Application of miR-345-3p in preparation of medicine for treating breast cancer
A technology of breast cancer and medicine, applied in the field of breast cancer treatment
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Embodiment 1
[0056] miR-345-3p is highly expressed in breast cancer tissues and breast cancer cells
[0057] In order to analyze the expression of miR-345-3p in breast cancer tissue and two types of breast cancer cells, this example collected breast cancer patients (from the Pathology Department of the First Affiliated Hospital of Chongqing Medical University) and paired In paracancerous tissues, the expression level of miR-345-3p in the tissue was detected by FISH experiment. The FISH experiment process was as follows: MicroRNA FISH probe and fluorescence in situ hybridization (FISH) kit (Shanghai Gemma) were used to detect miR-345-3p. 3p expression in human breast cancer tissue and paired normal breast tissue, paraffin sections were dewaxed with xylene and graded alcohol, digested with proteinase K and denatured at 78°C for 8 minutes, and then incubated with pre-denatured miR at 37°C -345-3p FISH probe was hybridized overnight; nuclei were stained with DAPI; photographed with a confocal ...
Embodiment 2
[0060] Study on the biological effect of knocking down miR-345-3p to inhibit the invasion, migration and proliferation of breast cancer cells.
[0061] As a negative control, negative control inhibitors were transfected in this example, and the effect of knocking down miR-345-3p on the proliferation, migration and invasion of breast cancer cells was detected by cck8, scratch test and tmaswell test.
[0062] cck8 experiment:
[0063] The Cell Counting Kit-8 (CCK8) (MCE) assay was used to determine the proliferative capacity of the cells. The miR-345-3p inhibitors were transfected in MDA-MB-231, and the negative control (NC) was used as the control. After 48 hours of treatment, the cell suspension was 5 × 10 3 Cells / well were seeded in 96-well plates. After 24 hours, 48 hours and 72 hours respectively, cells were incubated with 10% Cell Counting Kit-8 (CCK8) for 1.5 hours at 37°C. Optical density (OD) values at 450 nm were read in a digital spectrophotometer. Each exper...
Embodiment 3
[0070] This example verifies that knocking down miR-345-3p in breast cancer cells can down-regulate the PI3K / AKT signaling pathway by regulating PPP2CA.
[0071] In order to find out the mechanism of miR-345-3p in breast cancer, this example predicted the downstream target genes of miR-345-3p through the three online prediction websites Targetscan, miRDB and miRtarbase, and found that PPP2CA is miR-345- The possible target genes of 3p function, and the 3'UTR site of PPP2CA that miR-345-3p binds to is predicted, and the binding site is mutated ( Figure 6 , Figure 7 ).
[0072] has-miR-345-3p: 3'-GAGGUCUGGGGAGC AAGUCCC G-5' (SEQ ID No. 1);
[0073] PPP2CA3'UTR wildtype: 5'-UUGUUCUUUUUAUU UUCAGGG A-3' (SEQ ID No. 2);
[0074] PPP2CA3'UTR mutant: 5'-UUGUUCUUUUUAUUGGACUUUA-3' (SEQ ID No. 3).
[0075] Next, use the dual-luciferase reporter system to detect whether the target site is functional. The dual-luciferase reporter experiment process is as follows: use Lipofectamin...
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