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A Genetic Variant in pre-miR-27a Is Associated with a Reduced Renal Cell Cancer Risk

A Genetic Variant in pre-miR-27a Is Associated with a Reduced Renal Cell Cancer Risk
A Genetic Variant in pre-miR-27a Is Associated with a Reduced Renal Cell Cancer Risk

A Genetic Variant in pre-miR-27a Is Associated with a Reduced Renal Cell Cancer Risk in a Chinese Population Danni Shi1,2,3.,Pu Li4.,Lan Ma1,2.,Dongyan Zhong2,3,Haiyan Chu1,2,Fu Yan2,Qiang Lv4,Chao Qin4, Wei Wang2,Meilin Wang2,3,Na Tong3,Zhengdong Zhang1,2,3*,Changjun Yin4*

1State Key Laboratory of Reproductive Medicine,Institute of Toxicology,Nanjing Medical University,Nanjing,China,2Department of Molecular&Genetic Toxicology,the Key Laboratory of Modern Toxicology of Ministry of Education,School of Public Health,Nanjing Medical University,Nanjing,China,3Department of Occupational Medicine and Environmental Health,Jiangsu Key Lab of Cancer Biomarkers,Prevention and Treatment,Cancer Center,Nanjing Medical University,Nanjing,China, 4Department of Urology,the First Affiliated Hospital of Nanjing Medical University,Nanjing,China

Abstract

Background:MicroRNAs(miRNAs)are a class of small non-coding RNAs to regulate cell differentiation,proliferation, development,and apoptosis.The single nucleotide polymorphism(SNP)rs895819is located at the terminal loop of pre-miR-27a.Here,we aimed to investigate whether SNP rs895819was associated with the development of renal cell cancer(RCC)in

a Chinese population.

Methods:In this case-control study,we recruited594RCC patients and600cancer-free controls with frequency matched by age and sex.We genotyped this polymorphism using the TaqMan assay and assessed the effect of this polymorphism on RCC survival.Logistic regression model was used to assess the genetic effects on the development of RCC and interactions between rs895819polymorphism and risk factors.

Results:Compared with AA homozygote,individuals carrying AG/GG genotypes had a statistically significant reduced susceptibility to RCC(adjusted OR=0.71,95%CI=0.56–0.90).Furthermore,AG/GG genotypes were associated with reduced RCC susceptibility in localized clinical stage(adjusted OR=0.71,95%CI=0.55–0.91),and similar effects were observed in well differentiated and poorly differentiated RCC(adjusted OR=0.71,95%CI=0.55–0.93for well differentiated,adjusted OR=0.51,95%CI=0.28–0.93for poorly differentiated).We also observed that rs895819had multiplicative interactions with age and hypertension.However,the polymorphism did not influence the survival of RCC.

Conclusion:Our results suggest that the pre-miR-27a rs895819polymorphism can predict RCC risk in a Chinese population.

Larger population-based prospective studies should be used to validate our findings.

Citation:Shi D,Li P,Ma L,Zhong D,Chu H,et al.(2012)A Genetic Variant in pre-miR-27a Is Associated with a Reduced Renal Cell Cancer Risk in a Chinese Population.PLoS ONE7(10):e46566.doi:10.1371/journal.pone.0046566

Editor:Rui Medeiros,IPO,Inst Port Oncology,Portugal

Received June19,2012;Accepted August31,2012;Published October30,2012

Copyright:?2012Shi et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Funding:This study was partly supported by the National Natural Science Foundation of China(81230068,30972444,81102089and81201571),the Key Program of Natural Science Foundation of Jiangsu Province(BK2010080),and National Science Foundation of Jiangsu Province(BK2011773and BK2011775),the Qin Lan Project of Jiangsu Provincial Department of Education,and the Priority Academic Program Development of Jiangsu Higher Education Institutions(Public Health and Preventive Medicine).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:drzdzhang@https://www.wendangku.net/doc/303027936.html,(ZZ);drcjyin@https://www.wendangku.net/doc/303027936.html,(CY)

.These authors contributed equally to this work.

Introduction

Renal cell cancer(RCC)accounts for more than90%of kidney carcinomas,and clear-cell renal carcinoma is the most common type in RCC[1,2].The incidences of RCC vary substantially worldwide,with higher rates in Europe and North America and lower rates in Asia and South America[1].Rates among females are generally about half of those among males[1].Though few risk factors are established for RCC,there are a number of predisposing conditions which are known to be related to the development of RCC,such as cigarette smoking,obesity, hypertension,diabetes,family history of cancer,and others [3,4,5].However,only a part of the individuals exposed to these risk factors will develop RCC in their life time,suggesting that individual differences including genetic susceptibility factors may be one of the most critical agents in renal cell carcinogenesis. MicroRNAs(miRNAs)are a class of endogenous,small and non-coding RNAs(,22nt),which are initially transcribed from genomic DNA to long primary transcripts(pri-miRNAs)and then are cleaved by nuclear Drosha into60–70nt hairpin-shaped precursor RNAs(pre-miRNAs)[6,7].Pre-miRNAs are exported to the cytoplasm by Exportin-5and are further processed into ,22nt mature miRNA duplexes by the cleavage of Dicer[8,9].In association with RNA-induced silencing complex(RISC),miRNAs can induce mRNA degradation or translational repression by binding to the39-untranslated region of their target genes at the posttranscriptional level[10].To date,it has been estimated that miRNAs modulate the expression of approximately30%of human genes[11].MiRNAs are involved in a wide range of

biological processes including cell cycle regulation,apoptosis and stem cell maintenance,development,metabolism and aging[11]. It has been shown that miRNAs participate in human carcino-genesis as either tumor suppressors or oncogenes[12,13]. Accumulative studies have suggested that single nucleotide polymorphisms(SNPs)or mutations could make a significant contribution to disease susceptibility and outcome.Genetic variants or mutations in miRNAs or pre-miRNAs may alter miRNA expression and/or maturation[14,15].

