miRNA marker related to malignant transformation of colitis and proctitis and kit

A marker and kit technology, applied in the fields of biotechnology and medicine, to achieve high specificity, high sensitivity, and simple operation.

Active Publication Date: 2014-08-06
江西佰延生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, studies have found that the occurrence of tumors is associated

Method used

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  • miRNA marker related to malignant transformation of colitis and proctitis and kit
  • miRNA marker related to malignant transformation of colitis and proctitis and kit
  • miRNA marker related to malignant transformation of colitis and proctitis and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Colitis-related cancer model Muc2 gene knockout mouse miRNA expression profile in colonic epithelial cells

[0040]Collection of intestinal epithelial cells from mice: select 3-month-old Muc2+ / + and Muc2- / -, each group includes four mice, kill the mice, wash the colon, cut it longitudinally, and place a section of each in 20ml ice-cold PBS; transfer the tissue into 30ml 15mM EDTA at 37°C; shake at 37°C, 220rpm, 30min, fully shake to remove intestinal tissue; centrifuge at 5000rpm, 5min at 4°C; first discard the supernatant, resuspend the cells in 5ml cold PBS, and aliquot Put in a 1.5ml EP tube; centrifuge at 4°C, 3000rpm, 5min; discard the supernatant, uncap the EP tube and place in liquid nitrogen; take it out and place it at -80°C overnight, and cap the EP tube the next day.

[0041] Extraction of total RNA: total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) according to the operation manual.

[0042] miRNA array (expression profile): T...

Embodiment 2

[0044] Example 2 Expression of cytokines and verification of differentially expressed miRNA in colonic epithelial cells of Muc2 mice

[0045] Mouse colonic epithelial cell RNA extraction is the same as in Example 1;

[0046] qRT-PCR was used to detect the expression level of cytokine mRNA, reaction system: forward primer (20 μm) 0.15 μl, reverse primer (20 μm) 0.15 μl, cDNA 0.7 μl, H 2 O9 μl, fluorescent dye SYBR Green (2×) 10 μl; reaction conditions: 50°C for 2min, 95°C for 2min; denaturation at 95°C for 15sec, annealing / extension at 60°C for 1min, a total of 40 cycles.

[0047] Cytokine mRNA expression levels as figure 2 Shown: Compared with the control, the expression levels of several cytokines IL-6, COX-2, IL-10, TNFα, IL-1β in the colonic epithelial cells of Muc2- / - mice were significantly increased.

[0048] IL-6:

[0049] Forward primer: TAGTCCTTCCTACCCCAATTTCC,

[0050] Reverse primer: TTGGTCCTTAGCCACTCCTTC;

[0051] COX-2:

[0052] Forward primer: TGAGCAACTATT...

Embodiment 3

[0104] Example 3 collection of human colon cancer tissue samples

[0105] 16 pairs of colorectal cancer tissues, 6 pairs of colitis tissues and their paired normal tissues were collected from the First Affiliated Hospital of Xinxiang Medical College and the Affiliated Central Hospital of Xinxiang Medical College, and the specimens were stored at -80°C.

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Abstract

The invention belongs to the field of biotechnology and medical technology and provides a miRNA marker related to malignant transformation of colitis and proctitis. The marker is a composition of miR-138-5p, miR-145-5p, miR-146a-5p and miR-150-5p. The invention further provides a primer of the miRNA marker, application of the miRNA marker in preparation of a rectal cancer early-diagnosis kit, and the rectal cancer early-diagnosis kit. The provided miRNA marker, including miR-138-5p, miR-145-5p, miR-146a-5p and miR-150-5p and related to malignant transformation of colitis and proctitis is used for early screening of the colon cancer and the rectal cancer and has the characteristics of easiness in operation, high specificity and high sensitivity.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medical technology, and in particular relates to the application of a miRNA marker related to malignant transformation of colorectal inflammation to early screening of rectal cancer. Background technique [0002] Colorectal cancer is the third most common malignancy and the second leading cause of cancer death. Like other malignant diseases, genetic factors are an important cause of colorectal cancer, however, only 20% of colorectal cancer cases can be traced to familial gene mutations. In fact, most sporadic colorectal cancers are closely related to environmental factors, the most common mutation of the tumor suppressor gene APC leads to the destruction of the Apc / GSK3β / Axin complex, thereby activating the Wnt / β-catenin signal transduction pathway . Abnormal activation of the Wnt / β-catenin signal transduction pathway not only promotes the proliferation of small intestinal epithelial cells, but...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 鲍永华杨万才郭永臣李泽信薛会朝朱绍辉徐红伟游焜韩荣飞贾慧婕李凯王倩
Owner 江西佰延生物技术有限公司
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