Application of miR-7 in preparation/screening of prostate cancer diagnosis and treatment products
A prostate cancer and mir-7 technology, applied in the field of biomedicine, can solve the problems of prostate cancer diagnosis, prognosis and poor treatment effect, and achieve the effect of inhibiting proliferation and promoting cell apoptosis
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Embodiment 1
[0028] 1. Expression of miR-7 in prostate cancer cells and cancerous tissues
[0029] 1.1. RNA extraction: Tiangen RNA extraction kit is used
[0030] 1) Three kinds of cells PC3, DU145, LNCaP and prostate epithelial cell PBS for prostate cancer are washed twice, and an appropriate amount of pancreatic enzyme is added for digestion;
[0031] 2) Digest for 1 min, add medium to stop digestion at 1000 rpm, centrifuge for 5 min;
[0032] 3) Resuspend cells with a full amount of 150-200 μL cell lysate per centrifuge tube;
[0033] 4) Incubate on ice for 15 min with vortex shaker mixed evenly;
[0034] 5) The solution was added to the filter column CS, centrifuged at 12000 rpm for 2 min, and the filtrate was collected;
[0035] 6) Add filtrate and other volumes of 70% ethanol, mix well and add to the adsorption column CR2, centrifuge at 12000 rpm for 1min, discard the waste liquid, and put the adsorption column back into the collection tube;
[0036]7) Add 350 μL of deproteinization sol...
Embodiment 2
[0092] miR-7 inhibits the glucose metabolism of prostate cancer cells and primary cells, as well as proliferation and cloning
[0093] 1. Detection of cellular glycolytic metabolism level
[0094] 1) Cells in the logarithmic growth phase are selected for experiments, miR-7 (overexpressed miR-7), miR-NC (blank control) transfected into LNCaP and prostate cancer primary cells are added to pancreatic digestion solution digestion cells after 36-48 h and counted. Cell density per well is at 5×10 3 Cells / 80 μL are inoculated in a 96-well test plate dedicated to the Seahorse XF96 analyzer at 37°C, 5% CO 2 Culture overnight in the incubator to adapt the cells to adherent growth;
[0095] 2) Replace the cell culture medium into a special glycolysis stress test medium without glucose, set up the ECAR detection procedure step by step, and test on the SeahorseXF96 analyzer. At this time, the ECAR value is the acidification rate produced by the non-glycolytic pathway. Cells produce CO through ...
Embodiment 3
[0131] Construction of miR-7 transgenic mice
[0132]1. Vector construction: According to the miR-7 sequence, select the appropriate carrier and construct the CAG-miR-7-Luc-PA plasmid. Sequence of pCAG-Gene-intron-BGHpa transgenic DNA fragments:
[0133]
[0134] 2. Mouse preparation:
[0135] 1) Preparation of embryonic donor mice: 5.0 IU of pregnant horse serum gonadotropin (PMSG) is injected intraperitoneally into mature female mice, generally PMSG is injected at 13 to 14 pm, and then 5.0 IU of human chorionic gonadotropin (hCG) is injected after about 48 h, and immediately merged with male mice. The next morning, the female mouse's vaginal plug is examined, and the successfully mated female mouse will be used to retrieve the embryo. In order to obtain a better over-row effect, female rats of 3 to 6 weeks of age are generally selected. Male rats reach sexual maturity at about 8 weeks of age and can be used until their reproductive performance declines (6 to 8 months of age).
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