One study has systematically identified323SNP in227known human miRNAs,and12SNPs are located within the miRNA precursors[14].The SNP rs895819is located at the loop of pre-miR-27a and involves an A.G nucleotide transition.Sun et al.[16] reported the polymorphism could lead to process variation,higher expression of miR-27a and eventually predisposition of gastric cancer[16].While Yang et al.[17]found that G allele of rs895819 might impair the maturation of the miR-27a,thus,was associated with reduced familial breast cancer risk.Moreover,Mertens-Talcott et al.reported that in breast cancer cells,transfection of antisense miR-27a lead to increased expression of Zinc finger and BTB domain containing10(ZBTB10)(a putative Sp repressor) and these responses were accompanied by decreased expression of Sp-dependent survival and angiogenic genes,including survivin, vascular endothelial growth factor(VEGF),and VEGF receptor1 (VEGFR1)[18].However,over-expression of survivin was frequently observed in different types of cancer,including RCC [19].To date,there is no study on the association between pre-miR-27a polymorphism and RCC susceptibility.

Based on our knowledge regarding the new polymorphism and biological function of miR-27a,we hypothesized that the pre-miR-27a polymorphism was associated with RCC susceptibility.To test this hypothesis,we genotyped this particular SNP rs895819and assessed the association with risk of RCC in our ongoing case–control study in a Chinese population.

Materials and Methods

Study subjects

This study comprised594patients and600cancer-free controls. All subjects in our study are ethnic Han Chinese with no genetic relationship.All the patients were newly diagnosed with histopathologically confirmed incident RCC.Those cases that received chemotherapy or radio-therapy before surgery or had other type of cancer were excluded from the present study. Consecutive RCC patients were recruited between May2004and August2010at The First Affiliated Hospital of Nanjing Medical University,Nanjing,China.Disease was classified according to World Health Organization criteria and staged according to the American Joint Committee on Cancer TNM classification.The Fuhrman scale was used to assess tumor nuclear grade[20].The controls were recruited from those who were seeking health care in the outpatient departments at the same hospital.The cancer-free controls were frequency matched by sex and age(65years)to the cases without individual history of cancer and family unrelated to the cases.A guided questionnaire on demographic and lifestyle factors was administered through face-to-face interviews by trained interviewers.Each patient donated5ml blood for genomic DNA extraction after a written informed consent obtaining from all subjects.This study was approved by the institutional review board of Nanjing Medical University.

For the survival analysis,296RCC cases enrolled in our ongoing cohort study from May2004to October2009were used. The patients were followed up prospectively every6months from the date receiving a confirmed diagnosis until death or last time of follow-up.The maximum follow-up time was72months(last follow-up in August2010)and the median follow-up time was19.3 months.

Genotyping

Genomic DNA was isolated from leukocytes of venous blood by proteinase K digestion and phenol/chloroform extraction.Geno-typing was performed with the TaqMan SNP Genotyping Assay using the384-well7900HT real time PCR System and the SDS 2.3software for allelic discrimination(Applied Biosystems,Foster City,CA).The sequence of primers and probes are summarized in Table S1.Genomic DNA of50ng and0.56mix(TaKaRa Bio, JPN)was used for each reaction and amplification was performed under the following conditions:50u C for2min,95u C for10min followed by45cycles of95u C for15sec,and60u C for1min.The polymorphism analysis was performed by two persons indepen-dently in a blinded manner.About10%of the samples were randomly selected for repeated genotyping for validation,and the results were100%concordant.The PCR products of the SNP with different genotypes were selected and verified by direct sequencing(Figure1).

Analysis of hsa-miR-27a expression

To further evaluate correlation between hsa-miR-27a expres-sion and rs895819polymorphism in vivo,25surgically removed normal renal tissue samples containing100%normal cells adjacent to tumors from patients of different genotypes were collected.Total RNA was extracted from these samples using Trizol Reagent(Invitrogen,Carlsbad,CA,U.S.)according to the manufacturer’s protocol.Applied Biosystems7900HT Real Time PCR System was used to perform real-time quantification PCR (Foster City,CA,U.S.)based on the SYBR-Green method (TaKaRa Bio,JPN).The primers used for amplification of hsa-miR-27a mRNA were59-ACACTCCAGCTGGGTTCA-CAGTGGCTAAG-39(forward)and59-TGGTGTCGTGGAGTCG-39(reverse),and the primers for U6were59-CTCGCTTCGGCAGCACA-39(forward)and59-AACGCTTCACGAATTTGCGT-39(reverse).All reactions were conducted in triplicate.Fold changes were normalized to the expression levels of U6.

Statistical analysis

Differences in the distributions of selected demographic variables and frequencies of genotypes between the cases and controls were evaluated by using the Student’s t-test(for continuous variables)or Pearson’s x2-test(for categorical vari-ables).Hardy-Weinberg equilibrium(HWE)of the controls’genotype frequencies was assessed by a goodness-of-fit x2test. The association between the SNP rs895819polymorphism and RCC risk were estimated by computing odds ratios(ORs)and their95%confidence intervals(CIs)from unconditional logistic regression analysis with the adjustment for possible confounders. The Kaplan-Meier method,log-rank test,univariate and multi-variate Cox regression analyses were used to evaluate the effects of pre-miR-27a genotypes on the overall survival of patients with RCC.P,0.05was considered statistically significant.All the statistical analyses were done with Statistical Analysis System software(9.1.3;SAS Institute,Cary,NC,U.S.)with two-sided P values.The statistical power was calculated by using the PS software(https://www.wendangku.net/doc/303027936.html,/twiki/bin/view/ Main/PowerSampleSize).

