Construction And Expression Assay Of Anti-miR-221 RNA Interference Vector And Screening An Effective Target Sequence

BackgroundHuman glioma is one of the most common and refractory intracranial primary tumors. Glioma, with the highest incidence, accounts for 35.26%-60.96% of intracranial tumors in our country, among which about 60% gliomas are malignant. Most gliomas localize deeply in the central nervous system and grow infiltratively with obscure boundaries so that the surgical resection and radiotherapy are potentially dangerous for the patients with gliomas. Therefore, improving the diagnosis and treatment of gliomas is especially urgent at present. In recent years, application of molecular and biological methods to inhibit the malignant progression of glioma becomes new and feasible. The development of molecular biology, especially the discoveries of small interfering RNA (small interfering RNA, siRNA) and microRNA provide a broad space for gene therapy of cancer.MicroRNAs(miRNAs) represent a class of short, non-codingand single-stranded RNA molecules with about 21～25 nucleotides. These miRNAs negatively regulate gene expression by identifying the specific target miRNA, promoting miRNA degradation and (or) inhibiting the translation on post-transcriptional level. MicroRNAs are intimately associated with ontogeny stem cell differentiation and the onset of disease. Tumor is thoughet to be a kind of disease with multi-gene abnormalities disease essentially, to proliferate autonomously and invasively through activating expression of one or several oncogenes and through deletion or mutation of anti-oncogenes. Therefore, researches on miRNAs are significant for exploring the pathogenesis, early diagnosis and new therapeutic strategies for all malignant tumors, including gliomas.Objective1. To constructRNA interference lentiviral vector plasmids of the target gene.2. To construct over-expression vector of the fusion protein.3. To screen the effective RNA interference vector of target gene.Methods1. Four RNA interference sequences targeting miR-221 were synthesized according to the designing principles. The best target was selected for the follow-up experiments. The double-stranded DNA oligo sequences including the interferential sequences were directly attached to RNA interference vector. Then the product was transducted into the prepared competent cells. The positive clones were identified byPCR and sequenced for a successful RNA interference lentiviral vector. 2.The target gene was fished by PCR from the cDNA library, and then the target gene and the vector were double digested by enzyme separately. Purified enzyme products were translated into bacterial competent cells after they were connected or recombinated directionally. The positive clones, which representing the targeted vector combined with the targeted gene, were identified by PCR. Its up-stream and down-stream primers were designed respectively on the vector and the target gene. The positive clones determined by PCR were sequenced, analysed and compared in order to acquire the successful expression vector containing the fusion protein.3. The successfully conducted RNAi plasmid vector containing target gene were co-transfected into 293T cells according to the instruction of Lipofectamine 2000 of Invitrogen Corporation. The transfected cells were observed under the fluorescence microscope after they were transfected for 24 hours.At the same time, protein was extracted from 293T cells transfected for 36 ~ 48 hours and analyzed to determine the interferential effect of different targets.Results1. Preparation of RNA interference lentiviral vector containing the target geneThe results of electrophoresis showed bands appeared on the double-stranded lane in contrast to the position of single-stranded lane, which represented that the double-stranded DNA oligo sequence was synthesized successfully. After digested and linearized by Age I and EcoRI, the plasmid vectors were detected by agarose gel electrophoresis. It showed that clear bands of plasmids without digestion appeared around the position of 5kb, while the bands of plasmids already digested appeared around the site of 8kb. The positive clones with vshRNA fragment were 340bp in size, while the size of clones without vshRNA fragment was 306 bp. Besides, positive clones had a band at the position of 340bp around.2. Construction of over-expression vector involving the target geneThe PCR products'size was 130bp, verified by agarose gel electrophoresis after they were digested by Mfe I/HpaI enzymes. The size of positive clones was 467 bp, while the size of self-ligation was 365bp. Finally, the positive clones were sequenced to assure wheather the objective fragments were cloned into the vector correctly.3. Screening of effective RNAi targetThe over-expression plasmid and siRNA vectors were cotransfected into 293T ells successfully, assayed by immunofluorescence analysis. The transfection rate was more than 70%. The knockdown effect of PscSI576 target is optimal at two different concentrations of plasmids, determined by Western blot analysis. Conclusions1. The human anti-miR-221 RNA interference lentiviral vector has been constructed successfully.2. The human RNA fusion protein of miR-221 gene over-expression vector has been constructed successfully.3. The effective RNA interference targets of human miR-221 gene have been screened successfully.