| Background;Breast cancer is the most common cancer in women worldwide.Approximately 5%-10%of breast cancer cases are inheritable and a majority of breast cancer cases are sporadic[1].Large scale genomic profiling has revealed the molecular landscape and breast cancer driver genes[5-6],which could not basically explain the occurrence and development of breast cancer.At present,genetic diagnosis and targeted therapy are progressively adopted and get good effects in specific breast cancer patients[7-8].The prognosis of some subtypes?such as basal-like breast cancer?is still poor[9 10],which still requires further research on the causes of breast cancer.Cancer is the manifestation of both genetic and epigenetic modifications?13-15?.There have been many studies on the genetic changes with breast cancer,including gene deletion,mutations,chromosomal variation,and aneuploidy.Low frequency and rare genetic variants with large effects may account for the missing heritability for cancer[12].Epigenetics is defined as heritable changes in gene expression activity and expression that occur without alteration in DNA sequences but which are sufficiently powerful to regulate the dynamics of gene expression[16].Epigenetic changes in cancer are generally thought to be brought about by alterations in DNA and histone modifications that lead to the silencing of tumor suppressor genes and the activation of oncogenic genes,and thus cell phenotype.In addition to genetic alterations such mutations in oncogenes and tumor suppressor genes,epigenetic alternations such as promoter methylation and histone modification could also lead to initiation,promotion,and metastasis of breast cancer[15]Recognizing that carcinogenesis involves both genetic and epigenetic changes has led to a better understanding of the molecular mechanisms that dominate the development of cancer and to improvements in diagnosing and predicting the outcome of cancer.Epigenetics may play a role in drug intervention and treatment.Emerging evidence suggest that epigenetic factors also contribute to the development of breast cancer[20-22]Sirtuins are a highly conserved family of nicotinamide adenine dinucleotide?NAD+?-dependent protein lysine modifying enzymes shown to play biological and physiological roles in diverse cellular processes such as metabolism,transcription,and DNA repair.SIRT2 is NAD+-dependent class ? deacetylases,a member of sirtuin family.and mainly localized to the cytoplasm,but also can move to the nucleus during mitosis[31].SIRT2 was found to deacetylate histone H4 lysine 16?H4K16?mediating chromatin condensation during G2/M transition[106 108]SIRT2 stabilizes N-MYC and c-MYC protein by downregulating ubiquitin-protein ligase NEDD4 expression[70].SIRT2 has been involved in mitotic progression,oxidative stress,metabolism,microtubule dynamics,cell migration,apoptosis and differentiation.To date,Increasing evidences have suggested that SIRT2 is involved in tumorigenesis[39-42].The expression of the SIRT2 gene were different in different tumors[56-58].t has been reported that SIRT2 has two opposing roles,either as a tumor suppressor or an oncogene[25 62].A deficiency of SIRT2 in a murine model causes the development of gender-specific tumorigenesis,with the development of mammary tumors in females,and hepatocellular carcinoma?HCC?in males[38].Although more and more studies further support the tumor-suppressive role of SIRT2,it need to mention that SIRT2 has been shown to exert dual functions where Sirt2 may also have tumor-promoter qualities[19 40].The level of SIRT2 was more frequently decreased in human breast tumors[38]It was also reported that SIRT2 genes are up-regulated[75].Several SIRT2 inhibitors have also been reported to have anticancer effects[39 104 109].The role of SIRT2 in the development of breast cancer is complex,The molecular mechanisms related to SIRT2 gene expression still remain unclear.Functional DNA sequence variants?DSVs?is a rare epigenetic phenomenon,the emergence of DSVs leads to changes in the function of downstream genes,affecting the occurrence and development of diseases.In previous studies,we have genetically and functionally investigated the members of sirtuin family in AMI patients,including SIRT1,SIRT3 and SIRT6.A number of functional DNA sequence variants?DSVs?within their promoters have been identified and linked to AMI[80-82].We also demonstrated that functional genetic variants within the SIRT2 gene promoter in acute myocardial infarction[83].Because of the complex relationship between SIRT2 and breast cancer mentioned above,We speculated