Results

Characteristics of the study population

The frequency distributions of selected demographic character-istics of the cases and controls are shown in Table 1.There were no differences between the patients and controls on age (P =0.642),body mass index (BMI)(P =0.074),sex (P =0.222),smoking status (P =0.127),pack-years of smoking (P =0.251)and drinking status (P =0.714).However,there were more hypertension patients (36.4%),and diabetics (13.5%)among the cases than among the controls (all P ,0.05).The majority of patients (84.0%)had conventional clear cell carcinoma.Other patients who had papillary carcinoma,chromophobe carcinoma and unclassified were counted ninety-five (16%).Approximately 63.5%of patients were in stage I,18.4%,6.7%,and 11.4%was found to be in stage II,III,and IV,respectively.In addition,the frequencies of nuclear grades from I to IV were 18.2%,48.0%,24.7%,and 9.1%,respectively.

Association between the pre-miR-27a polymorphism and RCC risk

Allele frequencies and genotype distributions of pre-miR-27a rs895819polymorphism in patients and controls are shown in Table 2.The observed genotype frequencies in the controls were consistent with that expected from HWE model (x 2=0.795,P =0.373).The frequencies distribution of G allele significantly differentiated from A allele among cases and controls (P =0.019).After adjusting for possible confounding factors (age,sex,smoking status,drinking status,hypertension,and diabetes),logistic regression analysis revealed that when comparing with AA homozygote,AG heterozygote was associated with a significantly

reduced RCC risk (adjusted OR =0.68,95%CI =0.53–0.87)and individuals with AG/GG genotype also had a reduced suscepti-bility to RCC (adjusted OR =0.71,95%CI =0.56–0.90).

Furthermore,in the stratified analysis by age,BMI,sex,smoking status,drinking status,hypertension and diabetes,we found that the reduced risk was more pronounced in young subjects (adjusted OR =0.56,95%CI =0.40–0.78),subjects with BMI #24(adjusted OR =0.67,95%CI =0.48–0.93),males (adjusted OR =0.66,95%CI =0.49–0.88),non-smokers (adjusted OR =0.71,95%CI =0.53–0.95),non-drinkers (adjusted OR =0.72,95%CI =0.55–0.95),subjects without hypertension (adjusted OR =0.61,95%CI =0.46–0.81)and subjects without diabetes (adjusted OR =0.69,95%CI =0.54–0.88)(Table 3).

Association between pre-miR-27a polymorphism and clinical characteristics of RCC

In addition,the association between pre-miR-27a rs895819polymorphism and the clinical characteristics of RCC was also examined.Results showed that AG/GG genotype was associated with reduced susceptibility in localized clinical stage (adjusted OR =0.71,95%CI =0.55–0.91)and similar effects were observed in well differentiated and poorly differentiated RCC (adjusted OR =0.71,95%CI =0.55–0.93for well differentiated,adjusted OR =0.51,95%CI =0.28–0.93for poorly differentiated)(Table 4).

Interaction analyses of rs895819polymorphism and risk factors

We have evaluated whether there were existence of interactions between rs895819polymorphism and age,BMI,sex,

smoking

Figure 1.DNA sequencing chromatograms of three different samples of PCR products confirmed rs895819polymorphism.(A)Double peaks labeled with an arrow represented the heterozygous genotype TC (AG).(B)Single peak labeled with an arrow represented the homozygous genotype TT (AA).(C)Single peak labeled with an arrow represented the homozygous genotype CC (GG).doi:10.1371/journal.pone.0046566.g001

status,drinking status,hypertension and diabetes.As shown in Table S2,compared with individuals who were both#57years and AA carriers,significantly reduced risk was observed in those who were#57years with AG/GG genotype(adjusted OR=0.57, 95%CI=0.41–0.79)and those who were.57with both AA and AG/GG genotypes(adjusted OR=0.64,95%CI=0.46–0.89for AA,adjusted OR=0.60,95%CI=0.42–0.85for AG/GG). Similar effects were found in AG/GG carriers which were ,24kg/m2,males,nonsmokers,nondrinkers,subjects without hypertension and subjects without diabetes when compared with AA carriers.We further observed rs895819had multiplicative interactions with age and hypertension(P interaction=0.035for age and P interaction=0.030for hypertension).

In addition,Kaplan-Meier curves and log-rank tests were used to assess the association between the genotypes and survival of RCC.However,we did not find the rs895819genotypes to be associated with the patients’overall survival.In the stepwise Cox regression,the results suggested that clinical stage was the best prognostic factor for RCC survival followed by tumor grade(data not shown).

Association of rs895819polymorphism with expression levels of hsa-miR-27a

We have collected25surgically removed normal renal tissue samples adjacent to tumors with different genotypes of rs895819, and the frequency distribution of the AA,AG,GG genotypes was 13,10,and2,respectively.We further assessed the association between the polymorphism and hsa-miR-27a expression levels using quantitative real-time RT-PCR.The samples with GG genotype was added to the samples of AG genotype for analysis. However,due to small samples of GG genotypes,we did not find any association between rs895819and the expression of hsa-miR-27a(P=0.08)(data not shown).Further functional studies were warranted when we collected large samples.

