[Abstract] Objective observed gene on tumor cell growth zfp637 proliferation. The semi-quantitative RT PCR detection zfp637 gene expression in mouse tissues and tumor cell lines. The design and synthesis of small molecules of double-stranded RNA and semi-quantitative RT PCR screening best interference effect sites. SiRNA transfected EMT6 cells transfected with 1 ~ 4d cell proliferation assay. Results zfp637 in most normal tissues showed low to moderate expression in peripheral blood mononuclear cells did not express. In mouse hepatoma H22, hepatoma cells Hepal in mice 6 of Lewis lung carcinoma LL /, melanoma tumor cells B16 lymphoma cells Yac 1 and breast cancer cells EMT6 showed a high expression. Screened siRNA 881 Best interference effector sites. Cell proliferation assay (MTT): the 4th day after transfection with siRNA EMT6 cell proliferation of the experimental group was significantly lower than that of the negative control group 3d experimental group of cell proliferation and negative group difference was not obvious. Conclusion zfp637 gene expression levels in different tissues and tumor cell lines. zfp637 is likely to be factors promote positive regulator of cell proliferation. Lowered the expression of the gene can be suppressed EMT6 tumor cell proliferation.
Key words zinc finger protein 637; tumor cells; chemically synthesized small interfering; proliferation
Primary Study of Mouse Zinc Finger Gene zfp637
REN Jing jing, ZHANG Jie, LIU Kai, WANG Xiu jie, YANG Hong liang, WANG Qi, XIONG Zhu juan, WU Ya ying, GAO Yan ping, LIN Ping
Division of Experimental Oncology, National Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China
Corresponding Author: LIN Ping, E mail: firstname.lastname@example.orgAbstract: Objective To investigate the impact of zfp637 on proliferation of cancer cells.Methods The expression of zfp637 in normal tissues and cancer cell lines was examined by semi quantity RT PCR analysis. Small double strands interfering RNA (siRNA) was synthesized, and then screen the best interfering site. After that, proliferation of RNAi treated EMT6 cells was analyzed via 96 well plate approach. The assay was performed on day 1 ~ 4 . Results zfp637 was expressed moderately in most normal tissues, highly expressed in Yac 1, EMT6, B16, H22, LL / 2 and Hepal 6, but not expressed in peripheral blood leukocyte. The zfp637 specific siRNAs substantially reduced the proliferation of EMT6 cells on the 4th day.Conclusion zfp637 was expressed at different levels in multiple tissues and various cancer cell lines. It probably functions as a positive regulator of cellular proliferation. Down regulation of its expression in EMT6 cancer cells by RNAi led to a remarkable reduction of proliferation.
Key words: Zinc finger protein 637; Cancer cell; Chemo synthesized small interfering RNA; Cell proliferation
Zinc finger protein family is a large family of genes, it is estimated that about 1% of the human genome, the gene encoding zinc finger protein, the largest subfamily Cys2/His2 type of zinc finger protein, the gene total number of more than 700 [1 ]. The study confirmed that the motif structure with classic C2H2 zinc finger protein mainly through interaction with DNA sky the role of the transcription factor. They determine the dominant characteristics of the cells through the regulation of gene expression. In higher organisms, cell proliferation, tissue differentiation, and plays a key role in the development [2 4].
Mice zfp637 gene, present only chromosomal localization and structure prediction, yet reported for its function. In this study, the gene expression in normal tissues and tumor cell lines and the growth of tumor cell proliferation were discussed.
1 Materials and methods
1.1 Materials and Reagents
The extracted tissue source for C57BL/6J female mice were purchased from the Experimental Animal Center of Sichuan University Huaxi Medical. Mouse ascites hepatoma cell line H22, mice with Lewis lung carcinoma cell line LL / 2, mouse hepatoma cell line Hepal-6 mouse melanoma cell line B16 mouse by lymphoma cells Yac-1 and mouse breast cancer cells strains the EMT6 by the room saved.
RNA extraction kit (Shanghai Huashun Sheng matter company). RT PCR two-step kit the (Chengdu Bo Ruike company). Lipofectamine2000 transfection reagent (Invitrogen); calf serum, fetal bovine serum (Wuhan Sunry); RPMI1640 (Gibco); trypsin, DMSO and tetramethyl tetrazolium salt [3-(4, dimetnylthiazole 2 y , 2,5 diphenyl tetrazolium bromide), MTT] (Sino-American Biotechnology Company).
