| Objective To construct the recombined lentiviral vector, which can efficiently express small molecule of mi R-508-5p, and to explore its impact on the process of tumor cells growth, that finally provides a new biomarker and molecular target for clinical cancer treatment.Methods 1. Collected poorly differentiated gastric adenocarcinoma patients which were formalin-fixed paraffin-embedded tissues(FFPE) in the General Hospital of Ningxia Medical University. we used q RT-PCR method to detecte the expression level of mi R-508-5p of FFPE tissues, and immunohistochemistry was used to detect SKP2 protein levels of gastric adenocarcinoma(HCC), we collected clinical data in order to confirm the microarray data.2. Referring to mi RBase database of humanized hsa-mi R-508-5P gene sequence(MIMAT0004778), we designed a pair of oligonucleotide strands, to form a precursor mi RNA sequence comprising the stem loop structure after annealing, and the Hpa 1and Xho 1 restriction sites were added to the ends of the sequences; the linearized p Sico R plasmid and the double-stranded mi R-508-5p DNA fragment were connected, to confirme by double digestion and sequencing after a large number of cloned.3. Following the Trans Lipid Transfection Reagent, three plasmid system(p Sico R/mi R-508-5p overexpression vector 1.5?g, p VSVG 0.45??g, 8.91 1.05?g) were transfected into HEK-293 T cells, to package lentiviral and the virus titers were determined in accordance with the principle of dilution fold ratio.4. Using the mi Randa, Targetscan and Pic Tar target gene prediction software to find the potential target genes of mi R-508-5p, which was the high degree of conservatism, combined with lower free energy, and related to innate immune response. We selected MAPK1?Skp2 as target genes of mi R-508-5p,and then building the firefly luciferase expression vector to verify the relationship between mi R-508-5p and MAPK1?Skp2 in 293 T cells. The MAPK1, Skp2 3'UTR and the mutant subclones were inserted into the downstream of p MIR-Report luciferase reporter gene plasmid, so that it can express recombinant fusion target gene 3'UTR luciferase, p MIR-Report/MAPK1, p MIR-Report/Skp2 and p Sico R/mi R-508-5p recombinant plasmid were co-transfected into 293 T cells by liposome transfection methods. After 48 hour incubation, the cells were harvested and subjected to dual Luciferase assay, and Renilla Luciferase used as internal control. Simultaneously mutant target genes were co-transfected, to verify whether its targeted site by knocking out.5. p Sico R/mi R-508-5p lentivirus and p Sico R/NC empty virus infected Hep G2, SGC7901, Hela and SW480 cells, after 48 h, cells were collected and total proteins and micro RNAs were extracted by RT-PCR, q RT-PCR and Western Blot, to detect the expression levels of mi R-508-5p and Skp2.6. p Sico R/mi R-508-5p lentiviral and p Sico R/NC empty viral infected tumor cells, stimulation after 24?48?72 h, MTT assay was used to horizontal proliferation, Hochest level was used to assay cells apoptosis, cell scratch was used to test cell migration and cell healing ability, transwell experiments was used to detect cell migration and invasion, flow cytometry was used to detect apoptosis and cell cycle.Results 1. The expression level of mi R-508-5p was significantly reduced in poorly differentiated gastric adenocarcinoma compared to the normal gastric mucosa by q RT-PCR detection, and with more serious pathological grade, the expression of a gradual downward with positive correlation trend, q RT-PCR results was consistent with the microarray data test results; display the expression level of Skp2 protein was significantly higher in poorly differentiated gastric adenocarcinoma, while there was almost no expression of Skp2 in normal gastric mucosa by immunohistochemistry.2. p Sico R/mi R-508-5p lentiviral vector was successfully constructed and high-titer lentiviral was observed, it showed that the recombinant lentiviruses can effectively promote endogenous mi R-508-5p expression by Real-Time PCR assay.3. Bioinformatics analysis showed that mi R-508-5p and MAPK1, Skp2 binding sites may exist, and it confirmed that mi R-508-5p can be targeted regulation MAPK1, Skp2 and inhibit the protein expression level through the dual luciferase reporter gene system and Western blot.4. Tumor cells with lentivirus infection model: lentiviral particles can significantly promoted the expression level of mi R-508-5p(P<0.05) in the tumor cell lines; while over expression of mi R-508-5p could inhibit Skp2 m RNA(P<0.05) and/or protein levels; whereas the transfection group of mi R-508-5p inhibitor, Skp2 m RNA(P <0.05)and/or protein levels were elevated. The above experiments showed that the specific targeting relationship was existed between mi R-508-5p and SKP2 genes.5. In the group of p Sico R/mi R-508-5p infection: the cell proliferation was inhibited effectively at levels by MTT detection; cells chromatin condensation was increased compared with the normal control group by hoechst staining, the late apoptosis was occurrenced; the level of migration of tumor cells was inhibited significantly by cell scratch assay; the tumor cell migration and invasion was inhibited significantly by transwell experiments.6. To explore the effection of mi R-508-5p on the regulation mechanism of tumor cells, we found that cells in group of p Sico R/mi R-508-5p, early apoptotic and late apoptotic cells was significantly increased by flow cytometry, the percentage of haploid G0/G1 cell was significantly higher, while the percentage of diploid G2/M phase cells was decreased accordingly. It showed that mi R-508-5p inhibited the division and differentiation stage of cells to G2/M phase, it contained cells that staying in the non-dividing G0/G1 phase, thereby inhibited the proliferation of malignant tumor cells.Conclusions we constructed the human p Sico R-mi R-508-5p lentivirus successfully, it improved the expression of small molecules of mi R-508-5p significantly, we verified that mi R-508-5p can negatively regulate the expression of MAPK1, Skp2 genes; further study confirmed that over-expression of mi R-508-5p play an important role in the metabolic activity of tumor cells, it will become a potential molecular target for cancer therapy. |
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