Discussion

In the present study,we investigated the association between the pre-miR-27a rs895819polymorphism and RCC risk.Considering relatively scarce frequencies of GG genotype,and similar scales of the ORs for heterozygote(AG)and homozygote(GG)the equality of that tested to observe a nonsignificant P-value of0.568 suggesting a dominant model,we combined AG and GG genotypes as a dominant genetic model in the following association analysis.We found that individuals with AG/GG genotypes were associated with a significantly reduced RCC risk,compared with those carrying AA genotype.In addition,the effect was even stronger in non-smokers,young subjects,males and individuals without hypertension or diabetes.Besides,the subjects carrying G allele also had a reduced susceptibility to localized clinical stage, well differentiated and poorly differentiated RCC.To our knowledge,this is the first study to investigate the role of the pre-miR-27a rs895819polymorphism in the risk of RCC. Nowadays,increasing studies have suggested that miRNAs, which play an important role in cancer progress as tumor oncogenes or tumor suppressors are involved in crucial biological processes,including development,differentiation,apoptosis and proliferation[12,21].Genetic variations in miRNAs have been reported to be related with many tumors,such as breast cancer [22],gastric cancer[23],colorectal cancer[24]and lung cancer [15].Though SNPs in miRNAs have been widely studied,the association between SNPs in miRNAs and renal cell cancer risk remains still unknown.Horikawa et al.defined7SNPs in7pre-miRNAs,and10SNPs in8pri-miRNAs,but none of them had a significant influence on RCC risk[25].Nevertheless,Horikawa et al.suggested that genetic polymorphisms of the miRNA-machin-ery genes might affect RCC susceptibility individually and jointly.

Table1.Distribution of selected variables between the RCC cases and control subjects.

Variables Cases

(n=594)

Controls

(n=600)P a n%n%

Age(years)(mean6SD)56.8611.957.8611.30.143

#5730551.430050.00.642

.5728948.730050.0

BMI(kg/m2)(mean6SD)24.062.823.863.00.094

,2429249.232654.30.074 $2430250.827445.7

Sex

Male37863.640267.00.222 Female21636.419833.0

Smoking status

Never37563.140467.30.127 Ever21936.919632.7

Pack-years of smoking

037563.140667.70.251

0–2015726.414123.5

.206210.4538.8

Drinking status

Never42771.943772.80.714 Ever16728.116327.2

Hypertension

No37863.643372.20.002 Yes21636.416727.8

Diabetes

No51486.556594.2,0.001 Yes8013.535 5.8

Stage

I37763.5

II10918.4

III40 6.7

IV6811.4

Grade

I10818.2

II28548.0

III14724.7

IV549.1

Histology

Clear cell49984.0

Papillary18 3.0

Chromophobe35 5.9

Others427.1

a Student’s t-test for age and BMI distributions between cases and controls;two-sided x2test for other selected variables between cases and controls.

SD,standard deviation;BMI,body mass index.

doi:10.1371/journal.pone.0046566.t001

SNP rs895819was found in pre-miRNA regions of hsa-miR-27a,which was in chromosome19,and it was located at position 40relative to the first nucleotide.It has been reported that a variation causing structural change in the crucial region of miRNA could affect the maturation and the process of miRNA[26,27]. We used the RNA-fold program to predict the most stable secondary structural of pre-miR-27a with two different sequences, but the G variation of rs895819affected neither the conformation nor the free energy of pre-miR-27a(data not shown).In a word,the processing and maturation of miRNA was more complex and subtle than predicted.Zeng et al.observed that Drosha selectively cleaves RNA hairpins bearing a large terminal loop.When the loop is shortened by mutation or deletions,the maturation process will be impaired[28].According to the predicted structure of pre-miR-27a,rs895819is located in the center of the terminal loop,the rs895819A.G transition may reduce the size of the loop. Consequently the cleavage by Drosha would be blocked,and the maturation process is thereby inhibited.In conclusion,rs895819

Table2.Genotype and allele frequencies of rs895819polymorphism among cases and controls and the associations with RCC risk.

Genotypes Cases Controls Crude OR(95%CI)Adjusted OR(95%CI)a P b n%n%

AA33456.228848.0 1.00(reference) 1.00(reference)

AG21335.926243.70.70(0.55–0.89)0.68(0.53–0.87)0.002

GG477.9508.30.81(0.53–1.24)0.78(0.51–1.21)0.274

AG/GG26043.831252.00.72(0.57–0.90)0.71(0.56–0.90)0.004

G allele30725.836230.20.81(0.67–0.97)0.019

P trend0.020

a Adjusted for age,sex,smoking,drinking status,diabetes and hypertension in logistic regression model.

b Two-sided x2test for either genotype distribution or allele frequency.

doi:10.1371/journal.pone.0046566.t002

Table3.Stratification analyses between the genotypes of rs895819polymorphism and RCC risk.

Genotypes(cases/controls)

Variables Cases/controls AA AG/GG AG/GG versus AA AG/GG versus AA P a

n%n%Crude OR(95%CI)Adjusted OR(95%CI)a

Total594/600334/28856.2/48.0260/31243.8/52.0

Age(years)

#57305/300164/11853.8/39.3141/18246.2/60.70.56(0.40–0.77)0.56(0.40–0.78)0.001

.57289/300170/17058.8/56.7119/13041.2/43.30.92(0.66–1.27)0.93(0.67–1.30)0.670 BMI

,24292/326160/14854.8/45.4132/17845.2/54.60.69(0.50–0.94)0.67(0.48–0.93)0.016 $24302/274174/14057.6/51.1128/13442.4/48.90.77(0.55–1.07)0.76(0.54–1.06)0.104 Sex

Male378/402211/18855.8/46.8167/21444.2/53.20.70(0.52–0.92)0.66(0.49–0.88)0.005 Female216/198123/10056.9/50.593/9843.1/49.50.77(0.52–1.14)0.80(0.54–1.18)0.258 Smoking status

Never375/404211/19556.3/48.3164/20943.7/51.70.73(0.55–0.96)0.71(0.53–0.95)0.023 Ever219/196123/9356.2/47.596/10343.8/52.60.71(0.48–1.04)0.69(0.46–1.03)0.068 Drinking status

