The Association Between The Genetic Variant In MSI1 Promoter And Risk Of Lung Cancer In A Southern Chinese Population

Background:Lung cancer is the most serious type of cancer with high morbidity and mortality. In the United States, a total of 220,000 new lung cancer incidences and 160,000 lung cancer deaths occurred in 2010. In China, lung cancer has already been the leading incidence of tumor among all malignant tumors, which ranked first in males and second in females. In recent decades, lung cancer incidence and mortality rate is rising year by year, which is having a serious threat to our country and the global residents'health.In 21st century, thanks to the implementation of the human genome project, our recognition of the causes of lung cancer has changed from environmental factors discussed in traditional epidemiology to the interaction between genetic factors and environmental factors, especially the genetic factors which play a major role. Many studies have reported that nicotinic receptor gene families located in human 13q23 is highly related to lung cancer, and variation of DNA double strand breaks (DSBs) repair enzyme (NBS1, RAD51 etc) can affect its repair ability, thus affect lung cancer susceptibility. Gene genetic variation exists widely in the human genome, for example the SNP, which can function diversely to induce one's susceptibility to lung cancer under the role of environmental carcinogen, through its influence on gene's expression, structure and function.Cancer stem cell research is new focus of cancer research. Its existence was confirmed for the first time since Disk et al. isolated leukemia cancer stem cells from human acute myeloid leukemia. Afterwards, lots of studies found similar cancer stem cells in breast cancer, lung cancer, melanoma and other systems, which provide strong evidence to the cancer stem cell theory. Cancer stem cell was defined as a kind of cell in tumor tissues with the ability to self-renew and can produce heterogeneity tumor cells by American Association for Cancer Research in 2006. Cancer stem cells have the same capacity to proliferate infinitely as stem cell; they can convert into tumor once they gained excessive proliferative ability through variation. Lots of research demonstrated that cancer stem cells plays an important role in tumor's occurrence, development, recurrence and metastasis for its ability to self-renew, proliferation and differentiation despite its small number. Cancer stem cells are the source of tumorigenesis, studying its happening mechanism will help elucidate cancer pathogenesis and provide scientific basis for cancer treatment.External factors can regulate the function of cancer stem cells by the special signaling pathway in vivo; it is similar to the stem cells. At present, studies has been reported that cancer stem cells are closed related with the Notch,Wnt/β-catenin,Hedgehog signal pathway. Musashi1 (MSI1) is one of the cancer stem cell markers reported in the latest study. It participated in Notch, Wnt, Hedgehog et al. signal transduction pathways, and closed with the tumorigenesis. MSI1 is a RNA-binding protein; it can regulate the expression of the tumor gene. Researches indicated that MSI1 can determine cell fate on several levels, include keeping health of stem cells, cell differentiation and occurrence of tumor. Wang et al. reported that MSI1 is capable of facilitating cell proliferation, inducing cancer metastasis and drug resistance of cancer cells, which would interfere with prognosis of tumor patients.The SNP of the promoter region can influence the gene expression by affecting the binding affinity of the transcription factors, further associate with the tumorigenesis. Based on the dbSNP database (http://www.ncbi.nlm.nih.gov/) we choose two site:-2696 T>C (rs7959801) and -2297 T>C (rs3742038) on the principle that the frequency of rare allele should not be less than 5 percent ,through the function analysis(http://snpinfo.niehs.nih.gov/), we found that the two SNP are the potential functional sites. We further calculated linkage disequilibrium (LD) between these two SNPs based on the genotypes data from dbSNP database, and found that -2696 T>C (rs7959801) and -2297 T>C (rs3742038) was in complete LD (D′=0.246 and r2=0.002). In order to elucidate the relationship between SNPs in promoter region of MSI1 and lung cancer incidence among southern Chinese population, we measured the genotype distribution frequency of -2696 T>C(rs7959801)and -2297 T>C(rs3742038)in promoter region, and investigated the association between these SNPs and the risk of lung cancer, then validated the results with functional assay in vitro.Objective:In order to provide scientific evidence for lung cancer prevention, diagnose and therapy, we investigated the association between the genetic variant in MSI1 promoter and the risk of lung cancer in a southern Chinese population, and analyzed the gene-environment interaction on the lung cancer risk, further we explored the biological essence of relationship between genetic variant and lung cancer incidence through functional assay.Methods:For the current case-control study, 1056 newly diagnosed breast cancer patients were recruited from Guangzhou City and surrounding regions in Southern China. Patients with histopathologically confirmed diagnosis were consecutively recruited between March, 2007 and March, 2009 at the urban hospitals. 1056 sex and age (±1 year) frequency matched cancer-free controls were randomly selected from a subject pool with more than 10,000 individuals who participated in the healthy checkup programs in the community health stations in Guangzhou City during the same time period when the cases were recruited. We used Taq-man method to genotype -2696 T>C (rs7959801) and -2297 T>C (rs3742038), different alleles were marked on the probe with 5'UTR tagged with perssad FAM (blue) and VIC (red) respectively, and 3'UTR tagged with fluorescence quencher persad. ABI 7500 was used to determine the genotype according to their fluorescence value. 