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Gene inhibitor for promoting apoptosis of colon cancer cells and inhibiting migration of colon cancer cells

A colon cancer cell and gene suppression technology, applied in the field of biomedicine, can solve problems such as unreported effects, and achieve the effect of promoting apoptosis, inhibiting migration and invasion

Active Publication Date: 2020-10-09
CELLYAN THERAPEUTICS WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the role of long non-coding RNA LINC02360 in colon cancer and other malignant tumors has not been reported

Method used

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  • Gene inhibitor for promoting apoptosis of colon cancer cells and inhibiting migration of colon cancer cells
  • Gene inhibitor for promoting apoptosis of colon cancer cells and inhibiting migration of colon cancer cells
  • Gene inhibitor for promoting apoptosis of colon cancer cells and inhibiting migration of colon cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The expression difference of LINC02360 in normal colon epithelial cell line FHC and colon cancer cells SW480, SW620, HCT116 and HT29 was detected.

[0023] 1. Extraction of RNA

[0024] (1) Inoculate FHC cells, SW480, SW620, HCT116 and HT29 in a 6-well plate. After culturing for 48 hours, wash the cells twice with PBS, then add 1mL Trizol to each well, and repeatedly blow and beat the cells to fully mix the Trizol and the cells. Transfer the mixture to an EP tube and let it stand at room temperature for 5 minutes;

[0025] (2) Centrifuge at 12000r / min at 4°C for 5 minutes, transfer the supernatant to a new RNase-free EP tube, add 200 μL of pre-cooled chloroform to each EP tube, invert and mix, and place at room temperature for 10 minutes;

[0026] (3) 4°C, 12000r / min, centrifuge for 5min, transfer the supernatant to a new RNase-free EP tube, add an equal volume of pre-cooled isopropanol, mix well, and let stand at 4°C for 10min;

[0027] (4) 4°C, 12000rpm / min, centrif...

Embodiment 2

[0047] Detection of the inhibitory effect of si-LINC02360 gene inhibitor on LINC02360

[0048] si-LINC02360 and Control siRNA sequences are described below:

[0049] Control siRNA 5'-UUCUCCGAACGUGUCACGU- 3' (SEQ ID NO.5)

[0050] 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO. 6)

[0051] si-LINC02360 5'-GAAUGAUGUGAAUGGUUAACC-3' (SEQ ID NO. 7)

[0052] 5'-UUAACCAUUCACAUCAUUCAG-3' (SEQ ID NO.8)

[0053] (1) Inoculate SW480 cells on cell plates, transfect according to the instructions of lip2000, and divide them into control group, controlsiRNA group, and si-LINC02360 group, with 3 replicates in each group;

[0054] (2) After 6 hours of transfection, the transfection medium was removed and replaced with normal medium. After 48 hours of culture, RNA was extracted, reverse transcription, and fluorescent quantitative PCR. The specific experimental steps refer to Example 1.

[0055] Experimental results such as figure 1 As shown, it can be seen from the figure that the control siRNA grou...

Embodiment 3

[0057] Effect of LINC02360 on colon cancer cell apoptosis detected by flow cytometry

[0058] (1) Inoculate SW480 cells on 6-well cell plates, transfect according to the instructions of lip2000, and divide them into controlsiRNA group and si-LINC02360 group, with 3 replicates in each group;

[0059] (2) Digest the cells with trypsin, count the cells, and take 1×10 6 cells, centrifuge at 1000 rpm for 10 min at 4°C, and discard the supernatant;

[0060] (3) Add 1ml of pre-cooled PBS, shake gently to suspend the cells, centrifuge at 1000rpm at 4°C for 10min, discard the supernatant, repeat 3 times;

[0061] (4) Resuspend the cells in 200 μL Binding Buffer, add 10 μL Annexin V-FITC, mix gently, and react at room temperature for 15 minutes in the dark;

[0062] (5) Add 300 μL Binding Buffer and 5 μL PI to the machine for detection.

[0063] Experimental results such as image 3 As shown, it can be seen from the figure that inhibiting LINC02360 can significantly increase the numbe...

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Abstract

The invention provides a gene inhibitor for promoting apoptosis of colon cancer cells and inhibiting migration of the colon cancer cells, and belongs to the technical field of biomedicine. Experimentsprove that the LINC02360 gene inhibitor can promote apoptosis of colon cancer cells, and migration and invasion of the colon cancer cells can be inhibited by regulating protein expression of E-cadherin and N-cadherin. Therefore, the siRNA of the LINC02360 can be used for preparing a medicine for promoting apoptosis of colon cancer cells and can also be used for preparing a medicine for inhibitingmigration and invasion of the colon cancer cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a gene inhibitor used for preparing colon cancer cell apoptosis and inhibiting colon cancer cell migration. Background technique [0002] Colon cancer is a common malignant tumor in the gastrointestinal tract. In recent years, with the changes in our diet structure and living habits, the incidence and mortality of colon cancer in my country have maintained an upward trend. Although the surgical treatment, adjuvant therapy, targeted therapy and other treatment methods of colon cancer have made great progress, the prognosis of patients with advanced colon cancer is still not very optimistic. At present, there is still a lack of specific therapeutic drugs. [0003] Non-coding RNA is a type of RNA molecule discovered in recent years that is ubiquitous in organisms and plays an important role in life activities. Unlike mRNA, tRNA and rRNA, it does not participate in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/713A61P35/04C12N15/113
CPCA61K31/713A61P35/04C12N15/113C12N2310/17
Inventor 唐正晓
Owner CELLYAN THERAPEUTICS WUHAN CO LTD
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