Never427/437238/20955.7/47.8189/22844.3/52.20.73(0.56–0.95)0.72(0.55–0.95)0.019 Ever167/16396/7957.5/48.571/8442.5/51.50.70(0.45–1.07)0.67(0.42–1.05)0.079 Hypertension

No378/433212/19156.1/44.1166/24243.9/55.90.62(0.47–0.82)0.61(0.46–0.81)0.001 Yes216/167122/9756.5/58.194/7043.5/41.9 1.07(0.71–1.61) 1.00(0.66–1.52)0.997 Diabetes

No514/565292/26856.8/47.4222/29743.2/52.60.69(0.54–0.87)0.69(0.54–0.88)0.003 Yes80/3542/2052.5/57.138/1547.5/42.9 1.21(0.54–2.69)0.82(0.32–2.07)0.673

a Adjusted for age,sex,smoking,drinking status,diabetes and hypertension in logistic regression model.CI,confidence interval;OR,odds ratio.

doi:10.1371/journal.pone.0046566.t003

might play an important role in the maturation of miR-27a. However,due to we only had2individuals with GG genotypes,we did not find any association between rs895819and the expression of hsa-miR-27a,these findings need to be confirmed by further studies.

To our knowledge,many human miRNA transcripts have target sequences in the39-UTR to which miRNAs bind and exert posttranscriptional repression.Liu et al.found miR-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin [29]and Chintharlapalli et al.suggested that oncogenic miR-27a was a target for anticancer agent in colon cancer cells[30].Our results showed that the wild genotype AA of pre-miR-27a had higher frequency in cases than in controls,whereas individuals carrying variant G allele had a reduced RCC risk,indicating that miR-27a was more likely to be an oncogene,which was consistent with previous studies[29,30].Mertens-Talcott et al.reported that in breast cancer cells,transfection of antisense miR-27a lead to increased expression ZBTB10and these responses were accom-panied by decreased expression of survivin,Moreover,survivin is a structurally unique member of the inhibitor of apoptosis protein family that suppresses apoptosis and regulates cell division.Despite the redundancy of cell death pathways,survivin appears to be required for cancer cell viability,and interference with survivin expression/function has been associated with catastrophic defects of mitotic transition and induction of mitochondrial-induced cell death[31].Over-expression of survivin mRNA and protein were detected in RCC cell lines but not in normal human kidney epithelial cell line[19].Elevated expression of survivin was also observed in RCC tissues compared with adjacent normal tissues [19,32].RCC patients with high survivin levels had a significantly shorter overall survival time than those with low levels[33,34].In other words,decreased miR-27a levels might reduce the incidence of RCC through suppressing the expression of survivn indirectly. That was why we focus on the pre-miR-27a polymorphism in RCC patients.

In this study,there were more hypertension patients and diabetics among the cases than among the controls,indicating that there was potential association between the two factors and RCC. We also found that rs895819had multiplicative interactions with hypertension.It has been reported that certain types of renal tumors and cancer treatment could cause hypertension[35,36] and a history of diabetes has been linked to renal cell cancer risk in several cohort studies[37],but its role independent of those of obesity and hypertension has not been demonstrated conclusively. Besides,our results indicated that the individuals with G allele have a reduced RCC risk in non-smokers.Cigarette smoking is the most consistently established causal risk factor for RCC,account-ing for approximately20%of cases of RCC[38].Compared to never smokers,risk increased about50%in male and20%in female smokers[39].Furthermore,after stratification for RCC clinical stage,it appeared that rs895819GG genotype had a decreased risk of RCC with localized clinical stage.Thus,it was plausible that the variation was involved in the lower stage RCCs. However,this hypothesis needs to be confirmed in further studies. As to the association between genotypes and overall survival of RCC,there was no significant result.The reason for this discrepancy may be that the mortality rate of our survival analysis is low due to our short follow-up time and the biological mechanisms that underlie the incidence of RCC may be different from those underlying prognosis,and even if the miR-27a polymorphism is involved in the etiology of RCC,that does not necessarily mean that it affects the outcome.

When interpreting our results,several limitations should be concerned.Firstly,our case-control study was a hospital-based study and it could not provide a good representation of overall populations.Nevertheless,the G allele frequency in our controls was similar to that in the HapMap database.Besides,the genotype distributions of the polymorphism in our controls conformed to HWE.Thus,the selection bias was unlikely to be substantial. Secondly,our sample size was relatively small.However,we had 83%power to detect a minimal OR of0.68with an exposure frequency of30%under the current sample size.Thirdly,we did not perform the gene-environment interaction analysis due to lack of the detailed information of environment factors,such as occupational exposures,diet,and physical activities.However,our study also had several strengths.Firstly,it was the first study to investigate the association between pre-miR-27a rs895819poly-morphism and susceptibility to RCC.Secondly,we matched cases and controls by age and sex,and we obtained the results by adjustment of age,sex,smoking,drinking status,hypertension,and diabetes,which could reduce the influence of confounding factors. In conclusion,our results provided the first insight into the contribution of pre-miR-27a rs895819polymorphism to the risk of RCC in Chinese population.Although the association appeared to

Table4.Associations between rs895819polymorphism and clinical characteristics of RCC.