that the DNA sequence variants?DSVs?in the SIRT2 gene promoter may influence expression of SIRT2 gene and be involved in the tumorigenesis of breast.Obj ective:SIRT2 gene promoter sequences were sequenced to investigate genetic sequence variation in breast cancer and healthy control groups in SIRT2 gene promoter,and explore the relationship between these DSVs and breast cancer.Then,by constructing SIRT2 gene variant sites and wild The pGL3-basic reporter vector of the promoter detects changes in gene expression levels caused by genetic variation within the SIRT2 gene promoter.The role of SIRT2 gene in the development of breast cancer was investigated by studying the genetic and functional variation of the SIRT2 gene promoter region.Methods:1.In this study,200 patients with breast cancer were enrolled in the experimental group,and 184 healthy controls were included in the control group.Clinical data were collected from these two groups,and then whole genome DNA was extracted.1.1 All female breast cancer patients?n=200,age range from 26 to 81 years,median age 50.00 years?were recruited during the period from April,2014 to June,2017,from Division of Breast Surgery,Affiliated Hospital of Jining Medical University,Jining Medical University,Jining,Shandong,China.Breast cancer patients were diagnosed by two senior pathologists,including different subtypes,42 luminal A,93 luminal B,33 HER2-Enriched,26 triple negative and 6 ductal carcinoma in situ?DCIS?.All breast cancer patients had no family breast cancer history?Table 1?.1.2 Ethnic-matched healthy female controls?n=184,age range from 23 to 82 years,median age 52.00 years?were recruited from the same hospital during the same period.The controls with family history of breast diseases were excluded from this study?Table 1?.2.The PCR primers were designed according to the promoter sequence of human SIRT2?NCBI:NC000019.10?provided by the NCBI gene database,and the transcription start site?TSS?of the SIRT2 promoter sequence was located at 38899862?+1??Table 2?.The 1292 base pair sequence selected upstream of the TSS was synthesized and sequenced,identified and analyzed.The target fragment of the SIRT2 gene promoter was amplified by PCR,and the gene sequence variation of SIRT2 was statistically analyzed after direct sequencing.To ensure the accuracy and reliability of the sequencing results,the selected promoter segments were truncated into two overlapping target fragments for sequencing.3.This study will analyze the SIRT2 gene promoter DSVs found in the breast cancer group in the JASPAR program?http://jaspar.genereg.net/?,and speculate whether these DSVs will affect the putative transcription factor binding site and thus influences tumorigenesis of breast cancer.4.The variant and wild-type SIRT2 gene promoter sequences were constructed by gene cloning into the luciferase reporter gene expression vector?pGL3-basic?,including empty pGL3-basic?negative control?,pGL3-WT?wild-type SIRT2 gene promoter?),pGL3-38900839C,pGL3-38900478A and pGL3-38900291G.After co-transfected into cultured human embryonic-kidney cells?HEK293?,human breast adenocarcinoma cells?MCF-7?and invasive ductal carcinoma of the breast cells?BT-474?by lipofectamine.The cells and the dual luciferase were collected,and the ratio of firefly luciferase activity/renorin luciferase activity was calculated using a dual fluorescence reporter system,and the wild type promoter was set based on the transcriptional activity of the wild type SIRT2 gene promoter,The transcriptional activity was 100%.and the relative transcriptional activities of SIRT2 gene variant promoters?DSVs?were analyzed by using dual-luciferase reporter assay system on a Promega Glomax 20/20 luminometer.Results:1.Sequencing resultsWe analyzed the occurrence of DSVs in breast cancer patients and controls.Eight DSVs,including three single-nucleotide polymorphisms?SNPs?,were identified in this study.Locations and frequencies of the DSVs and SNPs were depicted in Figure 3 and summarized in Table 3.Two novel heterozygous DSVs?g.38900839G>C and g.38900478G>A?were identified in two breast cancer patients?41 and 50 year old,respectively?,but in none of controls.Both the breast cancer patients were luminal B subtype.The DNA sequencing chromatograms of these novel DSVs were shown in Figure 3.Three heterozygous DSVs,g.38900413A>C,g.38900030G>A and g.38899852C>T,were only found in controls.In addition,three SNPs,g.38901007delT?rs10713585?,g.38900291C>G?rs2053071?and g.38900145C>T?rs116900177?,were found in both breast cancer patients and controls.The SNP,g.38900145C>T?rs116900177?,was