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Serum microRNA liver cancer diagnosis marker and kit

A technology for diagnostic markers and diagnostic kits, applied in the field of biomedicine, can solve the problems of inability to fully and objectively reflect circulating microRNAs, difficult to explain markers for diagnostic markers, imperfect control settings, etc., so as to save time, labor costs, avoid The previous complicated detection, the effect of mature experimental methods

Active Publication Date: 2016-06-15
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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  • Serum microRNA liver cancer diagnosis marker and kit
  • Serum microRNA liver cancer diagnosis marker and kit
  • Serum microRNA liver cancer diagnosis marker and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Collection and preparation of serum samples

[0051] 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.

[0052] 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 .

[0053] 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.

[0054] Validation group 2: 139 serum samples from healthy people ...

Embodiment 2

[0063] Embodiment 2: qPCR Array and its data analysis

[0064] Serum samples from 6 patients with liver cancer before surgery 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).

[0065] Table 3. Clinicopathological characteristics of specimens used for qPCR Array analysis

[0066]

[0067] The present invention adopts AppliedBiosystems company's The ArrayHumanMicroRNA method screens microRNAs that differ between liver cancer and chronic hepatitis B, and detects the levels of 754 known human microRNAs. See the AppliedBiosystems website for specific steps. After the obtained raw data 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 (Table 4). ...

Embodiment 3

[0071] Example 3: Real-time fluorescence quantitative PCR detection training group specimen microRNA level

[0072] 1.1 Extraction of serum RNA

[0073] 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:

[0074] 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.

[0075] 2. Add 100 μl of pre-cooled chloroform, shake and mix well, and centrifuge at 12,000 g for 15 min at 4°C.

[0076] 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: has-miR-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa-miR-192 and hsa-miR-505. The hepatocellular carcinoma diagnosis marker combination and the kit can be used for liver cancer diagnosis.

Description

technical field [0001] The 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-145, hsa-miR-29a, hsa-miR-29c, hsa-miR-133a, hsa -miR-192 and hsa-miR-505, and a kit for the diagnosis of liver cancer and its application. Background technique [0002] Primary liver cancer is a common malignant tumor with high morbidity and mortality 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 cases. Due to the lack of effective scr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
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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