Study On Circulating MicroRNA As Biomarker For NNK Induced Lung Carcinogenesis

Background:As is known to all, cigarette smoke contains substantial amounts of lung carcinogens, including4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), polycyclic aromatic hydrocarbons (PAHs), metals and miscellaneous organic compounds etc. Of these lung carcinogens, NNK is a tobacco-specific N-nitrosamine and the most important lung carcinogen. Although many carcinogens in cigarette smoke contribute to lung cancer, the key compound NNK plays a major role in lung carcinogenesis. Many studies showed that NNK is a potent lung carcinogen in rats, mice, and hamsters. NNK induced lung tumorigenesis have remarkable organospecificity. The systemic administration of NNK to rats is a reproducible and robust method for the induction of lung tumors.Lung cancer is one of the most common cancers and a leading cause of cancer death in the world. Many studies indicate that cigarette smoking causes most lung cancers. Despite great efforts to improve smoking cessation and the treatment of patients with lung cancer, the5years survival rate for lung cancer patients has not significantly improved over the past30years. Scarcity of diagnostic biomarkers for early stage lung cancer is the most important causes. Discovery and use of efficient biomarker for early stage lung cancer will be able to improve the5years survival rate of lung cancer.MicroRNAs (miRNAs) are a class of small endogenous noncoding RNA (about19-25nt) that usually post-transcriptional negative regulate the expression of mRNA. Previous studies showed that miRNAs involve in a variety of biological processes including cancer development and progression, cell growth and proliferation, apoptosis etc. Increasing evidences have demonstrated that expression of miRNAs is dysregulated in a variety of cancers, such as lung cancer, pancreatic cancer, prostate cancer, breast cancer and hepatocellular carcinoma etc. MiRNAs differential expression profile can classify variety of cancers. Several recent studies indicated that miRNAs are stable and detectable in serum or plasma. These data consistently indicate that circulating miRNAs are potential biomarker for cancer.Previous several studies declared that chemical carcinogens can cause the alteration of miRNAs expression. Male F344rats continuously fed with NNK up to20weeks resulted in alteration of miRNA expression in lung tissues of rat. To our knowledge, relative study on alteration of circulating miRNAs expression in carcinogenesis induced by chemical carcinogens is not still reported. Chemical carcinogenesis usually includes four stages:initiation; progression; development and transformation. During NNK-induced lung carcinogenesis, the alteration of serum miRNAs expression is not still unclear. We hypothesized that the levels of serum miRNAs were change and associated with lung carcinogenesis induced by NNK. Previous studies showed that male F344rat is highly sensitive to NNK-induced lung carcinogenesis. The dose-response relationship indicated that administration of low dose NNK mainly induce lung carcinogenesis. Therefore, in the present study, NNK was systemically administered to rats to induce lung carcinogenesis. Using this animal model, Blood of rats were collected at1,5,10,20,40,60,80and95weeks after NNK treatment and sera of rats were isolated. Serum miRNAs differential expression profile was analyzed by small RNA solexa sequence. Using Quantitative real-time PCR (qRT-PCR), the differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study. The expression levels of miR-206and miR-133b were identified in rat serum at different stages of NNK induced lung carcinogenesis. The potential of miR-206and miR-133b as biomarker for lung cancer was identified in rat sera, rat lung tumors, lung cancer lines and human sera, respectively. Our study will provide important data for explore the biomarker of chemical carcinogens induced lung carcinogenesis. We predicted the barget genes and functions of miR-206and miR133b by computational bioinformatic analysis. These studies will provide experimental evidences for further study on the functions of circulating miRNAs.Objective:(1) Male F344rats were administered with subcutaneous injections of low-dose NNK (3times weekly for20weeks), and animals were maintained at SPF animal house until the95th week to induce lung carcinogenesis.(2) MiRNAs differential expression profile of rat serum in control and NNK treatment group was analyzed by small RNA solexa sequencing and candidate miRNAs were screened.(3) Using Quantitative real-time PCR (qRT-PCR), the differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study.(4) The expression levels of miR-206and miR-133b were identified in rat serum at different stages of NNK induced lung carcinogenesis.(5) The potential of miR-206and miR-133b as biomarker for lung cancer was identified in rat sera, rat lung tumors, lung cancer lines and human sera, respectively. Our study will provide important data for explore the biomarker of chemical carcinogens induced lung carcinogenesis.Methods:(1) The construction of rat model for lung cancer induced by NNK:Rats from control group and NNK treatment group were respectively administered with subcutaneous injections of0.3ml saline and NNK (1.15mg/kg,0.0055mmol/kg) in0.3ml saline (newly prepared before each injection). Each rat was given injections3times weekly for20weeks. Afterward, animals were maintained at SPF animal house until the95th week to be sacrificed.