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Application of mir-29a gene in detecting liver cancer and liver fibrosis, and construction method of gene conditional knock-in mice

A mir-29a, 1.mir-29a technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, genetic engineering, etc.

Pending Publication Date: 2020-09-11
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no international reports on the function or application of miR-29a in Schistosoma japonicum liver fibrosis

Method used

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  • Application of mir-29a gene in detecting liver cancer and liver fibrosis, and construction method of gene conditional knock-in mice
  • Application of mir-29a gene in detecting liver cancer and liver fibrosis, and construction method of gene conditional knock-in mice
  • Application of mir-29a gene in detecting liver cancer and liver fibrosis, and construction method of gene conditional knock-in mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Analysis of the expression of mir-29a in human liver cancer tissues and liver cancer cell lines and its effect on liver cancer cells

[0042] 1. Detection of miR-29a expression level in human normal liver tissue / hepatocellular carcinoma / paracancerous tissue specimens and liver cancer / normal liver cell lines

[0043] Human liver samples were collected and divided into normal liver tissue (n=12), liver cancer group (n=18) and paracancerous group (n=18). RT-PCR method was used to detect the expression level of miR-29a in human normal liver tissue, liver cancer and paracancerous tissue samples, and GraphPadPrism 5 software was used to analyze and count whether there were significant differences in the relative expression levels among the four groups; the liver cell lines were cultured under the above conditions (L02, HepG2, Huh7, SMMC7721), RT-PCR method was used to detect the expression level of miR-29a in normal liver cells LO2, liver cancer cell lines HepG2, Hu...

Embodiment 2

[0058] Example 2 Effect of mir-29a acting on hepatic stellate cells on liver fibrosis in Schistosoma japonicum mice

[0059] 1. Establishment of liver fibrosis model of Schistosoma japonicum mice

[0060] C57BL / 6 mice (6-8 weeks), 10 in each group were randomly divided into experimental groups (infection 4W, 6W, 8W, 12W) and control groups (infection 0W), the infection operation steps were as follows: (1) The snails with cercariae were placed under light, water temperature water, The cercariae can be seen escaping and floating on the water surface. (2) Pick a little water containing cercariae from the front end of the inoculation loop and put it on the cover glass, and count (16±1) under the dissecting microscope. (3) Remove 1×1cm of mouse abdomen 2 Rat hair, attach the slide to the abdomen for 15-20 minutes. The control group was similarly treated with sterile saline. (4) Raised in a P3 animal experiment center.

[0061] 2. Pathological staining of liver tissue of mo...

Embodiment 3

[0070] Example 3 Preparation and application of mir-29a gene conditional knock-in mice (H11-MIR29A-cKI mice)

[0071] 1. Preparation of mir-29a gene conditional knock-in mice (H11-MIR29A-cKI mice)

[0072] 1. Construction of human MIR29A-H11-cKI donor vector

[0073] 1) Synthesize the mir-29a gene sequence (NCBI Reference Sequence: NR_029503.1), as shown in SEQ No.1;

[0074] 2) Acquisition of the connecting skeleton: construct the basic skeleton of the H11 site (the Hipp11 (H11) locus is located in the intergenic region between the Eif4enif1 and Drg1 genes on mouse chromosome 11), and name it VB027; use the AsiSI / PacI double enzyme Cut the VB027 vector backbone, and recover the 11.5kb fragment by gel;

[0075] 3) Acquisition of the ligated fragments: use EcoRI to digest the Mir-29 gene synthesis product to synthesize a plasmid, and recover the fragments from the gel;

[0076] 4) Use In-fusion ligase to connect the fragment + backbone according to the operation guide of NEB...

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Abstract

The invention provides an application of a mir-29a gene in detecting primary hepatocellular carcinoma, and an application of the mir-29a gene in detecting Schistosoma japonicum liver fibrosis. The invention also provides an application of mir-29a gene conditional knock-in mice in the study of primary hepatocellular carcinoma and / or Schistosoma japonicum liver fibrosis. The invention confirms thatthe expression of mir-29a is significantly reduced in liver cancer, paracancerous and liver cancer cell lines, and the result suggests that the low-level expression of mir-29a may be related to the occurrence and development of liver cancer cells. In addition, the mir-29a conditional knock-in mice can be used as animal models for the study of liver fibrosis caused by primary hepatocellular carcinoma and chronic schistosomiasis infection or advanced schistosomiasis, and thereby the evidence and basis are provided for the follow-up study of drugs for treating liver fibrosis caused by hepatocellular carcinoma and schistosomiasis.

Description

【Technical field】 [0001] On the one hand, the present invention belongs to the field of gene function and application, and particularly relates to the application of mir-29a gene in detecting primary hepatocellular carcinoma and Schistosoma japonicum liver fibrosis; its application. 【Background technique】 [0002] The MiR-29 family (including miR-29a, miR-29b, and miR-29c) is cut from the corresponding shape of the precursor miRNA stem-loop structure or pre-miRNA hairpin structure, mostly from the 3' of pre-miRNA One arm in the (3p) end, a small part comes from its 5' (5p) end. The MiR-29 family is distributed on two chromosomes, miR-29b2 and miR-29c are distributed on 1q32, and miR-29b1 / miR-29a are distributed on 7q32. Among the three family members, miR-29a was first discovered by Lagos-Quintana et al., followed by extensive cloning studies that revealed the production of mature miR-29a. [0003] Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is the most commo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6883C12N15/85C12N15/90C12N15/66A01K67/027
CPCC12Q1/6886C12Q1/6883C12N15/8509C12N15/907C12N15/66A01K67/0278C12Q2600/158C12Q2600/178C12N2800/107A01K2207/15A01K2217/072A01K2227/105A01K2267/03
Inventor 黄加权宋启琴陶然范翔雪孔红言何济南向丹丹余海静陈广
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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