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An expression up-regulator of lncrna MALAT1 and its application

A HCT116, low-dose technology, applied in the field of tumors, can solve the problems of large cell loss, difficult MALAT1 overexpression, MALAT1 cell level expression regulation and other problems

Active Publication Date: 2021-08-20
THE FIRST HOSPITAL OF HEBEI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the long length of the gene, after the traditional construction of the expression plasmid, because the plasmid is larger than 10kb, it is difficult to achieve the overexpression of MALAT1 in the cells by ordinary transfection methods, and it can only be transfected by high-voltage electroporation (electroporation). transfection method, and the loss of cells after transfection is large, more than half of the cells will die during the electroporation process, and only about 1 / 3 of the cells can survive after the electroporation operation
And this method requires the use of electroporation apparatus and electroporation cell, and the cost of transfection is very high
The expression regulation of MALAT1 at the cellular level is relatively difficult, which is one of the main reasons for limiting the research on the regulatory mechanism of MALAT1

Method used

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  • An expression up-regulator of lncrna MALAT1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 Thapsigargin on the impact of tumor cell survival rate

[0019] Cells in the logarithmic growth phase were digested with trypsin and suspended in complete medium for counting. Cells were seeded into 96-well plates, 9000 cells per well. After adhering to the wall, the complete medium containing thapsigargin was replaced with the concentration of 0 μM (Control group), 0.1 μM (10x group), 0.01 μM (1x group), 100 μl per well. Put the 96-well plate in 37 °C 5% CO 2 After culturing in the incubator for 24 hours, add 10 μl Cell Counting Kit-8 reagent to each well, incubate again for 3 hours, measure the absorbance (450nm wavelength), and calculate the survival rate of the cells in each group.

[0020] Such as Figure 1-2 As shown, 0.01 μM (1x group) thapsigargin treatment does not cause cell apoptosis, and 0.1 μM (10x group) thapsigargin treatment can cause 30%-40% cell apoptosis.

Embodiment 2

[0021] Example 2 Effect of thapsigargin on the expression of tumor cell MALAT1

[0022] Cells in the logarithmic growth phase were digested with trypsin and suspended in complete medium for counting. Cells were seeded into 6-well plates, 5*10 per well 5 cell. After adhering to the wall, the complete medium containing thapsigargin in the specified amount was replaced, the concentrations were 0 μM (Control group), 0.01 μM (1x group), 2 ml per well. Put the 6-well plate in 37 °C 5% CO 2 After culturing in the incubator for 24 hours, the total RNA was extracted and reverse-transcribed into cDNA, and the relative expression of MALAT1 was detected by Real-time PCR (the internal reference gene was GAPDH).

[0023] Such as Figure 3-4 As shown, 0.01 μM (1x group) thapsigargin treatment can significantly promote the expression of MALAT1.

Embodiment 3

[0024] Example 3 Preparation of Cell Migration / Invasion Promoter Kit

[0025] Cell Migration / Invasion Promoter Kit, Contains Promoter and Diluent. The accelerator is thapsigargin with a concentration of 1uM, the solvent is DMSO, the total volume is 255 μL, and the specification of the diluent is 26 mL. The components of the diluent are shown in Table 1.

[0026] Table 1 Diluent Component List

[0027]

[0028]

[0029] The 100X enhancer in the cell migration / invasion promoter kit is stored at -20°C and is valid for one year, and the dilution is stored at 4°C and is valid for one year. This kit can effectively promote cell migration and can be used for cells with poor migration ability Migration and invasion experiments, the experimental procedure is as follows:

[0030] (1) Inoculate the cell suspension in a 24-well plate, and place the culture plate in an incubator for pre-cultivation for 12-24 hours (incubator at 37°C, 5% CO 2 );

[0031] (2) Take 445 μL of the dil...

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Abstract

The invention discloses an expression up-regulator of LncRNA MALAT1. The up-regulator is thapsigargin. Thapsigargin can regulate the expression of MALAT1, solve the technical problem of high expression of MALAT1 in cells, reduce costs, and can promote the metastasis of tumor cells, thereby improving the success rate of cell metastasis models, which is beneficial to the study of tumor publishing mechanisms and The establishment of tumor metastasis model is of great significance.

Description

technical field [0001] The invention belongs to the technical field of tumors, and in particular relates to an upregulator for promoting the expression of LncRNA MALAT1 in tumor cells and an application thereof. Background technique [0002] Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a long non-coding RNA, which is one of the first long non-coding RNAs found to have functions. The function of MALAT1 has been reported by most researches in recent years. In the field of tumors, the expression of MALAT1 is positively correlated with the proliferation and metastasis of various malignant tumors (lung cancer, breast cancer, gastric cancer, colorectal cancer, prostate cancer, etc.). Recently, MALAT1 was also found to be involved in the occurrence of Parkinson's, atherosclerosis and diabetic vascular lesions. However, the mechanism by which MALAT1 regulates the occurrence and progression of diseases has not yet been fully elucidated. [0003] The human MAL...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/30
Inventor 姜霞尹亚娟
Owner THE FIRST HOSPITAL OF HEBEI MEDICAL UNIV
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