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miRNA marker related to liver cancer sorafenib resistance and application of miRNA marker

A sorafenib and fenib-resistant technology, applied in the fields of biology and oncology, can solve problems such as unclear mechanism, and achieve the effect of strong applicability, reliable source and easy detection

Inactive Publication Date: 2020-06-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite efficacy, sorafenib resistance develops soon after initial treatment, but mechanism remains unclear

Method used

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  • miRNA marker related to liver cancer sorafenib resistance and application of miRNA marker
  • miRNA marker related to liver cancer sorafenib resistance and application of miRNA marker
  • miRNA marker related to liver cancer sorafenib resistance and application of miRNA marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 miRNA screening of liver cancer sorafenib resistance

[0046] Total RNA Extraction

[0047] (1) Add Trizol to Sorafenib-resistant Huh7 cell line and normal Huh7 cell line, and store at room temperature for 5 minutes

[0048] (2) Add 0.2ml of chloroform, mix thoroughly after shaking, and place at room temperature for 5-10min

[0049] (3) Centrifuge at 12,000 rpm for 15 minutes at high speed, transfer the upper aqueous phase to another new centrifuge tube, and be careful not to absorb the protein between the two aqueous phases. Transfer to a new tube, add an equal volume of -20 ℃ pre-cooled ℃ pre-cooled isopropanol, fully invert and mix, and place on ice for 10 minutes;

[0050] (4) After centrifuging at 12000rpm for 15min at high speed, carefully discard the supernatant, add 75% DEPC ethanol at a ratio of 1ml / ml Trizol to wash the precipitate (store at 4°C), wash the precipitate, shake and mix, and centrifuge at 12000rpm at 4°C for 5min at high speed;

[005...

Embodiment 2

[0054] Example 2 QPCR verification of differentially expressed miRNAs

[0055] (1) According to the detection results of the miRNA chip, miRNA378a-3p, miRNA-486-3p, miRNA-328-3p, miRNA-378a-5p, and miRNA-671-3p were selected for QPCR verification on cell lines. According to the total RNA extraction method in Example 1, Sorafenib-resistant Huh7 and normal Huh7, Sorafenib-resistant HepG2 and normal HepG2,2 were subjected to total RNA extraction from liver cancer cell lines.

[0056] (2) reverse transcription RNA

[0057] Mix 10pg-1μg of total RNA template with 2μl 10* buffer, 2μl dATP (10mM), 0.5μl polyA polymerase, 0.5μl ribonuclease (RNase) inhibitor, and RNase free water (RNase free water), volume last 20 μl and incubated at 37°C for 1h. Then add 1 μl 0.5 μg / μl Oligo (dT)-specific RT primer (purchased from GeneCopoeia, Cat. No. QP017 / QP018) to the reaction tube, incubate at 70°C for 5 min and immediately incubate on ice for at least 2 min to interrupt the interaction betwee...

Embodiment 3

[0062] The inventor collected liver cancer tissue specimens and adjacent normal liver tissue from 40 liver cancer patients in Run Run Shaw Hospital affiliated to Zhejiang University School of Medicine. All 40 patients were treated with sorafenib. The expression level of miRNA-486-3p in liver cancer tissues and adjacent tissues was detected.

[0063] After liver cancer tissue and paracancerous tissue were homogenized and crushed, total RNA was extracted according to the total RNA extraction step in Example 2. Follow the above steps to perform reverse transcription of RNA into cDNA, QPCR reaction, and result analysis. The result is as image 3 As shown, the expression of miRNA-486-3p in liver cancer tissues treated with sorafenib was significantly lower than that in adjacent normal tissues.

[0064] The relationship between miRNA-486-3p and survival and prognosis of liver cancer was analyzed in the liver cancer database. Such as Figure 4 As shown in Table 1, it was found t...

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Abstract

The invention discloses a miRNA marker related to drug resistance of liver cancer sorafenib, wherein the miRNA marker is miRNA-486-3p. In the invention, liver cancer cell lines Huh7 and hepG2 are firstly induced by sorafenib for a long term to make the cell lines Huh7 and hepG2 become liver cancer cell lines resistant to the sorafenib, then differentially expressed miRNA in normal and drug-resistant liver cancer cell lines is screened by miRNA chip analysis, through QPCR verification, a target band is determined by dissociation curve analysis and electrophoresis, a deltadeltaCT method is usedfor relative quantitative analysis, and finally the miRNA marker miRNA-486-3p is selected. The marker and detection reagents can be used to prepare a diagnostic kit for the early diagnosis of the liver cancer sorafenib resistance.

Description

technical field [0001] The present invention relates to the fields of biology and oncology, in particular to a miRNA marker related to liver cancer sorafenib resistance and its application; in particular to liver cancer-derived miRNA-486-3p and its role in liver cancer sorafenib resistance application in medicine. Background technique [0002] Liver cancer is the most common malignant tumor in the world, causing more than 700,000 deaths every year. In China, liver cancer is the fourth most common malignancy and ranks third among tumor-related fatal diseases. Due to the prevalence of hepatitis B virus in China and the large number of liver cancer patients, about 50% of the new liver cancer patients in the world in 2012 came from China. Patients with early liver cancer have a better prognosis after receiving appropriate treatment, with a 5-year survival rate of 60%-80%. However, due to the lack of effective early diagnosis, many patients have already reached the advanced st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/113
CPCC12Q1/6851C12Q1/6886C12Q2600/106C12Q2600/158C12Q2600/178C12Q2531/113C12Q2521/107C12Q2563/107C12Q2525/207
Inventor 蔡秀军徐俊杰季琳林中杰岑栋梁霄姜是
Owner ZHEJIANG UNIV
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