Application of NDUFA4L2 in drug resistance of herceptin

A drug resistance and drug technology, applied in the field of genetic engineering, can solve problems such as the reduction of antibody drug potency, drug resistance in patients, and disease progression

Pending Publication Date: 2021-08-27
JIANGSU CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] ① Increased expression of HER-1, HER-2 or HER-3 on the surface of tumor cells leads to a decrease in the potency of antibody drugs;
[0009] In order to reverse the drug resistance of Herceptin, there are currently clinical drugs developed for the drug resistance mechanism, such as HER-2 tyrosine kinase inhibitor (TKI) lapatinib, blocking HER-2/HER-3 Pertuzumab formed by heterodimers, etc. These drugs can be combined with Herceptin to improve the efficacy of Herceptin, but patients will eventually develop drug resistance and lead to disease progression
[0010] Existing studies can only partially clarify that the a

Method used

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  • Application of NDUFA4L2 in drug resistance of herceptin
  • Application of NDUFA4L2 in drug resistance of herceptin
  • Application of NDUFA4L2 in drug resistance of herceptin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Detect the expression of differential genes in BT474 and BT474HR cells:

[0031] (1) Method: Real-time PCR, Western blot and cell immunofluorescence;

[0032] (2) Determination of results: if Figure 1-A to Figure 1-D shown.

[0033] (3) Analysis of results: In Herceptin-resistant cells BT474HR cells, the expression of NDUFA4L2 was significantly increased, mainly located in the mitochondria of cells.

Embodiment 2

[0035] Use CyQuant kit to detect the effect of BT474, BT474HR and BT474, SKBR3 overexpressing NDUFA4L2 on sensitivity to Herceptin:

[0036] (1) Cell inoculation: digest monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with DMEM culture medium containing 10% fetal calf serum, inoculate 4000 cells per well in a 96-well culture plate, and Pore ​​volume 200ul.

[0037] (2) Culture cells: move the culture plate into CO 2 In an incubator at 37°C, 5% CO 2 : and relative humidity conditions, culture 24-96h;

[0038] (3) Color development: After 24-96 hours of culture, add 100ul of 2×CyQuant reagent to each well, and incubate at 37°C for 1 hour.

[0039] (4) Colorimetry: use 480 / 535nm wavelength to measure the optical density value (OD), each group has 3 duplicate wells, and the experiment is repeated three times. Calculate the survival rate. Cell proliferation rate=OD value of experimental group / OD value of control group×100%.

[0040] BT474&BT474HR...

Embodiment 3

[0044] The changes of ATP after overexpression of BT474, BT474HR and BT474 NDUFA4L2 were detected.

[0045] (1) Inoculation of cells: Digest monolayer cultured cells with 0.25% trypsin, prepare a single cell suspension with DMEM medium containing 10% fetal bovine serum, and inoculate 10,000 cells per well in a 6-well culture plate.

[0046] (2) Culture cells: move the culture plate into CO 2 In an incubator at 37°C, 5% CO 2 : and relative humidity conditions, culture 24-96h;

[0047] (3) Cell lysing: collect the same number of cells, add cell lysate, after fully lysing, extract 10ul supernatant from each group and test on the machine;

[0048] (4) Colorimetry: Make a standard reaction curve as the background fluorescence value, subtract the background value from the value in the sample, and draw the corrected result for the control sample;

[0049] BT474, BT474HR, and BT474 overexpressed NDUFA4L2 were planted on 6-well plates as above, 10,000 cells / well. After treatment wi...

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to an application of NDUFA4L2 in preparation of medicines for reversing breast cancer herceptin resistance. By constructing a breast cancer stable cell strain resistant to herceptin, NDUFA4L2 can play a role in reversing the drug resistance of HER-2 positive breast cancer cells to herceptin at the cellular level. According to the invention, firstly, whole genome analysis is carried out on herceptin sensitive or drug-resistant cells, 487 probes are adopted in total, FDR correction is carried out, 453 differentially expressed genes are found, NDUFA4L2 is the gene with the most significant difference, and the sensitivity to herceptin is regulated and controlled through energy metabolism and oxidative stress level of NDUFA4L2. When being applied clinically, the herceptin gene can contribute to formulation of a treatment scheme of a clinician, prognosis judgment and become a target for research and development of related drugs in the future, the curative effect of herceptin is improved, and the herceptin has a good clinical popularization value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to the effect of overexpression of NDUFA4L2 participating in reversing the drug resistance of HER-2 positive breast cancer cells to Herceptin. Background technique [0002] Breast cancer is the most common malignant tumor in women, and its incidence rate is increasing year by year. It ranks first in the death of female tumors. Among them, HER-2 positive patients account for 20-30%, and the prognosis of these patients is poor. [0003] Herceptin is a humanized monoclonal antibody targeting the extracellular domain of the receptor tyrosine kinase encoded by HER-2, and is currently the most commonly used clinical drug for treating HER-2 positive breast cancer patients. [0004] Although Herceptin improved the survival of patients with HER-2-positive breast cancer, more than 70% of patients developed drug resistance within 1 year after treatment. Therefore, the drug resist...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/02
CPCC12Q1/6886C12Q2600/106C12Q2600/158G01N33/5011
Inventor 袁渊娄可心张喆
Owner JIANGSU CANCER HOSPITAL
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