The Study Of SET Mediated Biological Behaviors Of Breast Cancer Cells In Vivo And Vitro

BackgroundBreast cancer is a common malignant tumor in women. The incidence of breast cancer has shown an rising trend in recent years in China and already took the first incidence of female malignant tumors in large cities. Breast cancer is a systemic disease, the occurrence and development of breast cancer is a complex process of multi factors and multi-step process, such as physical factors, chemical factors and gene mutation and chromosome abnormality, etc. Growth factors and cytokines, hormones and other bioactive substances involved in breast carcinogenesis and cell transfer process. The abnormal of these bioactive substances mediated signal transduction pathway will cause the amplification of some genes excessively, which will result in normal cell receive abnormality signals of proliferation and differentiation and finally lead to cancer. Despite the incidence of breast cancer is high, the death rate is falling. The reason is not only the established in breast cancer screening and early diagnosis system of female, but with the development of molecular biology technology and the rising level of comprehensive standardization diagnosis and treatment.Epigenetics study of breast cancer mainly includes:histone modification, DNA methylation and non-coding RNA. Histone is alkaline protein that exists in eukaryotic cell chromatin and containing arginine and lysine and other alkaline amino acid. There are many kinds of histone modifications, including histone terminal phosphorylation ubiquitination acetylation and methylation of glycosylation modification. DNA methylation refers to the CG two nucleotides cytosine of DNA adding methyl selectively and forms five-methyl cytosine through catalyzed by methyltransferase. Non-coding RNA (microRNA and miRNA) is composed of 18 to 24 nucleotides of non-coding single small molecule RNA and regulate development phase. It exists in animals and plants and participated in a series of important process in the course of life. Non-coding RNA is widespread in the animal and plant body and participated in a series of important process in the process of life such as cell proliferation and differentiation and the regulation of gene expression.The development of breast cancer involves multiple signal transduction pathways, mainly include the Notch signal transduction pathway, ER signal transduction pathways, receptor tyrosine kinase (RTK) mediated signal transduction pathways and the Wnt signaling pathway. Notch signal pathway is composed of receptors, ligands and CSL-DNA binding protein. Notch is a both simple and complex signal pathway. The change of Notch signaling will lead to the occurrence of tumor and its role varies in different tissue cells, and even the effect of the different development stages in the same tissue cells is different. ER is one of the steroid hormone receptor superfamily member, including the classic ER-a and newly discovered ER-β. ER mediated signal pathways can regulate the growth and development and cell proliferation of breast. RTK exists in all multicellular animals, participate in a variety of regulating cell activity, especially involved in cell growth and differentiation. RTK family was divided into many sub categories:EGFR family, insulin receptor (IGF), vascular endothelial growth factor receptor (VEGFR), etc. VEGFR family has the closest relation with breast cancer. Wnt signal pathway not only participated in the development of mammary gland in early embryonic, but resulted in breast cancer when it was abnormally activated and promote cell proliferation, migration and invasion of tumor cells, resistance to apoptosis, and also related to the tumor cell resistance to chemotherapy drug.SET protein is found in a leukemia patients named SE in 1992 for the first time. SET has multiple names such as template activation factor-1beta (TAF-1β), protein phosphatase-A inhibitor (I2PP2A), histocompatibility leukocyte antigen II related protein (PHAPII), DNA enzyme activation inhibitors activated by telomerase A (IGAAD), and SET is the generic name. SET protein has many important functions due to its highly conserved structure characteristics and evolution mechanism. SET is widely expressed in different tissues and human cell lines refer to lung, brain. embryonic tissues, and uterus and so on. SET cDNA have been cloned successfully in those tissue cells and the expression level is similar. SET can inhibit the activity of the protein phosphatase 2A (PP2A) efficiently and specifically. PP2A is an important tumor suppressor involved in many kinds of cell biological events such as cell cycle, DNA replication, signal transduction, cell differentiation and cell malignant transformation and so on. SET could complete many biological functions through inhibit PP2A. SET can inhibit histone acetylation and promote some nuclear receptor-mediated gene transcription. This inhibition process is mainly due to the SET combined with TAF-1α and pp32 formed INHAT complexe, then combining INHAT structure domain with histones and prevent the union of histones and histone acetyltransferase, and finally inhibit histone acetylation. SET protein can promote adenovirus DNA replication, which involved in the combination between host SET and naked DNA and nucleoprotein complexes. As a protein molecular chaperone, SET still has the function of nucleosome assembly and plays an important role in chromatin transcription process. SET is involved in apoptosis, which