1.2 cells and cultured
EMT6, Yac 1 containing 10% fetal calf serum, 100u/ml penicillin 100u / ml streptomycin complete medium RPMI 1640. Hepal 6, LL / 2, B16 containing 10% fetal bovine serum 100u/ml penicillin 100ug/ml streptomycin DMEM (high glucose) complete medium, more than at 37 ° C, 5% CO2 saturated humidity the incubator. Recovery H22 cells and inoculated C57BL/6J mouse ascites hepatoma cell H22.
1.3 RNA extracted
Tissues and cells, RNA was extracted in accordance with the the Shanghai Hua Shunsheng matter company organization manual RNA extraction. The analysis of the results of RNA electrophoresis gel imager determine the integrity of the UV spectrophotometer RNA purity of RNA quantitation.
1.4 Semi-quantitative RT PCR detection zfp637 expression in normal tissues and tumor cell mRNA levels
Primer design: (1) amplification primers S zfp637 gene fragment 5 ‘CTG CGG of TTC at the TCC AGA C 3’, A 5’CTC the TTG the ATT ACT GAG GGC TTT 3 ‘, the target gene product of 955bp. (2) Activity β muscle protein as an internal reference, S: 5 ‘CAC CAC ACC TTC TAC AAT GAG C 3’, A: 5’GTG the ATC the TCC TTC TGC the ATC CTG T-3 ‘, amplification product of 659bp. The reaction conditions of 94 ° C denaturation 3min at 94 ° C for 30s, 60 ° C for 45s at 72 ° C for 1min, 30 cycles at 72 ° C for 5min. Reaction products on a 1.2% agarose gel electrophoresis 65V 40min. Gel imaging system (Bio Rad) mining Figure the Quality One 4.4.0 software for image analysis. Calculated zfp637 / β actin density ratio (%) to 30%, and 60% as low, medium, high standard cut-off point.
The 1.5 Best interference screening effector sites
Design four different sites of target gene siRNA (Sheung Hai Jima biotechnology company design and synthesis), as follows:
Take the logarithmic growth phase the EMT6, count 1.5 × 105 per well were seeded in 6-well plates. 1d after transfection, each well 250μl serum-free 1640 medium without antibiotics diluted 4μgsiRNA 250μl of serum-free and antibiotic-free 1640 medium diluted 8μl lipofectamine 2000, the specific operation is carried out in accordance with the Lipofectamine2000 (Invitrogen) instructions. 48h after transfection, extracted RNA gene was amplified by two-step RT PCR reagents Description, expression detection zfp637mRNA level. PCR conditions: 94 ° C denaturation 3min at 94 ° C for 30s, 60 ° C for 45s at 72 ° C for 1min, 25 cycles at 72 ° C for 5 min. The amplification product in a 1% agarose gel electrophoresis, and gel imager grayscale scanning and density analysis, and calculating the interference efficiency. The interference efficiency (%) = [the negative group zfp637 / β actin density ratio – treated zfp637 is / of β actin density ratio] / (negative group zfp637 / β actin density ratio) to filter out most siRNA interference effect.
1.6 Cell proliferation assay (MTT)
The day before transfection, 1640 medium with no antibiotics to adjust the concentration of EMT6 cells 1 × 104/ml, seeded in 96-well plates, each well 200μl, cultured overnight. The next day in accordance with each hole the siRNA: liposomes (lipofectamine2000) = 100ng: 0.3μl, the establishment of the blank control group, negative control group and the experimental group, n = 5 Vice hole. The transfection with reference reagent instructions. 1 ~ 4d after transfection daily MTT assay: each well was added MTT10μl (5mg/ml), culture was continued to 4h after the supernatant was discarded, join 150μlDMSO, shock for 5 ~~ 10min, the microplate reader with a 570nm wavelength measured hole absorbance (A value). Calculated as follows: the cell proliferation inhibition rate (%) = (untreated group A value treatment group value of A) / A, in untreated group × 100% The experiment was repeated three times.