(2) Small RNA solexa sequencing of rat serum:Equal volume serum from each rat of control or NNK treatment group at the60th week was polled together, respectively. Pooled serum samples of control and NNK treatment group were send to Beijing Genomics Institute at Shenzhen for small RNA solexa sequencing and miRNAs differential expression profile was analyzed.(3) Candidate serum miRNAs expression analysis for rat individuals by qRT-PCR:According to miRNAs differential expression profile to select candidate miRNAs, Using Quantitative real-time PCR, The differential expression serum miRNAs were identified in each rat individuals. Significant differential expression miRNAs (miR-206and miR-133b) were selected for further study.(4) qRT-PCR analysis of the expression of miR-206and miR-133b at different stages of NNK treatment:Using Quantitative real-time PCR, miR-16was selected as endogenous control gene and the level of miR-206and miR-133b were detected in rat sera of1,5,10,20,40,60,80and95week.(5) The expression of miR-206and miR-133b in lung tissues of rats:Using Quantitative real-time PCR, RNUB6was selected as endogenous control gene and the level of miR-206and miR-133b were detected in lung tissues of rats.(6) The expression of miR-206and miR-133b in lung cancer cell lines:Using Quantitative real-time PCR, RNUB6was selected as endogenous control gene and the level of miR-206and miR-133b were detected in lung cancer cell lines.(7) The level of miR-206and miR-133b in human sera of health control and lung cancer patients:Using Quantitative real-time PCR, miR-16was selected as endogenous control gene and the level of miR-206and miR-133b were detected in human sera of health control and lung cancer patients.(8) Bioinformatic analysis:To predict the functions of miR-206and miR-133b, we predicted the target genes of miR-206and miR-133b from http://www.microrna.org/microrna/. The common target genes of miR-206and miR-133b were analyzed to find those target genes that are associated with carcinogenesis. The functions of miR-206and miR-133b are also predicted by http://acgt.cs.tau.ac.il/fame/index.html. Pathways of cancer or lung cancer were analyzed from http://www.genome.jp/kegg/pathway/to find those target genes involving in cancer pathway.(9) Statistical analysis:Statistical analysis was performed using SPSS software (version13.0, IL, USA).Normality test was performed for data of each group using the Shapiro-Wilk normality test. For parametric comparisons, two independent samples t-test (for two independent samples), one-way analysis of variance (ANOVA) for multiple samples and paired t-test (for two paired samples) were employed respectively. For nonparametric comparisons, the Mann-Whitney test (for two nonparametric groups) and the Kruskall-Wallis test (for more than two nonparametric groups) were used respectively. Fisher’s exact test was performed to compare the incidences of lung tumors between control and NNK treatment. Spearman correlation analysis was used to evaluate the correlation of two variables. In order to evaluate the predictive value of serum miRNAs, the receiver operating characteristic (ROC) curve were established for discriminating health control and patients with lung cancer. The area under the ROC curve (AUC) was calculated in order to better identify the potential of serum microRNAs as a diagnostic marker. All p-values are two-sided and less than0.05was considered statistically significant.Results:(1) The construction of rat model for lung cancer induced by NNK:At the95th week,19lung tumors were found in10rats of NNK treatment group, only1lung tumor was found in7rats of control group. No pathological changes were observed in lung tissues of rats of control. Most of lung tumors were moderately differentiated adenocarcinoma. Compared to control group, the incidence of lung tumor in NNK treatment group was statistical significance (Fisher’s exact test, P=0.004).(2) Serum miRNAs differential expression profile:We detected387known miRNAs of Rattus norvegicus (reference to miRBase Release13). Of these miRNAs,181miRNAs could be detected in control serum or NNK treatment serum and82differential expression miRNAs have significantly difference between control serum and NNK treatment serum. Compared with control serum,37miRNAs expression were up-regulated;45miRNAs expression were down-regulated;99miRNAs expression were equal. Of these181miRNAs,20miRNAs specifically expressed in the serum of control group;8miRNAs specifically expressed in the serum of NNK treatment group;153miRNAs commonly expressed in the serum of control and NNK treatment.