may be completed through inhibit the activation of DNA enzyme NM23-H1 and the assembly function of nucleosome. SET is associated with many and high expression in various tumor tissues.RNA interference (RNA interference, RNAi) is a new technique developed in recent years and is a specific, efficient and economic method of inhibiting gene expression. RNAi mainly through RNAase family nuclear complex enzyme Dicer cut dsRNA into small interfering RNA, siRNA could mediate the homologous mRNA molecules expression and result in gene silencing. As a new experiment means, RNAi technology has been rapidly applied in gene therapy and functional genomics research and so on. RNAi has several characteristics:sequence specificity, high stability, double interference system, transitivity and hereditary.To identify the role of SET protein in breast cancer, this study detected SET protein expression in breast cancer tissues firstly. Then we tested the effect of stable knockdown SET on growth and migration of breast cancer cells. This study further analyzed the biological effect of SET silencing on animal through nude mice model. Our study would help to reveal the molecular mechanism of the occurrence and development of tumor, and provide markers and therapeutic targets for the diagnosis and treatment of tumor.MethodsⅠ. The SET protein expression in clinical breast cancer tissue samples1. Western blot detect SET expression in tissuesApply appropriate patient tissue samples, liquid nitrogen grinding after adding protein lysate, after fully cracking centrifugal and collection supernatant protein, protein quantitative according to the kit instructions, add appropriate proportion of the sample buffer, boil degeneration. SDS-PAGE electrophoresis, transfer film, closed with 5% skimmed milk powder, take GAPDH as internal reference protein, add the first antibody and incubation overnight a 4℃. Wash membrane with TBST at the second day, plus the second antibody and incubation for 1 hour, wash membrane with TBST, take picture with the CCD imaging system and analyze the density of the band with ImageJ software, compare the changes of protein expression.2. Immunohistochemical (IHC) detect SET expression in tissuesApply appropriate patient tissue samples, fixed with 4% paraformaldehyde, gradient ethanol dehydration, paraffin embedded tissue samples. Cut into 5 micron tissue sections, xylene dewax, gradient alcohol rehydration, hydrogen peroxide neutralize the catalase of tissues, repair antigen in heated citrate buffer, normal goat serum closed, add the first antibody and incubation overnight a 4℃. The next day add the avidin binding second antibody according to the antibody instructions, DAB staining, hematoxylin staining, neutral gum seal, air-dried and viewed with microscope.Ⅱ. The construction of SET stable knockdown in breast cancer cell lines1. Construction and identification of shRNA expressing vectors targeting SET geneDesign SET specific siRNA target sequence after find the SET mRNA sequences in GENBANK, confirmed the specificity with BLAST homology analysis, synthesis two shRNA single-stranded DNA template, connected to the double enzyme cutted pLVX-shRNA1 vector after annealing. The product transformed by enzyme digestion and sequence identification. Amplification and extraction of recombinant plasmid after identification correctly.2. Packaging recombinant plasmid with LentivirusLentivirus packaging with 293T cells according to the lentivirus packaging kit instructions, collecting virus supernatant, titer detection.3. Construct SET stable knockdown breast cancer cell linesTransfer virus liquid into target cells in proper ratio mixture with cell culture medium, screening for 15 days with puromycin. Extracted cell protein and quantitative, Western blot detect SET protein expression. Extracted cell total RNA, reversed transcription for cDNA, determinated SET mRNA expression by RT-PCR.Ⅲ. Effect of knockdown of SET expression by RNA interference on biological behavior of breast cancer cell lines1. Determined cell growth by MTT methodSeeded each group of cells in 96-well plates, drawing standard curve by MTT method, establish linear relationship. Testing for 7 days and drawing cell growth curve, calculate the cell doubling time according to the formula.2. Detected cell apoptosis with Flow cytometrySeeded each group of cells in 6-well plates, collect cells after adherence, processing cells according to Annexin V-FITC cells apoptosis detection kit, detecting apoptosis with Flow cytometry.3. Cell scratch experiment tested the migration of tumor cellsSeeded each group of cells in 6-well plates, vertical scratch lines after cell adherence, observe and take photo at 0 h,12 h, and 24 h respectively.4. Cell invasion detectionCulture each groups of breast cancer cell lines, using Transwell chambers vaccination cells, through counting cell number those pass through cell membrane and detection the change of invasion of tumor cells.