Construction Of Eukaryotic Expression Vector For Human MiR-147a And Its Effects On Proliferation And Invasion Of Lung Cancer Cells

miRNA(microRNA) is an important post-transcriptional control factors for eukaryotic cells. In the cells and biological body, it plays an important role in proliferation, apoptosis, development, differentiation processes, its dysfunction can cause a variety of pathological state, including the occurrence and metastasis of tumor. In many tumors were found specificity abnormal expression of miRNAs. At present, We hold the opinion that, in the process of tumorigenesis many miRNAs play the role as the tumor-suppressor gene down-regulated the activity of proto-oncogene, or as the tumor gene down-regulated the activity of proto-oncogene, or play a role as the tumor metastasis adjustment factor, its mutation, deletion, translocation, and abnormalities of the mutual regulation can also lead to abnormal expression of related genes. So the miRNA plays an important regulatory role in human tumorigenesis and development, and can be used as a new molecular marker of tumor diagnosis. miRNA plays an important role in the development of lung tissue, its abnormal expression is closely related to the development of lung cancer. So screening and identification specific miRNA of lung cancer, clarifing it’s molecular mechanisms in the development of lung cancer has considerable application value for lung cancer diagnosis, prognosis and target therapy. In preliminary work, we used gene chip to analyse the influence of9-cis-retinoic acid for miRNA expression pattern of lung cancer cell line A549, found that9-cis-retinoic acid can up-regulate the expression of miR-147a more than two times, and confirmed by real-time fluorescent quantitative PCR. At that, We hypothesized miR-147a has the role of tumor inhibition. Accordingly, we build the expression vector of miR-147a, pSilencer-147a, in addition preliminarily research its expression and functions in lung cancer cell A549.Objective:Build the miR-147a eukaryotic expression vector of human beings, Detect its expression in A549lung adenocarcinoma cancer cells, and the influence to A549cell of proliferation and invasive ability.Methods: 1. Construction of recombinant plasmid vector, pSilencer-147a:Total RNA of A549cells as templates, RT-PCR amplification precursor sequence of miR-147a(pri-mir-147a), Inserted into pSilencer-CMV neo expression vector, to build the pSilencer-147a reorganization carrier.2. qRT-PCR direct detection the expression of exogenous pri-miR-147a:Extend the A549cells to new cell culture medium in cell culture bottle, using X-tremeGENE HP transfection reagents, to transfer pSilencer-147a reorganization carrier into A549cells, using pSilencer empty carrier as contrast. After48h we collecting A549cells to extract RNA, qRT-PCR to detect the expression of exogenous miR-147a.3. Construction of recombinant plasmid vector, pMIR-147aT:Synthetic147a target sequence147aT, Inserted into pMIR-REPORTTM Luciferase vector, to build the pMIR-147aT reorganization plasmid vectors.4. Reporter gene analysis to indirect detection the expression of miR-147a:Extend the A549cells to new cell culture medium in24hole cell culture plate, transfer pSilencer-147a and pMIR-147aT reorganization carrier into A549cells,①using same amount of pSilencer-147a and pMIR-REPORT empty carrier,②using same amount of pSilencer and pMIR-147aT carrier,③using same amount of pSilencer and pMIR-REPORT empty carrier as contrast. After48h we collect A549cells, detect the relative luciferase activity of cell lysis solution.5. Determined exogenous miR-147a excessive expression had effect on A549cell proliferation ability by MTT assay:Respectively deal with A549cells:①Do not do any processing,②Only use X-tremeGENE HP transfection reagent processing,③use X-tremeGENE HP to transfer pSilencer plasmid,④use X-tremeGENE HP to transfer pSilencer-147a plasmid. Then inoculate the handled cells into96-well plates and continue to cultivate24-96h. Detect cell proliferation by MTT method.6. Determined exogenous miR-147a excessive expression had effect on A549cell invasion ability by Matrigel invasion experiment:After transfection, we inoculated A549cells into the Transwell room with serum free medium, continue to develop48h, wipe the unacrossed cells, use methanol to fix the cell at the bottom of the chamber which has been through the Matrige, and dyeing by Giemsa stain. Calculation the efficiency of invasion.7. Construction of recombinant plasmid vector, pGL4-AP1.ect:Synthetic tumor signal response element, such as AP1, ELK1, cMYC, STAT3, FOXO, TCF, Inserted into pGL4.23[luc2/minP] Vector, to build the tumor signal response element luciferase report gene reorganization plasmid vectors, pGL4-AP1, pGL4-ELK1, pGL4-cMYC, pGL4-STAT3, pGL4-FOXO, pGL4-TCF.8. Reporter gene analysis to detection the miR-147a’s influence on related to lung cancer tumor signaling pathways:To detect the influence of pSilencer-147a to EGFR-RAS-MAPK signal pathway, using X-tremeGENE HP transfection reagents, we transfer pGL4-AP1, pGL4-ELK1, pGL4-cMYC, pGL4-STAT3, pGL4-FOXO, pGL4-TCF recombinant vectors of tumor signaling pathways into A549cells with pSilencer-147a, simultaneously transfer pSilencer empty carrier and pGL4-AP1, pGL4-ELK1, pGL4-cMYC, pGL4-STAT3, pGL4-FOXO, pGL4-TCF as control. After48h we collect A549cells, detect the relative luciferase activity of cell lysis solution.Results1. Structured pSilencer-147a recombinant plasmid, confirmed by restriction enzyme digestion and DNA sequencing of the build is successful.2. After A549cells infected by pSilencer-147a, qRT-PCR detected obvious exogenous miR-147a expression.3. Structured pMIR-147aT recombinant plasmid, confirmed by restriction enzyme digestion and DNA sequencing of the build is successful.4. Reporter gene analysis results proved that, in A549cells, pSilencer-147a can effective express functional miR-147a.5. MTT results revealed that, the over-expression of miR-147a can obviously restrain proliferation of A549cells. After transfected48h,72h,96h, the inhibition effects were13%,18%and30%compared with the control group.6. Matrigel invasion assay results revealed that, the over-expression of miR-147a can obviously restrain invasive ability of A549cells. After transfected48h, the experimental group cells invaded to the under bottom membrane surface of the Transwell Chambers was decreased by72%compared with the control group cells which were transfected with pSilencer4.1.7. Structured pGL4-AP1, pGL4-ELK1, pGL4-cMYC, pGL4-STAT3, pGL4-FOXO, pGL4-TCF recombinant plasmid, confirmed by restriction enzyme digestion and DNA sequencing of the build is successful.8. Reporter gene analysis results revealed that, the over-expression of miR-147a can obviously down-regulate luciferase activity of pGL4-AP1(down-regulated50%), pGL4-ELK1(down-regulated57%), pGL4-cMYC(down-regulated58%).Conclusion:We successfully constructed human miR-147a eukaryotic expression vector, and effectivly expressed in lung adenocarcinoma cancer cells A549. Its inhibitory effect on the proliferation and invasion of A549cells, maybe relative to miR-147a for inhibition of EGFR-RAS-MAPK pathway.