ZNF695 as marker and treatment target of prostate cancer bone metastasis
A ZNF695, prostate cancer technology, applied in the field of biomedicine, can solve the problems of reducing the quality of life and life expectancy of PCa patients, and clinical treatment difficulties
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Embodiment 1
[0034] Example 1, mRNA and protein detection of ZNF695
[0035] 1.1 ZNF695 mRNA detection
[0036] Real-time quantitative PCR method was used: ① RNA was extracted using Trizol reagent, and 2 μg of RNA was subjected to reverse transcription reaction. After the reaction was completed, the cDNA was stored at -20°C for later use. ② Prepare the SYBRGreen mix (Roche) required for DNA amplification according to the instructions of the detection kit, as well as ZNF695 detection primers and internal reference primers. The design and synthesis of the primers are from Guangzhou Ruibo Biotechnology Co., Ltd. In the Bio-rad real-time quantitative PCR instrument, the amplification reaction conditions were set according to the requirements of the kit instructions, and 40 cycles of reactions were performed. The GAPDH gene was used as an internal reference, and at least 3 auxiliary wells were set for all samples. ③ The relative expression of target gene DNA passed 2 -ΔΔCt calculated by the ...
Embodiment 2
[0043] Example 2. Low expression of ZNF695 inhibits bone metastasis ability of prostate cancer cells in vivo
[0044] 2.1 In vitro culture of PCa bone metastasis cells
[0045] PCa bone metastasis PC-3 cell line was cultured in CO 2 The concentration is 5%, and the temperature is in an incubator at 37°C. Cells were grown in Ham's F-12 medium (10% fetal bovine serum). Observe the cell state and cell density, and replace the medium regularly (generally about 2-3 days), and it is suitable for experiment use when the cell density reaches 80%.
[0046] 2.2 Transfection
[0047] The RNA interference plasmids (siZNF695#1, siZNF695#2) and their control plasmids (vector) used to down-regulate the expression of ZNF695 were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. Before transfection, prostate cancer PC-3 cells were incubated with 10 5 Each well was inoculated on a culture plate, and diluted RNA interference plasmids, control plasmids and liposomes were ad...
Embodiment 3
[0054] Example 3. Low expression of ZNF695 inhibits bone metastasis of prostate cancer cells in vitro
[0055] 3.1 In vitro culture of PCa cells
[0056] PC-3 cell line and VcaP cell line were cultured in CO 2 The concentration is 5%, and the temperature is in an incubator at 37°C. Cells were grown in Ham's F-12 medium (10% fetal bovine serum). Observe the cell state and cell density, and replace the medium regularly (generally about 2-3 days), and it is suitable for experiment use when the cell density reaches 80%.
[0057] 3.2 Transfection
[0058] In the same way as in 2.2, siZNF695#1 and its control plasmid were transfected into prostate cancer PC-3 cells or VcaP cells, respectively, to obtain prostate cancer cells with low expression of ZNF695.
[0059] 3.3 Migration and invasion experiments
[0060] ①Use 50mg / L matrigel to coat or not coat the polycarbonate filter membrane of the transwell chamber. ② Take 100 μL of serum-free medium containing about 5×104 PCa cells...
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