Ⅳ. Effect of stable knockdown SET expression by RNA interference on nude mice model1. The preparation of nude mice modelNude mice were randomly grouped, sterile injection each groups of breast cancer cell lines, measuring tumor volume every five days after the success of the tumorigenic, after 30 days euthanized mice and took out the tumor tissue and measured the weight of tumors tissues.2. Western blot detect SET expression in tumor tissuesApply appropriate tumor tissue samples, liquid nitrogen grinding after adding protein lysate, after fully cracking centrifugal and collection supernatant protein, protein quantitative according to the kit instructions, add appropriate proportion of the sample buffer, boil degeneration. SDS-PAGE electrophoresis, transfer film, closed with 5% skimmed milk powder, take GAPDH as internal reference protein, add the first antibody and incubation overnight a 4℃. Wash membrane with TBST at the second day, plus the second antibody and incubation for 1 hour, wash membrane with TBST, take picture with the CCD imaging system and analyze the density of the band with ImageJ software, compare the changes of protein expression.3. Immunohistochemical (IHC) detect SET expression in tumor tissuesApply appropriate tumor tissue samples, fixed with 4% paraformaldehyde, gradient ethanol dehydration, paraffin embedded tissue samples. Cut into 5 micron tissue sections, xylene dewax, gradient alcohol rehydration, hydrogen peroxide neutralize the catalase of tissues, repair antigen in heated citrate buffer, normal goat serum closed, add the first antibody and incubation overnight a 4℃. The next day add the avidin binding second antibody according to the antibody instructions, DAB staining, hematoxylin staining, neutral gum seal, air-dried and viewed with microscope.Ⅴ. Statistical analyzeThe results was analyzed through SPSS13.0 software, result data stated with ± S, single factor analysis of variance and multiple comparison method which based on the analysis of variance were used to analyze our results, took p< 0.05 as statistical significance.ResultsⅠ. The SET protein expression in clinical breast cancer tissue samplesWith liquid nitrogen grinding method to extract the protein of breast cancer tissues. The result of Western-blot shown that SET protein expression is higher in cancerous tissues and parcancerous tissues compared with the normal breast tissues (p<0.001). Immunohistochemical results show that SET is expressed both in the cytoplasm and nucleus. A large number of brown granules could be seen in the cancerous tissues and parcancerous tissues tissue, but only a small amount of pale yellow particles in normal breast tissues.Ⅱ. The construction of SET stable knockdown in breast cancer cell lines1. Constructed shRNA expression vector targeting SET gene successfullyDesigned siRNA target of SET cloned the annealed double chain oligonucleotide fragment into pLVX-shRNA1 vector, the result is correct after enzyme identified and sequenced of positive colonies.2. The drop degree testing of LentivirusPacked Lentivirus with 293T cells, collecting virus supernatant, virus titer detection showed that virus titer degree is high.3. The successful construction of SET stable knockdown in breast cancer cell linesCompared with the control group in breast cancer cell lines, SET protein expression was significantly suppressed after the transduction of interference fragment shRNA virus (p<0.001), the results of Western blot and RT-PCR are in consistent.Ⅲ. Effect of knockdown of SET expression by RNA interference on biological behavior of breast cancer cell lines1. The cell growth rateThe standard curve drawn by MTT method shown good linear relationship. Cell growth curve shown that SET stable knockdown cell growth become slowly. The result of cell doubling time calculated according to the formula indicated the SET silencing extended cell doubling time.2. Cell apoptosisCompared with the control group cell apoptosis, the cell apoptosis rate of psiRNA-SET cell group increased significantly (p<0.001).3. The migration of tumor cells through cell scratch methodMeasuring the scratch width and calculate the cell mobility according to the formula. The results shown that the psiRNA-SET cell migration rate were significantly inhibit compared with the control group (p<0.001).4. Cell invasion detectionThe cell number those pass membrane of control group and pLVX bland plasmid group were not significant difference, and psiRNA-SET group was significantly lower than the other two groups (p<0.001).Ⅳ. Effect of stable knockdown SET expression by RNA interference on nude mice model1. The development and growth of tumorAfter inoculation of breast tumor cells, all nude mice live in good condition and no accidental death during the testing process. The tumor growth curve showed that the tumor cell growth rate of psiRNA-SET group were significantly lower than the control group and pLVX blank plasmid group of breast cancer cells (p<0.001). Mice were euthanized after 30 days, the weight of stripped plant tumor shown the tumor weight of psiRNA-SET group were significantly lower than the control group.2. The SET protein expression in clinical tumor tissuesBy liquid nitrogen grinding method to extract the tumor tissues protein, The result of Western-blot shown that SET protein expression is lower in SET silencing group compared with the control group and pLVX bland plasmid group. The results of Immunohistochemistry are consistent with Western-blot.Conclusion1. This study shown that SET protein expression is higher in cancerous tissues and parcancerous tissues compared with the normal breast tissues.2. This study successfully constructed stable knockdown SET breast cancer cell lines and provided cell lines model for subsequent experiment study.3. This study shown that SET silencing in breast cancer cell lines could inhibit cell growth and promote apoptosis to some extent, the migration and invasion were suppressed significantly.4. This study shown that SET stable knockdown was able to inhibit the tumor ability of cancer cells in nude mice and could remain SET silencing in vivo.