Application of miR-204 and target gene thereof to diagnosis and treatment of osteosarcoma
A technology of 1.mir-204 and 2.mir-204, applied in the application field of miR-204 and its target gene in the diagnosis and treatment of osteosarcoma, can solve the problems of unclear function and mechanism of miR-204, Achieve the effect of inhibiting proliferation and migration, convenient operation and high sensitivity
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Embodiment 1
[0043]Example 1 Expression of miR-204 in osteosarcoma cells and regulation of Runx2 protein expression
[0044] 1. Expression of miR-204 in osteosarcoma cells
[0045] 1. Extraction of sample tissue RNA
[0046] ①Pre-cool the mortar with liquid nitrogen first, cut the samples of osteosarcoma paracancerous tissue and cancer center tissue into small pieces, and grind them into powder in liquid nitrogen. Add the powder into a 1.5mL centrifuge tube, add 1mL of Trizol solution per 100mg of tissue powder, shake vigorously, and mix well.
[0047] ②Leave at room temperature for 3-5 minutes, add 200 µL of chloroform, and shake vigorously for 30 seconds.
[0048] ③Centrifuge at 4°C and 12,000 rpm for 15 minutes, take the supernatant aqueous phase, add 0.5 to 1 times the volume of isopropanol, and gently invert to mix. Place at room temperature for 10 minutes, and then centrifuge at 4°C and 12,000 rpm for 10 minutes.
[0049] ④ Discard the supernatant, add 1mL of 70-75% ethanol, and ...
Embodiment 2
[0075] Example 2 Effect of miR-204 on the proliferation and migration of U2OS and MG63 cells
[0076] Cell proliferation experiments were performed by the MTT method. Specifically: 48 hours after the cells were transfected, the culture medium was changed to serum-free but containing 0.5g / L MTT. Then the culture plate was incubated at 37°C for 4h. Aspirate the medium and add dimethyl sulfoxide (DMSO). Incubate at 37°C for 10 min, and then detect at 570 nm on a microplate reader using full wavelength.
[0077] Such as image 3 As shown, the results showed that compared with the Normal group and the RNA-control group, the proliferation of U2OS and MG-63 cells in the miR-204 group was significantly reduced.
[0078] Such as Figure 4 As shown, the results showed that, compared with the Normal group and the RNA-control group, the migration ability of U2OS and MG-63 cells in the miR-204 group decreased.
[0079] The above results indicate that overexpression of miR-204 in tumo...
Embodiment 3
[0081] A diagnostic kit for osteosarcoma, comprising a reverse transcription primer and a PCR amplification primer pair, the sequence of the reverse transcription primer is shown in SEQ ID NO:1, and the sequence of the PCR amplification primer pair is as SEQ ID NO:2 ~shown in SEQ ID NO:3.
[0082] Reverse transcription primer (SEQ ID NO:1): 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGCAT-3'
[0083] PCR amplification primer pair:
[0084] Forward primer (SEQ ID NO:2): 5'-CGGCGTTTGTCATCCTATG-3'
[0085] Reverse primer (SEQ ID NO:3): 5'-GTGCAGGGTCCGAGGT-3'
[0086] The kit also includes RNA extraction solution, PCR reaction solution, positive control solution and negative control solution; the RNA extraction solution is composed of Trizol solution, isopropanol, 70-75% ethanol and absolute ethanol, and the volume ratio is 1 : 0.5~1:1:1; the PCR reaction solution includes 12.5 μL 2×SYBR Premix Ex Taq, 2 μL PCR amplification primer pair and 8.5 μL RNase-free H 2 O.
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