Variables AG/GG AA AG/GG versus AA AG/GG versus AA P a

n%n%Crude OR(95%CI)Adjusted OR(95%CI)a

Controls(n=600)31252.028848.0 1.00(reference) 1.00(reference)

Cases(n=594)

Stage

Localized(I+II)21343.827356.20.72(0.57–0.92)0.71(0.55–0.91)0.006 Advanced(III+IV)4743.56156.50.73(0.47–1.11)0.73(0.47–1.11)0.142 Grade

Well differentiated(I+II)17344.022056.00.73(0.56–0.94)0.71(0.55–0.93)0.010 Moderately differentiated(III)6846.37953.70.80(0.55–1.14)0.80(0.55–1.16)0.235 Poorly differentiated(IV)1935.23564.80.50(0.28–0.90)0.51(0.28–0.93)0.029

a Adjusted for age,sex,smoking,drinking status,diabetes and hypertension in logistic regression model.

doi:10.1371/journal.pone.0046566.t004

be statistically significant in our population,these findings should be further validated by larger,preferably prospective studies.For polymorphisms often vary in different ethnic groups,additional studies are also needed to clarify the association of this polymorphism with RCC risk in diverse ethnic populations. Supporting Information

Table S1The sequences of the primers and probe used to genotype the rs895819polymorphism.

(DOC)Table S2Interaction analyses of rs895819polymor-phism and risk factors.

(DOC)

Author Contributions

Conceived and designed the experiments:DS ZZ CY.Performed the experiments:DS PL LM DZ HC FY.Analyzed the data:DS HC FY WW MW.Contributed reagents/materials/analysis tools:NT PL CQ MW QL. Wrote the paper:DS PL LM ZZ CY.Critical review of manuscript:ZZ CY MW.

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对音乐的认识大学音乐鉴赏论文

浅谈对音乐的认识 系部:工程管理 班级:B120803 姓名:XXX 学号:B12080329 联系方式:157XXXXXXXX

一直以来我对音乐有种说不清道不明的情愫,音乐是人类最古老最具有普遍性和感染力的艺术形式之一。音乐以其独特、美妙的艺术魅力伴随人类历史的发展,伴随我们每一天的生活。聆听一场音乐会,会使我们陶醉其中,观看一场歌舞晚会,会使们激动万分。演奏家精湛的表演,令我们羡慕不已,歌唱家动听的歌声,令我们跃跃欲试,这是因为音乐是美的艺术。追求美是人类的天性,高层次的审美追求会使人的精神得到升华,情操受到陶冶,生活也将变得更加丰富而充实。 音乐是一种令人心旷神怡的艺术语言。音乐是情感的艺术,任何音乐都有它的情感色彩。音乐以其强烈的感情来撩拨人的情思,陶醉人的心灵。它通过声音的高低、强弱、长短、快慢、连断等来抒发变化多端的内心感情。如表现欢乐的情绪,音乐的节奏就明快;表达烦乱、惊惧的情绪,音乐的音响就呈现出不连贯、不规则的样态等。音乐,就是将几个不同的音符巧妙的连接起来,就组成了不同的灿烂之章。节奏是“音乐的生命”。韵律则是在节奏基础上的变化,给节奏以一定的情调贯穿而形成的。韵律在音乐中表现为起伏变化的旋律线条,使音乐节奏更具有鲜明性。同时,节奏感限制了音乐的特征,协调了音乐的内涵。 音乐与我们的生活是密切相关的,它不以地域而存在,也不以时间而消亡,它弥漫于天地间,参透于人群里。音乐是一种高雅的艺术,也是声音美味的品尝。音乐来源于生活,也反映着生活。音乐在生活中有着很大的作用,就好像人们需要用语言来与人沟通一样。很难想像,没有了音乐世界不知会变成什么样子?音乐已经在人们的日常生活中占据了一定的位置,音乐可以调剂紧张、单调的生活,使人们的神经得到放松,有益于身体健康。音乐可以给人听觉以愉悦,净化人的灵魂,使人们的精神得到一种高度的享受。音乐还能帮你排除忧愁和悲伤,使你从痛苦中解脱。医学上还用音乐来辅助治病,能起到药物所起不到的效果。德国伟大的音乐家贝多芬认为:音乐是比一切智慧、一切哲学更高的启示。谁能说透音乐的意义,便能超脱常人无以振拔的苦难。说明音乐具有感化人、塑造人、拯救人的作用。人们在进行强体力劳动时,为了减轻精神上的负担,发出“杭唷!嗨唷!”的声音,特别是在集体劳动时,更有用歌唱的节奏来统一步伐和着力点的作用。劳动号子就是这样产生的。另如持续时间较长的重复性劳动,为避免单调及精神上的疲劳,人们也会自然地发出种种歌声来调剂精神。如采茶、放牧、摇船、插秧等等,虽节奏并不一定与劳动动作合拍,但因有了歌唱的调节,就会使人感到轻松和减少寂寞感,枯燥感。当我们在非常愉快的时候,会一面唱着歌,一面手舞足蹈地跳着舞。当我们在非常郁闷时,忽然一支优美动听的旋律飘至耳畔。烦恼、不快立刻烟消云散,无有踪迹。 音乐是心灵的语言,它可以放射出人类追求光明灿烂理想的光芒。一首好的乐曲,像一幅美丽的图画,也像神话般的故事,启迪鼓舞着孩子们的心灵,追求光明,创造幸福,创造未来,创造新世界。音乐能够表现出一定的音乐形象。当我们给孩子唱摇篮曲的时候,孩子会安静地合着小手将头侧睡在小手上;当我们给孩子唱欢快的歌曲时,孩子会不自觉手舞足蹈,随着音乐而起舞;当我们给孩子唱一些进行曲时,孩子会大步走向前,迈着坚定而有力的步。 音乐是我们生活中传递情感的纽带。如著名的小提琴协奏曲——《梁祝》梁祝小提琴协奏曲是陈钢与何占豪就读于上海音乐学院是的作品,作于1958年冬,翌年5月首演于上海获得好评,首演与俞丽拿担任小提琴独奏。题材是家喻户晓的民间故事,一跃居中的曲调为素材,综合采用交响乐与我国民间戏曲音乐表现手法,依照剧情发展精心构思布局,采用奏鸣曲式结构,单乐章,