(3) Candidate serum miRNAs expression analysis for rat individuals by qRT-PCR:Compared with control serum, the expression level of miR-206(U=21.00, P=0.012), miR-133b (U=26.00, P=0.030) and miR-382(U=24.00, P=0.021) were up-regulated significantly in NNK treatment group (Mann-Whitney tests). MiR-365, miR-34c, miR-20a, miR-29b, miR-30e, miR-183, miR-331and miR-195have no significances between rat sera of control and NNK treatment group.The expression of miR-206and miR-133b was closely associated in serum of rat individuals. Spearman rank correlation analysis show that the expression of miR-206and miR-133b are positive correlation, Spearman r=0.758,95%CI=0.339-0.926, P=0.003.(4) qRT-PCR analysis of the expression of miR-206and miR-133b at different stages of NNK treatment:Compared with control serum, the levels of miR-206and miR-133b expression were significantly up-regulated in rat serum of NNK treatment group at1,5,10,20,40,60and80weeks, respectively. Fold change up-regulated of miR-206were respectively1.43±0.11(t=-6.744, P=0.003);1.59±0.32(t=-3.215, P=0.032);2.17±0.35(t=-5.735, P=0.029);4.82±0.88(t=-7.497, P=0.017);3.23±0.99(t=-3.876, P=0.018);2.41±0.15(t=-16.250, P=0.004);1.85±0.12(t=-12.035, P=0.007). Fold change up-regulated of miR-133b were respectively1.08±0.10(t=-1.485, P=0.276);1.45±0.08(t=-9.208, P=0.011);2.12±0.25(t=-7.774, P=0.016);2.68±0.03(t=-97.887, P<0.001);2.33±0.17(t=-13.238, P<0.001);1.82±0.16(t=-8.953, P=0.001);1.30±0.16(t=-3.156, P=0.034). At95weeks after NNK treatment, miR-206and miR-133b were significantly down-regulated compared to rat serum of control. Fold change were2.09±0.64(t=5.769, P=0.004);2.78±0.30(t=26.813, P=0.001), respectively.(5) The expression of miR-206and miR-133b in lung tumors tissues of rats: In9rat lung tumor tissues and corresponding normal lung tissues. Compared with normal lung tissues, miR-206and miR-133b was significantly low-expression in9paired lung tumor tissues (paired t-test). Meanwhile, we examined the levels of miR-206and miR-133b expression in7normal rat lung tissues of control group. Statistically significant differences were analyzed using ANOVA. Among three groups, the levels of miR-206(F=16.376, P<0.001) and miR-133b (F=11.001, P<0.001) have significantly difference. Compared with normal lung tissues of control, miR-206(P=0.001) and miR-133b(P=0.019) were low-expression significantly in normal lung tissues of NNK treatment group and lung tumor tissues of NNK treatment group (p<0.001). Compared to normal lung tissues of NNK treatment group, miR-206(P=0.048) and miR-133b (P=0.031) were low-expression significantly in paired lung tumor tissues of NNK treatment group.(6) The expression of miR-206and miR-133b in lung cancer cell lines:The level of miR-206was significantly down-regulated28.341±3.435,97.638±16.922,33.999±11.882,33.055±16.138,23.424±3.899and31.688±12.306fold in A549, QG56, H446, H1299,95-D and16HBE-T cell relative to16HBE cell, respectively. The level of miR-133b was down-regulated1.793±0.410,3.926±2.014,2.894±2.135,3.768±0.734,2.176±0.710and2.652±1.186fold in A549, QG56, H446, H1299,95-D and16HBE-T cell relative to16HBE cell, respectively.(7) The level of miR-206and miR-133b in human sera of health control and lung cancer patients:Compared with health controls, the expression of serum miR-206and miR-133b were significantly down-regulated in lung cancer patients (miR206-U=61.00, miR133b-U=80.00, P<0.001, Mann-Whitney test). Receiver operating characteristic (ROC) curve analyses show that the areas under curve (AUC) of serum miR-206and miR-133b were0.9024(95%CI=0.8125-0.9923) and0.8720(95%CI=0.7734-0.9706), respectively.(8) Bioinformatic analysis:has-miR-206and has-miR-133b were predicted7201and5314target genes, respectively. And of these target genes,2577genes are common target genes of miR-206and miR-133b. By functions prediction, we found that miR-206involve in6KEGG pathways, including VEGF signaling pathway, p53signaling pathway, Nicotinate and nicotinamide metabolism, SNARE interactions in vesicular transport, Glutathione metabolism, Olfactory transduction. MiR-133b involve in2KEGG pathways, including Notch signaling pathway, Sphingolipid metabolism. Of these pathways, P53signaling pathway and notch signaling pathway are closely related to carcinogenesis.Conclusions:(1) Small RNA solexa sequencing of rat serum at the60th week indicate that NNK can induce the alteration of the expression of serum miRNAs.(2) qRT-PCR analysis of differential expression miRNAs in sera of rats individuals showed that miR-206and miR-133b were significantly up-regulated in rat sera of NNK treatment group. Moreover, the expression of miR-206and miR-133b was positive correlation. The expression of miR-206and miR-133b in lung tumors and lung cancer cell lines has significantly covariant. They may involve in NNK induced lung carcinogenesis as tumor suppressor.(3) The level of serum miR-206and miR-133b at different stages of NNK induced lung carcinogenesis indicated that alteration of serum miRNAs expression followed a two-step evolution. At early stage, the level of miR-206and miR-133b was progressively increased and the peak of expression occurs at the20th week. Afterward, the level of miRNAs was progressively decreased and low expression was observed at the95th week.(4) By identifying the expression of miR-206and miR-133b in rat sera, rat lung tumors, lung cancer cell lines and sera of lung cancer patients with smoking history, serum miR-206and miR-133b may be potential biomarker for lung carcinogenesis induced by NNK.