Application of micromolecule RNA has-miR-29c in preparing medicine treating liver cancer

A rnahas-mir-29c, a technology for the treatment of liver cancer, applied in the field of biotechnology research, can solve problems that do not involve miR-29 liver cancer research

Inactive Publication Date: 2011-09-21
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of these studies involved the study of miR-29 in HBV-related liver cancer, so it is of great significance to conduct related research

Method used

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  • Application of micromolecule RNA has-miR-29c in preparing medicine treating liver cancer
  • Application of micromolecule RNA has-miR-29c in preparing medicine treating liver cancer
  • Application of micromolecule RNA has-miR-29c in preparing medicine treating liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Detection of the expression level of has-miR-29c in liver cancer cell lines

[0023] The total RNA of liver cancer cell lines HepG2 and HepG2.2.15 cells were extracted by Trizol method. Wash the cells twice with PBS, add 1ml Trizol to each bottle, pipette to mix, and incubate at room temperature for 5min; transfer the cell solution into a DEPC-treated 1.5ml centrifuge tube, add 0.2ml chloroform to each tube, shake vigorously for 15s to mix, Stand on ice for 3-5 minutes to separate the layers; centrifuge at 12,000 rpm for 15 minutes at 4°C; carefully absorb the upper layer liquid, transfer it to another 1.5ml centrifuge tube treated with DEPC, add an equal volume of isopropyl alcohol that has been pre-cooled at 4°C Alcohol, upside down and gently mix for 15-30s, let stand on ice (room temperature) for 5-10min; 4°C, 12000rpm, centrifuge for 15min; discard the supernatant, add 1ml 75% ethanol, gently wash the precipitate twice; 4°C , centrifuge at 7500rpm for 1...

Embodiment 2

[0027] Example 2: Prediction of has-miR-29c target genes

[0028] Through bioinformatics methods, using online databases (TargetScan: http: / / www.targetscan.org / ; Pictar: http: / / pictar.bio.nyu.edu / ; miRanda: http: / / microrna.sanger.ac.uk / ) to predict the target gene related to has-miR-29c, tentatively determined that the target gene of has-miR-29c is tnfaip3.

Embodiment 3

[0029] Embodiment 3: Construction of has-miR-29c expression vector

[0030] First, according to the miRNA data included in the Sanger miRBase database, primers (miR-29c-F and miR-29c-R) were designed according to the pri-miR-29 sequence, and the endonuclease BamHI was overhanging at the 5′ end of miR-29c-F. Restriction site and protective base; the restriction site and protective base of another endonuclease HindIII are overhanging at the 5' end of miR-29c-R. Then perform PCR amplification, and the PCR reaction system (50ul) is as follows: 2× reaction buffer 25ul, 10uM upstream primer 1ul, 10uM downstream primer 1ul, DNA 2ul (less than 1ug), ddH 2 O 21ul. Reaction conditions: 94°C for 3min; 32 cycles of 94°C for 30s, 55°C for 40s, and 72°C for 40s; 72°C for 5min; storage at 4°C. After amplification, perform double enzyme digestion on the PCR product, the enzyme digestion system is (50ul); 10×K Buffer 5ul, BamHI 2.5ul, HindIII 2.5ul, PCR product 15ul, ddH 2 O 25ul. The enzy...

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Abstract

The invention discloses a RNA has-miR-29c related to Hepatitis B Virus (HBV) and liver cancer and its uses in inhibiting HBV infection and preventing liver cancer from generation. The invention is directed to the field of biology technology research. Based on qRT-PCR technology, it is found that compared to a hepatocyte lineage HepG2, the has-miR-29c has a down-regulation of expression in a hepatoma cell line. A bioinformatics method and double fluorescence detection are used to determine that the target gene of has-miR-29c is Tnfaip3. The has-miR-29c expression carrier is externally transfected. It is observed that miR-29c can inhibit the growth of tumor cells for a short period and has a certain inhibition effect to the duplication and expression of HBV DNA. The target gene Tnfaip3 has a down-regulation of expression both on mRNA and the level of protein. The research result shows miR-29c possibly participates in the generation and development process of liver cancer related to HBV through its target gene Tnfaip3. Therefore, miR-29c can be used as a regulation and control molecule for inhibiting HBV infection and preventing liver cancer from generation, thereby providing a theoretical and experimental basis for clinic prevention and treatment.

Description

technical field [0001] The invention relates to the use of a small molecule RNA has-miR-29c related to HBV-related liver cancer in inhibiting HBV infection and preventing the occurrence of liver cancer, and belongs to the field of biotechnology research. Background technique [0002] microRNAs (miRNAs) are a class of non-coding endogenous small RNA molecules ubiquitous in eukaryotes, and their length is usually 21-25nt. Hundreds of microRNA molecules have been identified in humans so far. miRNAs do not encode proteins or polypeptides, but interact with the 3'-UTR (3' non-coding region) of the mRNA of the complementary or partially complementary target gene to degrade the mRNA or mediate its translational inhibition, thus playing a role in the regulation of gene expression. play an important role in [1] . Studies have shown that miRNAs participate in various biological processes such as development, proliferation, differentiation, and apoptosis by regulating the expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7105A61P1/16A61P31/20A61P35/00
Inventor 刘玉刚王春梅王燕范春光孙汶生
Owner SHANDONG UNIV
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