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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

Inactive Publication Date: 2017-02-22
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, LIMK1, as a new research hotspot in tumors, is receiving extensive attention. Some research reports have proved that LIMK1 is overexpressed and its activity is enhanced in the cells and tissues of malignant tumors such as prostate cancer and breast cancer. Related studies have found that in prostate cancer, metastasis-related The chromosomal locus is located at 7ql1.2, while the human LIMK1 gene is located at 7ql1.23, suggesting that the two are related; but it does not reveal its correlation with osteosarcoma

Method used

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  • Tumor marker LIMK1 and application thereof
  • Tumor marker LIMK1 and application thereof
  • Tumor marker LIMK1 and application thereof

Examples

Experimental program
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Effect test

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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Abstract

The invention relates to a tumor marker LIMK1 and application thereof, in particular to an LIMK1 gene and application of an expression product thereof in diagnosis and treatment of osteosarcoma. A fluorescent quantitative polymerase chain reaction (PCR) technology is used for detecting cells of the osteosarcoma and tissues of patients, so that the overexpression of the LIMK1 gene is shown. Furthermore, a plurality of groups of siRNA are designed so as to silence the expression of the LIMK1 gene. The tumor marker LIMK1 provides a new thought for research of the osteosarcoma on the one hand, and provides a potential new target point for the diagnosis and the treatment of the osteosarcoma in a gene level on the other hand.

Description

technical field [0001] The present invention relates to tumor marker LIMK1 and its application, more specifically to the application of LIMK1 gene and its expression product in the diagnosis and treatment of osteosarcoma. The invention detects osteosarcoma cells and patient tissues through fluorescent quantitative PCR technology, and shows the overexpression of LIMK1 gene. Further, multiple sets of siRNA were designed to silence LIMK1 gene expression. On the one hand, the invention provides a new idea for the study of osteosarcoma, and on the other hand, it provides a potential new target for the diagnosis and treatment of osteosarcoma at the gene level. Background technique [0002] Osteosarcoma is a common osteogenic malignant tumor, accounting for about 20% of all bone tumors. Its high degree of malignancy is more likely to occur in adolescents, easy to relapse and transfer, and the prognosis is poor. With the improvement of molecular biology theory and technical level...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/574G01N33/573A61K45/00A61P35/00
CPCA61K45/00C12Q1/6886C12Q2600/158G01N33/573G01N33/57407G01N33/57484G01N2333/912
Inventor 王岩
Owner JILIN UNIV
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