more seen in controls than breast cancer patients?P<0.05?.Sequencing chromatograms of these DSVs and SNPs were shown in Figure 3.2.JASPAR program analysis resultsTo determine whether DSVs affect putative biding sites for transcription factors,the SIRT2gene promoter was analyzed with JASPAR program?http://jaspar.genereg.net/?.The DSVs identified in breast cancer patients may abolish,create or modify the putative binding sites for transcription factors.Based on the analysis results of JASPAR program,we hypothesized that the low frequency DNA sequence variation found in the SIRT2 gene promoter may alter some of the original transcription factor binding sites or affect the efficiency of binding of related transcription factors,thereby affecting the expression level of SIRT2 gene and participating in the development of breast cancer.3.The reporter gene pGL3-basic vector was successfully constructed in this experiment.The vector types were:empty pGL3-basic?negative control?,pGL3-WT?wild type SIRT2 gene promoter?,pGL3-38900839C,pGL3-38900478A and pGL3-38900291G.?SNPs with similar frequency of occurrence in the two groups were>0.05?.The results are shown in Figure 5 and Figure 6.4.The wild-type and variant SIRT2 gene promoter sequences were cloned to construct a luciferase reporter gene expression vector?pGL3-basic?to generate an expression vector,including empty pGL3-basic?negative control?,pGL3-WT?wild type?SIRT2 gene promoter),pGL3-38900839C,pGL3-38900478A and pGL3-38900291G.Cell lines cultured by liposome transfection in vitro,HEK-293 cells?human embryonic kidney cells?,MCF-7 cells?human breast cancer cell line?and BT-474 cells?breast cell invasive ductal carcinoma cell line?.After that,the cells and the dual luciferase were collected,and the ratio of firefly luciferase activity/renorin luciferase activity was calculated by a dual fluorescence reporter system,and the wild-type SIRT2 gene promoter was used as a reference,and the transcriptional activity was 100%,The relative transcriptional activity of other DSVs of the SIRT2 gene promoter was calculated?Figure 7?.?1?The DSVs?g.38900478G>A and g.38900839G>C?that were identified only in breast cancer patient significantly decreased activity of the SIRT2 gene promoter?P<0.01 for both DSVs?in HEK-293 cells.The SNP,g.38900291C>G?rs2053071?,found in both breast cancer patients and controls with similar frequencies did not also change activity of the SIRT2 gene promoter?P>0.05??Figure7?.?2?To further investigate the effects of the DSVs in breast cancer cells,we examined the transcriptional activity of the DSVs found in breast cancer patients?g.38900839G>C and g.38900478G>A?in BT-474 cells and MCF-7 cells.Both the DSVs significantly decreased the activity of the SIRT2 gene promoter in BT-474 cells and MCF-7 cells?P<0.01?.Similarly,the SNP,g.38900291C>G?rs2053071?,did not significantly alter the activity of the SIRT2 gene promoter?P>0.05??Figure 7?.Taken together,the DSVs identified in breast cancer patients altered the activity of the SIRT2 gene promoter in HEK-293 cells,MCF-7 cells and BT-474 cells,indicating that their effect on SIRT2 gene promoter were not tissue-specific.Conclusion:In this study,genetic and functional analysis of the human SIRT2 gene promoter in breast cancer patients and healthy controls was performed.Two novel DVSs of the SIRT2 gene promoter were identified in breast cancer patients.The novel DSVs identified in breast cancer patients significantly altered the transcriptional activity of the SIRT2 gene promoter in cultured human embryonic kidney cell lines and breast cancer cell lines,indicating that the effects of these two novel DSVs on the SIRT2 gene promoter are not tissue-specific.This experimental data provides supporting evidence that SIRT2 may act as a breast tumor suppressor.The SIRT2 gene promoter DSVs may be a rare risk factor for the development of breast cancer by altering the transcriptional activity of the SIRT2 gene promoter and altering the expression level of SIRT2.Our experimental results expand the risk factors for breast cancer from the perspective of epigenetics,and provides a genetic basis for translational and targeted therapeutic studies for breast cancer patients.These results also require a larger sample to study the occurrence of SIRT2 gene promoter DSVs in breast cancer patients.Our laboratory is further investigating how DSV affects the regulation of SIRT2 gene expression.The expression and regulation mechanism of SIRT2 in tumorigenesis of breast cancer needs further research. |
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