Application of TMIGD2 gene in diagnosing and treating rectal adenocarcinoma
A rectal adenocarcinoma and gene technology, applied in gene therapy, medical preparations containing active ingredients, microbial measurement/inspection, etc., can solve problems such as tumor metastasis
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Embodiment 1
[0061] Example 1 Screening for gene markers associated with rectal adenocarcinoma
[0062] 1. Collection of samples
[0063] 8 cases of rectal adenocarcinoma non-metastatic tissue and rectal adenocarcinoma metastatic tissue samples were collected, and all the above samples were obtained with the consent of the organizational ethics committee.
[0064] 2. Preparation of RNA samples (operated using QIAGEN tissue RNA extraction kit)
[0065] 1) Tissue extraction
[0066] In a clean area with less RNase interference, use a mortar containing an appropriate amount of liquid nitrogen to weigh the isolated intervertebral disc nucleus pulposus tissue sample, grind it to powder with a pestle, and then transfer the sample to a RNase-free 2ml centrifuge tube middle. Add 300 μl of lysate, place in a homogenizer, grind thoroughly for 5 minutes, centrifuge at 12,000 g at 4°C for 10 minutes, and transfer the supernatant to a new 1.5ml centrifuge tube. Add 600 μl of RNase-free water, mix w...
Embodiment 2
[0084] Example 2 QPCR sequencing verification of differential expression of TMIGD2 gene
[0085] 1. According to the detection results of high-throughput sequencing, the TMIGD2 gene was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 80 cases of non-metastatic rectal adenocarcinoma tissue and 80 cases of metastatic rectal adenocarcinoma tissue were selected.
[0086] 2. The RNA extraction steps are the same as in Example 1.
[0087] 3. Reverse transcription: use the reverse transcription kit of TAKARA company to operate. Specific steps are as follows:
[0088] (1) Take 1 μg of total RNA for reverse transcription, add 2 μl of Oligo(dT), and mix well. Immediately after 5 min in water bath at 70°C, ice bath for 2 min.
[0089] (2) Construct a 25 μl reaction system, including 5 μl of 5× reverse transcription buffer, 5 μl of dNTP (2.5 mM), 40 U / μl of RNasin, 200 U / μl of M-MLV, and make up to the expected volume with nuclease-...
Embodiment 3
[0110] Embodiment 3 suppresses TMIGD2 gene expression
[0111] 1. Cell culture: human rectal adenocarcinoma cell line SW480, in DMEM medium containing 10% calf serum and 1% P / S at 37°C, 5% CO 2 , in an incubator with a relative humidity of 90%. The medium was changed once every 2-3 days, and 0.25% trypsin was used for routine digestion and passage.
[0112] 2. siRNA design
[0113] siRNA sequences against TMIGD2:
[0114] siRNA1-TMIGD2:
[0115] The sense strand is 5'-UUUGCUUUUUCUUCACAAGCC-3' (SEQ ID NO.7);
[0116] The antisense strand is 5'-CUUGUGAAGAAAAAGCAAAAA-3' (SEQ ID NO.8),
[0117] siRNA2-TMIGD2:
[0118] The sense strand is 5'-UUUUUUGUGUGUACCUAUGGG-3' (SEQ ID NO.9);
[0119] The antisense strand is 5'-CAUAGGUACACACAAAAAAAGG-3' (SEQ ID NO.10),
[0120] siRNA3-TMIGD2:
[0121] The sense strand is 5'-UUUUUGUUUUUUUUGAGACGG-3' (SEQ ID NO.11);
[0122] The antisense strand is 5'-GUCUCAAAAAAAAACAAAAAGC-3'(SEQ ID NO.12)
[0123] Negative control siRNA sequence (si...
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