[Abstract] Objective To construct the recombinant plasmid inhibit Wnt5a gene expression in rats, to inhibit H157 cells the Wnt5a expression of the purpose. Rat Wnt5a gene sequence were designed and synthesized two oligonucleotide fragments containing hairpin structures cloned into the vector plasmid PAVU6 +27 annealed to form double-stranded, positive recombinants were digested and sequencing transfected a normal H157 cells, and Western blot analysis Wnt5a protein expression level changes. Results The recombinant plasmid double digested target bands appear, suggesting the Wnt5a interference fragments have been cloned to PAVU6 +27 plasmid DNA sequencing results showed that insertion sequence with pre-designed exactly the same. Recombinant plasmid transfected H157 cells by G418 successful positive clones, Western blot analysis results show that the the Wnt5a protein level was significantly decline. The Wnt5a gene RNA interference is successfully constructed expression plasmid significantly inhibited Wnt5a expression in H157 cells, for in-depth study of the gene in non-small cell lung cancer and metastases laid the foundation.
Key words Wnt5a H157 transfected with expression
The Wnt5a gene is a tumor metastasis potential related to tumor metastasis related genes [1,2], which is located on chromosome 3p14 ~ p21. Wnt5a gene expression in a variety of tumor tissue were analyzed and found Wnt5a mRNA expression and tumor metastasis. The study found that Wnt5a gene expression with the majority of tumor metastasis positively correlated to play a catalytic role of tumor metastasis indicates that Wnt5a increased lymph node metastasis and poor prognosis. But there are studies have shown that the Wnt5a gene as a tumor suppressor gene delay part of the development of malignancies. Wnt5a not consistent with the biological role of the gene in different tumors. For in-depth study of the gene’s role in the occurrence of non-small cell lung cancer invasion and metastasis, and to describe its mechanism of action, we constructed Wnt5a gene RNA interference expression vector, and its specific inhibition of gene expression H157 Wnt5a.
1 Materials and methods
1.1.1 strains and plasmids E.coli DH10B as room saved. In plasmid PAVU6 +27 Engelke professor for the University of Michigan presented . PAVU6 +27 expression vector full length 6 154bp, its multicloning site with Sal Ⅰ and Xba Ⅰ two restriction sites, the carrier containing the ampicillin resistance gene and G418 selection gene.
1.1.2 siRNA designed according to Genbank human Wnt5a gene sequence (Wnt5a gene is 5 855bp cDNA clone MGC: 71 588, IMAGE: 30.3462 million) in the coding regions were designed for two dispersed in different critical areas Wnt5a gene siRNA fragment (402,403) as the RNAi Objective target sequence.
BLAST comparison of these sequences to avoid homology with other genes. Sal Ⅰ and Xba Ⅰ identify sites both ends of the oligonucleotide contains sequence can be directly connected to the carrier and digested by Sal Ⅰ and Xba Ⅰ by Shanghai Sangon Biological Technology Co., Ltd., synthetic siRNA oligonucleotides (5 ‘end is Sal Ⅰ restriction sites, 3 ‘end Xba Ⅰ restriction sites, see Figure 1). This experiment with restriction enzyme Sal Ⅰ and Xba Ⅰ pAVU6 +27, digested plasmid, linearized, interference and then annealing the fragment and the linearized plasmid pAVU6 +27, to give the new plasmid, named pAVU6 siWnt5a. The extracted plasmid pAVU6 siWnt5a, obtained an approximately 400bp target fragment with BamH Ⅰ and Hind Ⅲ digestion. The experimental reference Promega Corporation and Omega Operations Guide.
Figure 1 siRNA design schematic diagram
1.1.3 Main reagents total cellular DNA extraction kit was purchased from Promega (USA), the liposomes were purchased from Roche (USA) PCR kit was purchased from Takara cell culture medium DMEM was purchased from Hyclone Company (USA) anti-Wnt5a antibody was purchased from Santa Cruz Company (USA) and various restriction enzymes were purchased from New England Biolabs, electrophoresis reagents were purchased from Sigma, other reagents were of analytical grade.
1.2 Experimental Methods
1.2.1 oligonucleotide annealing and the connection will be a synthetic oligonucleotide was dissolved in double distilled water at a concentration of 1μg/μl. Depicting 1μl Add 5μl 10 × T4DNA ligase buffer, to fill with double distilled water to 50μl. Annealing in accordance with the following conditions:
Denaturation at 95 ℃ for 5min; 95 ° C to 70 ° C cooling of 0.1 ° C / s; 70 ° C insulation 10min; 70 ° C to 30 ° C cooling of 0.2 ° C / 30s. DNA removed after the annealing, with a carrier (PAVU6 +27, Sal Ⅰ and Xba Ⅰ digested) connected to transform competent E. coli DH10B.
1.2.2 Identification of the recombinant plasmid selection of transformants, in accordance with the instructions of the kit DNA extraction, after Hind Ⅲ and BamH Ⅰ (Sal Ⅰ and Xba Ⅰ only 55bp), 0.8% agarose gel electrophoresis. Preliminary identification of recombinants were sequenced to determine the expected inserted fragment amplified recombinant plasmid transfected H157 cells.