For gene mRNA 2.1 zfp637 expression profiling
The gene in normal tissues showed low to moderate expression in brain, lung, kidney, liver and spleen moderate expression, heart, thymus, pancreas and bone marrow as low expression, no expression in peripheral blood mononuclear cells, see Figure 1. Showed a high expression in the tumor cell line H22, Hepal-6, LL / 2, B16, YAC 1, EMT6 in Figure 2. Table 1 four different sites of siRNA sequence
2.2-specific siRNA EMT6 cells zfp637 mRNA levels
Measured by RT PCR electrophoresis and gel imager density, interference efficiency of zfp637 siRNA 881 67.14%, the strongest interference effect, as shown in Figure 3.
2.3 zfp637 siRNA 881 EMT6 cells transfected
A cell proliferation assay measured value the SPSS13.0 software diversity of this mean variance analysis results showed: 1 day, 2 days, the experimental group and negative group (P are 0.040), P close to 0.05, due to The number of samples was not sure the difference between meaningful. Three days, the experimental group and the negative group (P = 0.039), and also can not draw firm conclusions. Four days, the experimental group and the negative group (P = 0.001), and the difference was statistically significant. 4 days cell proliferation inhibition rate alone t-test found that the experimental group was significantly higher than the negative control group (P <0.01), as shown in Figure 4.
Zinc finger transcription factor genes is an important part of the vertebrate genome . C2H2 zinc finger are usually arranged in series, most of the transcription factor of three or more than three zinc finger structure the the common specific recognition of DNA or RNA . Zinc finger protein N-terminal usually has a different domain to regulate the function of the transcription factor, these domains BTB, KRAB, FAX, POZ, SCAN, and LeR .
Studies have shown that some C2H2 zinc finger protein can not only regulate normal organ development, and has a potential molecular mechanism in tumor development . The PLAG1 through the expression of the raised IGF Ⅱ growth factor, stimulates cell proliferation tumorigenic effects . ZNF268 play a role in embryonic blood cell development and leukemia . Bsg6 with cerebral organ formation and tumor formation . Nolz and striatal neuronal development . Hzf1 induced differentiation of erythroleukemia play a role . ZT2 with rat bone tissue and differentiation . Znf217 appear amplified in epithelial tumors weakened apoptosis signal, resulting in abnormal telomere promote neoplastic transformation . Expressed in cells through increased of E2F expression of cyclin E activity ZZaPK can initiate cell growth , while Lotl you can reverse the process of apoptosis and cell cycle arrest .
Bioinformatic analysis zfp637 position on chromosome 6, the cDNA of 1114bp, open reading frame encoding 272 amino acids (GeneID: 232337). Structure prediction SMART (http://smart. Embl heidelberg.de) to with C2H2 the classic zinc fingerprints sequence structure. NPS @ PROSCAN of the integrated multiple protein structure and function prediction software the analysis of the EXPASY, EMBL, of the EBI well Interproscan, found zfp637 typical seven C2H2 domain protein kinase C phosphorylation sites, may have tyrosine kinase phosphorylation of sites, N-terminal myristoylation sites and EGF like domain. May also Krueppel type C2H2 zinc finger protein (EMBL EBI), G2 / M transition factor (NCBI Conserved domain) cut. But were not related experiments confirmed.
This experiment studied the gene expression distribution zfp637 rendered low to moderate expression in normal tissue, or even does not express, and in tumor cell lines showed high expression, suggesting that zfp637 gene may be associated with tumor formation have a certain relationship.
RNA interference (RNA interference, RNAi) is a sequence-specific gene silencing of the mRNA levels caused by double-stranded RNA (double stranded RNA, dsRNA), is a powerful tool for an understanding of gene function. mRNA degradation is a key link in the regulation of gene expression [18 20]. Efficiency, the study of gene function and anti-tumor research hotspot in recent years due to the simplicity of its operation and cut target gene expression. This experiment down zfp637 gene expression by interfering found significantly inhibited proliferation of tumor cells 4 days after transfection, inferred zfp637 genes may have a role in promoting cell proliferation. The role of small interfering RNA-based transient transfected cells 4 days after the effect will diminish and even disappear, have not been able to carry out long-term behavior of cell biology. But the interference by screening small fragments, in-depth study of an experimental basis for the future will be constructed to the carrier, and screening stable interfere with cell lines.