交响乐心得体会

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人的轻快的美妙的旋律。歌词也没在我脑海中留下太多的印象。我想这就是音乐的神秘处吧,她让你记住的只是她的“美的所在”。 然而,在开始的歌剧欣赏上,我感觉在课程的学习过程中,最让我感觉枯燥的内容是那些我听不懂得语言和那些听起来还没京剧那么好听的旋律。再加上有些没有中文翻译,更使我看不太懂,只能看出几个人在那儿吼。但是,这并没有影响我对音乐知识渴求的欲望,后来反而增加了我学习的兴趣。那便是我着重去听它的旋律和节奏,我不会再因为听不懂语言而否定了那首曲。因为我知道,一种情绪是通过旋律和声音及演唱者的整体舞台表现来传递给观众的。 在课上,通过了解,我知道了歌剧是将音乐、戏剧、文学、舞蹈、 舞台美术等融为一体的综合性艺术,通常由咏叹调、宣叙调、重唱、合唱、序曲、间奏曲、舞蹈场面等组成。由歌剧的定义我们就能知道歌剧其实就是个“集大成者”,它集各种艺术与一身,要想练好一部歌剧情节远比练好一首流行音乐要难多了。它也可以说是电影与音乐的结合,电影里的故事基本上是用评述的语言来表达的,而在歌剧中则是用“唱”的形式和观众交流的。那是一种由美声开始的心灵的交流。时间过得很快,一个学期的选修课就要结束了。我认为我对歌剧艺术欣赏的能力还是有限的,还是有不足之处的。当然,这学期我也学到了我想学的知识。虽然学习的时间不是很长,但我的收获却非常多。最后,我想说:“能把选修课上的比专业课还好,也只有黄老师您了。篇二:交响乐鉴赏学习心得 交响乐鉴赏学习心得

交响乐欣赏论文-浅谈对古典音乐的理解

交响乐欣赏论文:浅谈对古典音乐的认识

摘要 蓝色多瑙河作为维也纳新年音乐会必奏的曲目,可以说是耳熟能详了,蓝色的多瑙河水已随着每个新年的第一声钟响流进了每一个热爱音乐的人的心中,但每一次听还是那么令人陶醉,尤其在阶梯教室这种新环境里,和众多听众一起,透过拉得不严实的窗帘投进几缕阳光,听着这首管弦乐曲,别有一番感受…… …… 曾经弹过钢琴王子理查德克莱德曼以第五交响曲改编的钢琴曲《命运》,单薄的声音,轻快的节奏,改编之后让第五交响曲失去了排山倒海的气势,丢失了高亢激昂的神韵。是的,任何改动都将破坏这部垂世之作,它是杰出的、无法超越的! …… 一千个读者就有一千个哈姆雷特,如此优美的音乐,无需深入剖析所谓的意境,闭上眼,静静聆听,思绪任飞,脑海里浮现出的各种画面和场景,无论是天鹅,亦或是少女,那便是这部美妙的乐曲带我们前往的精神乐土…… …… 关键词:西方音乐古典音乐交响乐蓝色多瑙河第五交响曲天鹅湖

蓝色多瑙河圆舞曲 作曲家:小约翰·施特劳斯 创作时间:1866年 把这首曲子放在第一个来写,一是因为这首曲子是这学期最先欣赏的曲子,先入为主,于是印象也最为深刻。还有一个原因,小施特劳斯是我最喜欢的天才音乐家,就连家里的钢琴都选的是strauss。言归正传,蓝色多瑙河作为维也纳新年音乐会必奏的曲目,可以说是耳熟能详了,蓝色的多瑙河水已随着每个新年的第一声钟响流进了每一个热爱音乐的人的心中,但每一次听还是那么令人陶醉,尤其在阶梯教室这种新环境里,和众多听众一起,透过拉得不严实的窗帘投进几缕阳光,听着这首管弦乐曲,别有一番感受。为了写这篇论文,我又戴上耳机,再一次从头到尾仔细聆听这部伟大的作品,试图第一次捋清它的脉络。整首乐曲好象是多瑙河的水波在轻柔地翻动,河水一般从宁静平稳到轩然大波,浪花飞溅,显示出多样化的变换。最开始听起来向悠扬的笛声从远处传来,呈现出一派生机勃勃的景象,明显的小提琴的声音给出了轻微徐缓的背景,仿佛朦胧晓雾中的潺潺流水,轻轻荡漾,辽阔开朗,引人遐想,简短和谐的呼应,似乎是远处的回想。音乐渐渐响亮高昂,似乎东方发白,随之而来的明亮的旋律,又像旭日冉冉升起,进而喷薄四射、光照大地,带来了光明和温暖,我似乎看到人们情不自禁地聚集在河岸上,满怀希望和欢欣,迎接黎明。我听了这首作品后,回味无穷。微微地闭上眼睛去感受那多瑙河的美景,看碧绿的碧波,在阳光的照耀下波光粼粼,微风吹来,夹杂着青草的香味……多么令人心旷神怡,这美景所有的一切在阳光的沐浴下充满了生机,充满了活力。的确,这曲蓝色多瑙河让我置身于静美的清晨,让我在那多瑙河旁,无所束缚地自由漫步,享受纯净的大自然赋予我们的惊奇和惊喜。 c小调第五交响曲 又名命运交响曲 作曲家:路德维希·凡·贝多芬 创作时间:1805年末至1808年初 提到交响乐,我脑海里首先浮现出的一定是命运交响曲,相信多数人都和我一样。正如老师上课播放这部作品之前介绍的,“此曲声望之高,演出次数之多,可谓交响曲之冠”。在我眼里,它就是古典音乐的标志,是这篇题为“浅谈对古典音乐的认识”的小论文中不可能不提及的内容。那由四个音构成的简短有力的片段,可以说是每个人都耳熟能详的旋律了,在交响曲一开始就轰然而呜。“这是命运在敲门”。用命运的敲门声去布下悲惨的罗网,不朽的命运的主题就展露无遗,展现出一幅去抗争、去搏斗、去与命运交战的宏伟画面。那充溢着含蓄的活力的部分好像略带点神秘感。似乎在多次抗争中,销声匿迹了,天地似乎处于一片寂静。这时人们屏住呼吸,等待着胜利的瞬间,也是人们对斗争中牺牲的英雄的无声哀悼。这个段落,最好的描述便是听得见的寂静。刹那间,静寂的长音在鼓点的伴衬下,送来渐强的力度,我真的没有想到一个作曲家在失去听觉的情况下,能写出这样有震撼力的交响曲。这里必定有他内心深处的思想发展,和他的隐忧、他被人抑制的愤怒。他将自己的对命运含愤的抗争、对幸福深情的憧憬,全部倾诉于音乐。于是,在终曲揭幕时,灿烂的阳光、潮涌的人群、鼎沸的笑声,迸发出震撼的胜利凯歌。太阳升起来了,凯旋的歌声响起来了,所有的忧郁在这种欢呼声的激流之前都消失得无影无踪了。从绝望到斗争,从斗争到平静,从平静到凯旋。我从未有听这部交响曲时的如此清晰而深刻的感受。尽管贝多芬的耳朵使他无法聆听自己音乐那撼人的音响,然而他能在头脑中更生动地想像的这一切。这一切如此准确