1.2.3 Cell culture and transfection of H157 cells donated by Professor Chen Zhengtang, Xinqiao Hospital oncology. Culture medium composition: DMEM, 10% fetal calf serum, 100U/ml penicillin, 50μg/ml streptomycin, and cultured at 37 ℃, 5% CO2. The cells were cultured until the logarithmic growth phase, harvested cells were transferred to 6-well plates were incubated for 24h after transfection. With cationic liposome Lipofectamine 2000 as transfection reagent, the recombinant plasmid and empty vector transfected H157 cells, 500μg/ml G418 screening four weeks and, ultimately, transfection positive cells for further experiments.
1.2.4 immunodetection positive cells collected after transfection, total cellular protein was extracted, ultraviolet spectrometer measured the protein concentration. The sample volume adjusted protein SDS-PAGE, polyacrylamide separation gel concentration of 12% (30% acrylamide 6.00 ml, 4 × Tris · Cl / SDS, 3.75 ml H2O 5.25ml, 10% over ammonium sulfate 0.05ml, of TEMED 0.01ml). The proteins were transferred using a semi-dry transfer apparatus (Bio Rad), 15V, 30min. The concentration of an antibody was diluted 1:2 in 500, and the second antibody dilution of 1:5,000.
2.1 RNA interference (RNA interference, RNAi) vector construction
Transformants produce visible after the annealed DNA and the vector and transformed into E.coli DH10B. From five randomly selected transformants, extracted DNA were digested. The electrophoresis showed: three positive clones. Sent to the Shanghai Biological Engineering Technology & Services Co., Ltd. DNA sequencing, the Wnt5a interference fragment sequences and inserted the direction of pre-designed exactly the same. The successful cloning, recombinant plasmid named PAVU6 siWnt5a, as shown in Figure 2.
2.2 Wnt5a protein in H157 cells in the suppression
Western blot method to detect Wnt5a protein expression in transfected cells, the results showed that: H157 cells transfected with the recombinant plasmid pAVU6 siWnt5a (402,403), Wnt5a protein expression significantly decreased, as shown in Figure 3.
Wnt gene cluster  is a highly conserved family of signal, widely distributed online beetle to numerous species of human, and their involvement in normal embryogenesis pattern formation and differentiation of the cell lines and tumors. The Wnt5a gene is one of the important members of the Wnt family, the gene encoding the cysteine-rich growth factor signaling involved in cell growth and differentiation process. Wnt5a proteins of human and mouse with 99% homology. And the the Xenopus Wnt5a protein has 87% homology, mouse and Xenopus Wnt5a cDNA can be used for human cells.
RNA interference [5,6] in recent years developed a new method-specific repressor of gene expression, the technology is a new gene silencing technology developed in recent years. Has been used as a powerful tool for the study of gene function, gene therapy and new drug research and development field of medicine. It is to be homologous to the endogenous mRNA coding region of the smaller fragment (19 ~ 23bp) exogenous double-stranded RNA (double stranded RNA) is introduced into the cell through a series of intracellular reactions lead to the degradation of the corresponding mRNA in the cells, resulting in a lot a technique for gene expression silencing (gene silence). RNAi has high specificity, specificity and the inhibitory effect of the targeted gene, the gene silencing, does not have an impact on other genes. RNAi technology to suppress the expression of target genes for the treatment of parasitic, viral infections, cancer, genetic diseases, and other diseases has opened new avenues of research. RNAi efficiently and specifically blocked the purpose of target gene expression, has been gradually as a simple and effective tool instead of knockout, more and more attention in the field of functional genomics research, gene therapy. RNA target sequence 19 21nt theoretically be any position in the mRNA gene, only because the target sequence serves as a guide, binding to mRNA after cutting the mRNA in the role of various exonucleases, thereby reducing the stability of mRNA until eventually completely degraded. Generally speaking, each target sequence design three to four pairs of siRNA, to choose the most effective follow-up study. In this study, the mRNA coding region of the human Wnt5a design two pairs shRNA.
Efficient and stable expression is the premise and foundation for the study of gene function, the carrier is an important means of delivery of exogenous DNA fragment in cloning, select the appropriate carrier is critical. In addition, the level of transfection efficiency has a direct impact on the expression of inserted into a plasmid, efficient transfection efficiency of cytological research purposes gene function is also crucial. Liposomes experimental study is more commonly taken one of the non-viral vectors with high transfection efficiency, low cytotoxicity, and simple operation.
Build the RNA interference pAVU6 siWnt5a plasmid experiments, we use the gift Engelke DR professor of the University of Michigan pAVU6 +27 plasmid, the plasmid full-length 6 154bp, with ampicillin and G418 selection marker can be used to stably transfected cells Screening. With dependent on the RNA Pol III promoter U6, the promoter has a clear transcription start and end, generated shRNA polyA tail, 3 ‘UU, which makes its structure is similar to the siRNA, the transcription termination signal TTTTT the second T at precise terminated.
Thus, we successfully constructed for the Wnt5a gene RNA interference by plasmid pAVU6-siWnt5a, after restriction analysis and sequencing using liposome transfection method successfully transfected into human lung squamous carcinoma cell line H157, H157 cells significantly inhibited Wnt5a expression of Wnt5a gene for further research on the invasion and metastasis of lung cancer and its biological behavior of laying the foundation.