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Application of targeted exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer (NSCLC)

A non-small cell lung cancer, PKM2 technology, applied in the biological field, can solve problems such as hindering the treatment effect, and achieve the effect of enhancing chemotherapy sensitivity, inducing cell apoptosis, and improving cisplatin resistance.

Active Publication Date: 2021-01-05
SHANGHAI UNIV OF MEDICINE & HEALTH SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, clinical drug resistance remains a challenge, greatly hampering therapeutic efficacy

Method used

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  • Application of targeted exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer (NSCLC)
  • Application of targeted exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer (NSCLC)
  • Application of targeted exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer (NSCLC)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Induction of cisplatin-resistant cells and detection of cisplatin resistance under hypoxic conditions

[0075] (1) Use non-small cell lung cancer cell A549 as the parental cell line, add cisplatin to the final concentration of 2ng / ml each time of subculture, and then repeat the subculture and dosing until the cells basically adapt to no death, and gradually increase the concentration of cisplatin , cisplatin-resistant cell line A549 / CR was obtained after 10 months.

[0076] (2) Detect the half inhibitory rate (IC50) of cisplatin in the sensitive cell A549 and drug-resistant cell A549 / CR. First, the survival rate and inhibition rate of each cell at each concentration are obtained by the CCK8 method, and then the cisplatin half inhibitory rate is obtained by Graphpad Prism 7 The software makes an inhibitory dose-response curve with a variable slope, and calculates the IC50 value. The results are shown in Figure 1A .

[0077] (3) The method for CCK8 to detect t...

Embodiment 2

[0079] Example 2 Overexpression of PKM2 in Sensitive Cells and Detection of Cell Proliferation

[0080] (1) The coding sequence of the PKM2 gene (SEQ ID NO: 1) was cloned into the pcDNA4TO-Flag vector, and a plasmid overexpressing PKM2 was obtained after confirmation by sequencing. The extraction of the plasmid was carried out according to the instructions of the reagent manufacturer.

[0081] (2) Sensitive cells A549 were planted in 6-well plate 4×10 5 Each cell / well was transfected with 2 μg of PKM2 overexpression plasmid or control empty plasmid, respectively. After 24 hours the cells were digested and the cells were pressed for 10 4 Cells / well were seeded into 96-well plates, and the cell proliferation was detected by the method of CCK8.

[0082] (3) Cell proliferation was detected by the clone formation method. The specific method was as follows: 200 cells / well of the sensitive cell A549 were planted in a 12-well plate, transfected with 1 μg of PKM2 overexpression plasm...

Embodiment 3

[0083] Example 3 Knockdown of PKM2 in drug-resistant cells and detection of cell proliferation

[0084] (1) Design two shRNA sequences targeting PKM2 (shRNA sequence #1 is SEQ ID NO:2 and SEQ ID NO:3, shRNA sequence #2 is SEQ ID NO:4 and SEQ ID NO:5), and synthesize DNA The fragment was inserted into the LV3 shuttle vector to obtain a shuttle plasmid carrying the PKM2 shRNA sequence. The shuttle plasmid and packaging plasmid (pGag / Pol, pRev, pVSV-G) were co-transfected into 293T cells, and the lentivirus in the supernatant was collected 72 hours after transfection and the virus titer was measured.

[0085] (2) The lentivirus carrying the PKM2 shRNA sequence was infected into drug-resistant cells A549 / CR, and the A549 / CR cell line stably knocking down PKM2 was obtained by puromycin selection.

[0086] (3) A549 / CR cells stably knocking down PKM2 and control cells were mixed at 10 per well 4 Cells were inoculated into 96-well plates for CCK8 experiments, and 200 cells per well ...

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Abstract

The invention relates to application of a targeted exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer (NSCLC). A tumor hypoxic region is often accompanied by drug resistance, as the expression of pyruvate kinase M2 type (PKM2) in cisplatin-resistant NSCLC cells is higher than that of sensitive cells, the tumor hypoxic region often accompanies drug resistance, the PKM2 gene is interfered in the cisplatin-resistant cells, the level of active oxygen in the cisplatin-resistant cells is increased, cell apoptosis is induced, sensitization of drug-resistant cell treatment is achieved, and he important clinical application value is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an application of targeting exosome PKM2 to improve cisplatin resistance in non-small cell lung cancer. Background technique [0002] Lung cancer is one of the malignant tumors that threaten human health and life, and about 85% of the patients are non-small cell lung cancer (NSCLC). Chemotherapy is the main treatment for lung cancer, and platinum-based chemotherapy has become the standard treatment for patients with advanced NSCLC. However, clinical drug resistance remains a challenge, greatly hindering therapeutic efficacy. Therefore, a more comprehensive understanding of drug resistance mechanisms has important implications for the treatment of NSCLC patients. [0003] Exosomes play an important role in the tumorigenesis of lung cancer. Exosomes are membrane-bound vesicles produced by late endosomes, ranging in size from 40–160 nm, that fuse with the plasma membrane and are rele...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N15/54A61K45/00A61K31/7105A61K48/00A61P35/00
CPCA61K31/7105A61K45/00A61P35/00C12N5/0693C12N9/1205C12N15/86C12N2510/00C12N2740/15043C12Y207/0104
Inventor 杨浩黄钢王栋梁解伟张坤驰孔平梁蓓蓓
Owner SHANGHAI UNIV OF MEDICINE & HEALTH SCI
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