Preparation and application of triple artificial miRNA in restraining of VEGFRs
A VEGFR2, artificial technology, applied in the field of biomedicine, can solve the poor prognosis of pancreatic cancer patients and other problems, achieve the effect of reducing growth, reducing cell migration and invasion ability, and promoting apoptosis
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Embodiment 1
[0033] 1) Large-scale pancreatic cancer tissue chips (including 168 pancreatic cancers and 40 benign pancreatic diseases), clinical data include gender, age, tumor diameter, location, degree of differentiation, metastasis, 5-year overall survival rate, etc. The samples are used by the hospital The ethics committee agreed. Paraffin tissue microarray sections were repaired with high-temperature antigens, and the primary antibody was incubated overnight at 4°C. According to the number of positive cells and staining intensity, they were divided into high expression, low or no expression. It was confirmed by statistical analysis that the three subtypes of vascular endothelial growth factor receptor The expression of VEGFRs in cancer tissues is higher than that in benign pancreatic tissues. VEGFR1 and VEGFR3 are not only highly expressed in tumor cells, but also have different degrees of positive expression in tumor stroma; the combined high expression of VEGFRs in cancer and pancrea...
Embodiment 2
[0052] The artificial miRNA (artificial miRNA, amiRNA) targeting the three target genes VEGFR1, VEGFR2, VEGFR3 were constructed in pcDNA TM 6.2-GW / miR vector, such as image 3 As shown, it is used to express a specific artificial miRNA, which is engineered to have 100% homology with the target gene sequence and can cause cleavage of the target molecule. The vector is transfected into the cell, and the engineered Pre-miRNA formed by transcription from the strong CMV type II (Pol II) promoter contains the flanking sequence and loop sequence of mouse miR-155, in which the 5' and 3' The flanking sequence makes the transcribed Pre-miRNA similar in structure to miR-155, and the efficiency of cutting the target gene is improved by artificially optimizing the loop sequence. Cloning of miRNA by quick ligation protocol and transfection can immediately express pre-miRNA, and the expressed premiRNA is processed by endogenous cellular machinery (including Drosha enzyme) in the nucleus, t...
Embodiment 3
[0080] The proliferation experiment of embodiment 3 cell count (CCK8)
[0081] For two cell lines, SW1990 and PANC-1, the cell concentration was adjusted to 5×10 4 cells / mL, inoculated in 96-well culture plate, 100 μL per well, cultured in 37°C, 5% CO2 incubator for 24 hours. Drugs with different concentration gradients were added to the 96-well plate, 150 μL per well, and each group had three replicate wells. After 6h, 12h, 24h, 48h, and 72h, CCK8 (product of Sigma Company) was detected. Add 15 μL of CCK8 per well at 37°C, 5% CO 2 Incubate in the incubator for 3 h in the dark. The OD value at the same time point was measured by an enzyme label detector (Thermo MK3 type) at a wavelength of 492 nm, and the effect of cell proliferation was analyzed using the measured OD value.
[0082] The result is as Figure 6 As shown, compared with the control cells, the triple artificial miRNA targeting VEGFRs inhibited the proliferation of PANC-1 and SW1990; the inhibition rate of SW1...
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