交响乐欣赏心得与感受

作者:王力学号:PB09210281 音乐,对于我来说的确是一个熟悉又陌生的东西。生活中, 无处不存在音乐,而对于生活在现代的我们更是沐浴在各式各样 的音乐中,交响乐、古典以及流行等等;但是没有乐感也没有任 何乐理知识,又让我觉得音乐是那么陌生那么遥不可及。抱着试 一试的态度这学期我选了交响乐欣赏这门课。经过了一学期的学 习在欣赏音乐方面我的水平有了很大的提高。 交响乐给我的第一感觉就是“吵”,感觉她不能让我静下心 来聆听,但慢慢地我发现交响乐就是没有杂质的艺术,那是天籁! 少了现代生活中的浮躁,进入那些艺术大师为我们构造的另外一 个世界的时候,音乐响起,我的内心也会不由自主地随着音乐的 旋律颤动,曾经,在哪里听过,似曾相识的感觉让人感觉美妙无 比,虽然不懂,但伴随着悦耳动听的声音,会不由自主地想象着 万马奔腾,想象着小泉叮咚,想象着情意绵绵,想象着心潮澎湃, 想象着一切可以想象的,天马行空…… 听得多了也就知道了应该怎么去欣赏了。我觉得音乐跨越了 时空,带着乐符和旋律来到我们身边,一首乐曲,就是一段心情。 乐曲中携着心情,心情中含着乐曲,套用王国维的那句话就是: 带着心情去听音乐,那音乐就全是抒发着自己的心情了;听着音 乐去找心情,那心情里面便全是音乐了,因此这时就不知何者为 心情何者为乐曲了。当我们真正进入了音乐之后,每一个音符的 跳动都会带来心灵最深处的颤动。作为二十一世纪的新新人类,

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交响乐赏析课程学习的总结与思考 序号:10 班级:车辆15-1 姓名:李云辉 学号:150602131 上课班级:周二晚7点—9点 实习:无缺课 手机: 日期:2016年6月18日

目录 第一节课:古希腊人音乐思想及前言 (1) 第二节课:巴洛克乐风 (4) 第三节课:音乐是人类本质精神 (6) 第四节课:古典主义精神 (9) 第五节课:奏鸣曲式(交响曲的第一章) (11) 第六节课:浪漫主义乐派 (13) 第七节课:浪漫主义(补充) (15) 第八节课:民族乐派 (16) 第九节课:交响诗与印象主义 (18) 第十节课:交响序曲与歌剧序曲 (20) 第十一节课:现代主义音乐 (23) 第十二节课:中国传统音乐审美特征 (25) 后记: (28)

古希腊人音乐思想及前言 艺术的本质是对事物本性的把握。正因为这样,艺术往往口述心传。艺术是一种只能意会不能言传的感知,不同于科学,可以是实体或者是一般自然规律或者自然现象。而与宗教相类比,两者都是追求精神愉悦,而不是物质满足。 大学音乐包括了三个性质:知识性、感受性、观念性。我们所学的是交响乐赏析,实际上是音乐鉴赏,并不是在专业学习的角度学习,而是从文化角度鉴别与鉴赏音乐,感知音乐之美。音乐鉴赏是从审美、历史、文化解析音乐,从人文艺术角度、从人性角度出发,欣赏与鉴别音乐。音乐由人创造,是从良好出发,具有历史心同时具有善的特性。音乐促进身心成长;音乐启迪人的智慧;音乐点亮人的生活。罗曼罗兰曾说过:艺术的伟大意义,基本上在于它能显示人的真正感情、内心生活的奥秘和热情的世界。 音乐的发源均有对应的古代文明,不仅包括城市文明,也包括了自然文明。欧洲音乐是有系统的、完善的技术体系与理论体系,是专业音乐的典范,孕育出的最高形式的音乐就是交响乐。而欧洲文明的发源就是希腊文明与罗马文明。交响音乐的精神品德追溯至古希腊,大概于十七世纪萌芽,十八世纪确立。交响乐简单来说就是数音和鸣。两个以上的单字音,多个音乐和谐共鸣。交响乐中包含了两个以上的音色、旋律、力度。“不同而和,和而不同。”交响乐来源于古希腊人的自由精神,是一种自由的艺

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