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Application of serum microRNA in diagnosis of liver cancer and diagnostic kit

A diagnostic kit, hsa-mir-192 technology, applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problem of inability to comprehensively and objectively reflect circulating microRNA, difficult to explain marker diagnostic markers, and lack of multi-center Validation and other issues to achieve the effect of saving time and labor costs, mature experimental methods, and easy access

Active Publication Date: 2019-12-17
江苏阔然生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the current research on circulating microRNA as a diagnostic marker for liver cancer still has the following deficiencies: (1) Most studies only select microRNAs that have been reported to be dysregulated in liver cancer tissues as candidate indicators, which may not be able to fully and objectively reflect the situation of circulating microRNAs ; (2) The sample size of some studies is small and lacks multi-center validation; (3) The control settings are not perfect, and it is difficult to explain that the identified markers are specific diagnostic markers for liver cancer

Method used

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  • Application of serum microRNA in diagnosis of liver cancer and diagnostic kit
  • Application of serum microRNA in diagnosis of liver cancer and diagnostic kit
  • Application of serum microRNA in diagnosis of liver cancer and diagnostic kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Collection and preparation of serum samples

[0056] The inventor collected healthy people (HC), hepatitis B carriers (IHC), chronic hepatitis B patients (CHB), liver cirrhosis patients (LC) and liver cancer patients (HCC) from August 2009 to August 2014. These populations met the inclusion criteria (Table 1), and according to the principle of gender and age matching, liver cancer and its control group specimens were set.

[0057] Training group: 257 serum samples collected from healthy people (51 cases), chronic hepatitis B patients (51 cases), liver cirrhosis patients (47 cases) and liver cancer patients (108 cases) from August 2009 to March 2012 .

[0058] Validation group 1: 352 serum samples from healthy people (60 cases), chronic hepatitis B patients (68 cases), liver cirrhosis patients (71 cases) and liver cancer patients (153 cases) collected from April 2012 to April 2013 specimen.

[0059] Validation group 2: 139 serum samples from healthy people ...

Embodiment 2

[0068] Example 2: qPCR Array and its data analysis

[0069] Serum samples from 6 patients with liver cancer before operation and 8 patients with chronic hepatitis B at least one year before the last examination were selected for qPCR Array screening. These patients were all male, and there was no significant difference in mean age and distribution (Table 3).

[0070] Table 3. Clinicopathological characteristics of specimens used for qPCR Array analysis

[0071]

[0072] The present invention adopts Applied Biosystems company's The Array Human MicroRNA method screens microRNAs that differ between liver cancer and chronic hepatitis B, and detects the levels of 754 known human microRNAs. See the Applied Biosystems website for specific steps. After the original data obtained were calibrated, the Significant Analysis of Microarray (SAM) analysis method was used to select differential microRNAs, and finally 19 candidate microRNAs for subsequent verification were screened (Tab...

Embodiment 3

[0076] Example 3: Real-time fluorescence quantitative PCR detection training group specimen microRNA level

[0077] 1.1 Extraction of serum RNA

[0078] The present invention uses Trizol reagent to extract, and obtains serum RNA through the method of phenol / chloroform extraction and purification, isopropanol precipitation, and glycogen-assisted precipitation. The specific steps are as follows:

[0079] 1. Take 200 μl of serum, preferably add an equal volume of double-stranded RNA mixed with cel-miR-67 (NC67, based on the mature body sequence of nematode miR-67 that has no homology with the human genome sequence, and the final concentration is 0.2 nM, the sequence is shown in Table 4) of Trizol lysate, fully vortexed and mixed, and ice-bathed for 15 minutes.

[0080] 2. Add 100 μl of pre-cooled chloroform, shake and mix well, and centrifuge at 12,000 g for 15 min at 4°C.

[0081] 3. Transfer the supernatant, add an equal volume of pre-cooled phenol / chloroform (1:1), shake and...

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Abstract

The invention discloses a hepatocellular carcinoma diagnosis marker combination composed of serum microRNA and a kit comprising the marker combination. The hepatocellular carcinoma diagnosis marker combination comprises following microRNA: hsa-miR-193a-5p, hsa-miR-143, hsa-miR-145, hsa-miR-29a, hsa-miR-133a, hsa-miR-505, hsa-miR-29c and hsa-miR-192. The hepatocellular carcinoma diagnosis marker combination and the kit can be used for liver cancer diagnosis.

Description

technical field [0001] The present invention belongs to the field of biomedical diagnosis, and in particular relates to a liver cancer diagnostic combination composed of serum microRNA, said serum microRNA including hsa-miR-193a-5p, hsa-miR-143, hsa-miR-145, hsa-miR-29a , hsa-miR-133a, hsa-miR-505, hsa-miR-29c, hsa-miR-192, and a kit for diagnosing liver cancer and applications thereof. Background technique [0002] Primary liver cancer is a common malignant tumor with high morbidity and high lethality worldwide, among which hepatocellular carcinoma (referred to as liver cancer, Hepatocellular Carcinoma, HCC) accounts for more than 90% of primary liver cancer. According to the "Global Cancer Report 2014" issued by the World Health Organization, in 2012, the new cases of liver cancer in the world ranked fifth among malignant tumors, and its fatality rate ranked second. Among them, the new cases and deaths of liver cancer in China were about accounted for half of the global c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 庄诗美林雪嘉方坚鸿郭智伟
Owner 江苏阔然生物医药科技有限公司
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