Tumor marker LIMK1 and application thereof
A technology of osteosarcoma and gene, applied in the tumor marker LIMK1 and its application field, can solve the problem of not revealing the correlation of osteosarcoma, and achieve the effect of reducing the invasion ability
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Embodiment 1
[0028] Example 1: Verification of the expression of LIMK1 in osteosarcoma cells
[0029] In order to verify the expression and characteristics of LIMK1 in osteosarcoma cells, two kinds of human osteosarcoma cells were routinely cultured, and the cell samples of each group were collected. At the same time, human osteoblast hFOB cell samples were collected as the control group, and fluorescence real-time quantitative PCR (qRT -PCR) for verification, the specific operation steps are as follows:
[0030] (1), RNA extraction
[0031] ①Take the tumor cells cultured to the logarithmic growth phase and wash them 3 times with pre-cooled PBS. Add 1ml Trizol to each bottle to lyse the cells and shake horizontally on ice for 5min. Use a 5ml gun to move it to the EP tube, and then use a 1ml gun tip to repeatedly suck the cells to completely disperse and suspend the cells in Trizol.
[0032] ② Add 1 / 5 volume of chloroform (0.2ml), vibrate the centrifuge tube vigorously, mix well, and pla...
Embodiment 2
[0053] Example 2: Verification of the expression of LIMK1 gene in osteosarcoma tissue
[0054] The present invention collects 6 osteosarcoma tissue samples and 6 adjacent normal tissue samples at the same time, and uses fluorescent real-time quantitative PCR (qRT-PCR) to verify the expression of LIMK1 gene in osteosarcoma tissue. The specific operation steps are as follows:
[0055] (1.) RNA extraction
[0056] ①Collect the tumor tissue and freeze it in liquid nitrogen. After taking it out, put the tissue into a pre-cooled mortar for grinding. After the tissue sample is powdered, add Trizol and leave it at room temperature for 5 minutes;
[0057] ② Add 1 / 5 volume of chloroform (0.2ml), vibrate the centrifuge tube vigorously, mix well, and place it at room temperature for 5min-10min;
[0058] ③After 12000rpm high-speed centrifugation for 15 minutes, suck the upper aqueous phase (absorb 70%) into another new centrifuge tube, and be careful not to suck the protein substances bet...
Embodiment 3
[0077] Example 3 RNAi inhibits LIMK1 gene expression
[0078] 1. siRNA construction and synthesis
[0079] The siRNA expression vector pSUPER contains the ampicillin resistance gene. According to the full sequence of LIMK1 mRNA Homo sapiens LIM domain kinase 1 (LIMK1) (NM_002314.2) in the NCBI database, and according to the siRNA design principle, three pairs of oligodeoxynucleotide chains (oligo) of LIMK1 SiRNA were designed and synthesized ( LY1, LY2, LY3) (Invitrogen), no homology with known human gene sequences other than LIMK1 was confirmed by BLAST Search. From the 5' end to the 3' end, there are BglⅡ restriction site, target sequence consisting of 19 bases, stem-loop structure, complementary sequence that can bind to the target sequence, transcription terminator, Hind III restriction site, etc. Six regions (BglⅡ+Sense+Loop+Antisense+termination signal+HindIII). At the same time, the pSUPER empty vector was used as a negative control. The sequence of the oligodeoxynu...
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