total amount of cDNA between different samples.The primers for amplication of the MALA1 gene were: forwardTGAGAGAAAGGACTACAGA， and reverseCTTCCC GTACTTCTGTCTT.

　　1.3 Statistical Analysis

　　Statistical Analysis was carried out with SPSS software system(SPSS for Windows Version 10.0).Difference in gene expression level between metastasizing and nonmetastasizing tumors was analyzed statistically with MannWhitney Utest.Cancerspecific survival curves were plotted using the KaplanMeier method and the logrank test was used to assess the statistic significance of differences between groups.Cox proportional hazards regression analysis was used to define gene expression level with independent predictive value with respect to cancer specific survival.
　　
　　2 Results

　　2.1 Cloning of MALA1 gene

　　Two hundred and twentyfive independent sequences were obtained totally in metastatic cDNA library after subtractive hybridization.Twentysix transcripts were found more than once.The most frequently identified transcript was a so far uncharacterized mRNA derived from chromosome 11q13 by DNA homology comparison in Genbank(Accession number AF203815).To obtain more of the sequence of this transcript， we used 5′ and 3′ Rapid Amplification of cDNA(RACE).The full length of this DNA sequence was 737 base pairs.Database searches revealed that this sequence had before been identified only as an EST(Expression sequence terminal).The sequence of the transcript is shown in Fig.1， in which the longest open reading frame for peptides of 52 amino acids(aa) was predicted in both directions.However， it is currently unclear whether this sequence codes for a peptide in vivo.We have named this RNA MALA1.To confirm the reliability of MALA1 expression， this transcript was analyzed in the pooled samples as well as in the subtracted libraries(metastasis minus nonmetastasis and nonmetastasis minus metastasis).These analyses demonstrated for MALA1 an about 3fold higher expression in the pooled sample from metastasizing tumors compared to the nonmetastasizing tumors.

　　2.2 Expression analysis of MALA 1 gene in patients

　　The issue of the association of MALA1 gene with metastasis was specific for certain histological subtypes or stages of disease was analyzed.When only stage Ⅰ and Ⅱ patients were included into the analyses， expression levels of MALA1(P=0.017) was significantly higher in metastasizing tumors， and another one was EIF4A1(Eukaryotic translation initiation factor A1)(P=0.031).Also， further analyses indicated that the association of MALA1 gene with metastasis was highly specific for histological subtypes.Twentysix tumors were histologically classified as adenocarcinomas.For three out of five genes(MALA1， EIF4A1， NPCRP) that we identified by our screening method， expressed in metastatic adenocarcinomas was several folds higher than in nonmetastatic adenocarcinomas.Interestingly， no significant differences in gene expression were found for squamous cell carcinomas(n=34).For large cell carcinomas(n=10) mixed results were obtained: whereas some of the genes were clearly enriched in metastasizing tumors(MALA1， EIF4A1， MDM2)， others were clearly not(NPCRP).These data provided evidence that the association of the identified genes with metastasis depended on the tumor′s histology.The relative expression of MALA1 in three types of lung carcinoma was shown in Fig.2.
　
　　To evaluate the relationship of the identified metastasisassociated transcripts and survival， all samples were classified for couples of gene as high or low expressers.High or low expression was determined according to the expression level of the samples with regard to median expression of all 70 samples ［11］.KaplanMeier plots indicated that high expression level of MALA1 was associated with significantly worse survival of patients with stage Ⅰ and Ⅱ disease(Fig.3).High expression of MALA1 was highly predictive for a poor prognosis in early disease.Indeed， only 2/22(9%) of low expressing stage Ⅰ and Ⅱ disease patients died during a fiveyear follow up period but more than 40%(12/28) of patients with high levels of MALA1 ultimately died.When a stepwise Cox regression analysis was performed for stage Ⅰ and Ⅱ patients， high levels of MALA1(P=0.02) emerged as independent prognostic parameters for patient survival(Fig.4).

　　3 Discussion

　　We identified genes that might be associated with metastasis of NSCLC.The direct comparison of subsequently metastasizing primary tumors with nonmetastasizing primary tumors could offer important advantages.Firstly， it avoided changes in gene expression and metastasis due to the different environments of tumor; secondly， for clinical patient management， the knowledge of differences between metastasizing and nonmetastasizing primary tumors can lead to better prognosis prediction and ultimately to risk adapted therapeutic strategies［12］.Several unknown genes were identified in our study.One of these， a novel transcript on chromosome 11q13， was the most frequently found transcript.To determine the 5′and 3′ends of the sequence we used 5′and 3′ RACE techniques and obtained a 737 base pair fragment that potentially encodes for a 52 amino acid peptide.This transcript was named as MALA1.Currently， it is unclear whether this sequence has peptidecoding potential in vivo， or whether its expression is merely an epiphenomenon for processes on 11q13 that are associated with the metastasisbearing potential of NSCLC tumors.The region 11q13 has been known to be relevant for tumorigenesis and metastasis ［13-15］， there were some important genes， such as CCND1(cyclin D1) and MEN1(Multiple endocrine neoplasia 1) located in this region as well.However， in most cases， 11q13 mutation was associated with lung carcinoma.It might hint that MALA1 in the region of 11q13 was translocated to a new area and affected other genes expression in that area.Nevertheless， even if MALA1 is nonfunctional， it bears strong prognostic potential in early stage NSCLC.
　　
　　Tumors of patients were grouped for each gene in high vs.low expressing ones based on the expression of tumor in comparison to the median expression of all patients.Kaplan Meier survival plots are shown for patients with adenocarcinoma or squamous cell carcinoma and stage Ⅰ or Ⅱ disease.The logrank test was used to calculate statistical significance
　　
　　Recently， Clark et al.［16］ published microarray data derived from a melanoma metastasis model.The thymosin β4 gene， identified by them， was also cloned by our strategy.Therefore， we tested to test whether the two other main genes identified by them were associated with metastasis in NSCLC.Thymosin β4 proved to be associated with metastasis and prognosis in our patient group， whereas no significant differences were found for either Rho C or fibronectin.These findings provided another strong hint that the stage and cell type specific gene expression profiles were associated with metastasis.

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