60 samples (10% of the total samples) were randomly selected from controls and patients for repeated assays and the results were 100% concordant. SAS 9.13 was used to analyze the different distribution of risk factor, such as sex, age, smoking, drinking, family history, BMI between patients and controls, and analyzed the difference of distribution of the number of mutant allele and the dose-response relationship. Logistic modeling was used to assess the main effect of MSI1 polymorphisms -2696 T>C (rs7959801) and -2297 T>C (rs3742038) and dose-response relationship was also analyzed between the number of alleles and the risk of lung cancer. All statistical tests were 2-sided and p<0.05 was considered statistically significant.The in vitro luciferase assays and reporter gene were similar as the reference described the function mechanism: the reporter gene contained 2950bp of the MSI1 promoter region(from +36bp～-2914bp relative to the translation start site ATG), the amplified fragments were then cleaved with the PGL3-basic vector, then Enzyme cut and sequencing detected . According to the SNP of people with screening by study, the difference allele reporter constructs were obtained from the p2696 difference allele by site-directed mutagenesis. H460 and A549 of human cell lines were seeded onto plates for transfections, and each well was transfected with 1μg of the vector DNA with the three SNPs's allele, using FuGENE HD Transfection Reagent (Roche Applied Science, Mannheim, Germany). Then luciferase activity was measured with a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and we use the pGL3-basic plasmid as the negative control, the Renilla luciferase gene as the actin. Differences in the expression levels of different constructs were determined by Student's t test.Results1.1 Demographics of the study subjectsThis study included 1056 lung cancer patients and 1056 cancer-free controls. Of the 1056 cases, there were 384(36.4%) cases of adenocarcinoma, 369(34.9%) squamous cell carcinoma, 43(4.1%) large cell carcinomas, 128(12.1%) small cell lung cancers, and 132 (12.5%) mixed-cell or undifferentiated carcinomas. Overall, the differences in distributions of sex and age between the cases and controls were not statistically significant (Page=1.000,Psex=0.931). In the single factor analysis, significant differences in smoking status and BMI were observed in the two study groups (Psmocking=0.028,PBMI<0.001), it suggested that smoking and the high BMI are risk factors to the early symptoms of lung cancer. Moreover, the differences in distributions of family history of cancer, family history of lung cancer and drinking between the cases and controls were not statistically significant (Pfamily history of cancer=0.942 , Pfamily history of lung cancer=0.150, Pdrinking=0.916), these factors may have nothing to do with the incidence of lung caner.1.2 The distributions of MSI1 genotypes and the risk of lung cancerThe distributions of the MSI1-2696 T>C (rs7959801) among the cases are 53.3% (TT), 36.5% (CT), 10.1% (CC), and 43.5% (TT), 46.6% (CT), 9.9% (CC) among the controls. The observed genotype frequencies of this polymorphism were in agreement with the Hardy-Weinberg equilibrium in the control subjects (P= 0.096). The distribution difference of MSI1 -2696 T>C in controls was significant (P=7.89×10-6). Compared with the most common -2696 TT genotype, carriers of the -2696 TC heterozygote had a decreased risk of lung cancer (adjusted OR=0.64, 95%CI=0.52-0.78, P=1.3×10-6); carriers of the CC genotype also had a decrease risk of lung cancer (adjusted OR =0.87;95%CI=0.62-1.21), but because of the lower frequency of this genotype in cases and controls, there was no significant difference. When -2696 TC and CC genotypes combined for further analysis, we found that carriers of -2696 C variant genotypes (-2696CT+CC) also had a decreased lung cancer risk (adjusted OR=0.81; 95%CI=0.70–0.94,p=0.0000073). There was a significant trend for an allele dose-effect on the decreased risk of lung cancer (Ptrend=0.0042).In the Stratification analysis, it was showed that the decrease risk of lung cancer was more evident in groups without family history of caner (adjusted OR=0.65; 95%CI=0.54-0.78,p<.0001), BMI (18kg/m2-25kg/m2)(adjusted OR=0.67; 95%CI=0.55-0.83,p=0.0001) and BMI (>25kg/m2)(adjusted OR=0.56; 95%CI=0.35-0.88, p=0.0134) in carriers of -2696 C variant genotypes.The distributions of the MSI1-2297 T>C (rs3742038) among the cases and controls are 90.9% (TT), 8.24% (CT), 0.85% (CC) and 88.5% (TT), 10.8% (CT), 0.66% (CC) respectively. The observed genotype frequencies of this polymorphism were also in agreement with the Hardy-Weinberg equilibrium in the control subjects (P= 0.092). However, the distribution difference of MSI1 -2297 T>C was not statistical significant (P=0.1220), further stratum analysis also did not find significant differences between cases and controls.1.3 luciferase activity detection Because of the association study showed that -2696 T>C polymorphisms was associated with lung cancer morbidity, so we constructed the reporter plasmids with -2696 T and C alleles successfully through in vitro cloning combined with point mutation. We used plasmids to transfect A549 and NCI-520 lung cancer cells, and then tested the luciferase activity. The results showed that the activity of the cells tranfected with -2696 C alleles was much higher than cells tranfected with -2696 T alleles (P=0.0006 and 0.0021), suggested that -2696 C variant genotypes can remarkably reduce the transcriptional activity of MSI1 promoter, and influence the expression of MSI1, it is concordant with the association study results.InnovationThis study discussed the relationship between stem cell biomarker Musashi1 and the lung cancer of southern people in our country for the first time, and the association between the promoter region -2696T>C of MSI1 gene and the people in south China in lung cancer, it fills a vacancy for domestic and foreign relevant research.DeficiencyThis study is lack of the association between the Musashi1 genotype and the protein expression levels in tissues (western Blot), and further analysis the difference between the hereditary variation of -2696T>Cand the ability of nuclear transcription factors by EMSA, in order to confirm the metamorphosis of lung cancer in molecular biology.ConclusionOur data suggested that the functional -2696C variant in the MSI1 promoter contributes to a decreased risk of lung cancer by decreasing the promoter activity and that the C variant may be a marker for susceptibility to lung cancer. Especially in BMI (18 kg/m2– 25 kg/m2or >25 kg/m2) and no family history of cancer, furthermore the functional test showed that -2696C variant allele can decrease the transcriptional activity of MSI1 promoter region, and influence the expression of MSI1, so it is association with the lung cancer mobidity.