WO2019000148A1 - Sirna of human abcb6 gene and use thereof - Google Patents

Abstract

Provided is an RNA interference fragment which efficiently and specifically inhibits the mRNA expression level of the ABCB6 gene, which can be used for the preparation of a medicament for treating a disease associated with abnormal expression of the ABCB6 gene.

Description

 Description: A human ABCB6 gene siRNA and its application

 [0001] The present invention belongs to the field of molecular genetics, and particularly relates to an siRNA capable of inhibiting the expression of a human ABCB6 gene and an application thereof.

 Background technique

 [0002] Adenosine triphosphate binding cassette transporter (ABC transporter) is a large class of transmembrane proteins that utilize the energy of hydrolyzing A TP to transport transmembrane transport of various endogenous and exogenous biomolecules in solute. Substrates include: sugars, amino acids, metal ions, peptides, proteins, cellular metabolites, and drugs. ABCB transporters are widely present in eukaryotic and prokaryotic organisms. To date, 49 ABC transgenic superfamily members have been identified in the human genome, which are divided into seven subfamilies of A-G.

 technical problem

 [0003] The ABCB family of genes is only found in the vertebrate genome, and the human genome has 11 members, of which AB CB1 is the first human ABC transporter to be discovered. Because multiple members are associated with multidrug resistance (MD R), the ABCB subfamily is also known as the ABC transporter MDR family. As one of them, A BCB6 is not only related to MDR, but also related to the pathogenesis of various tumors. However, the function and role of ABCB6 in it have not been clarified so far, and further research is needed.

 [0004] The phenomenon of RNA interference (RNAi) was first discovered by Jorgensen et al. It is a highly efficient and specific gene blocking technology, which has developed rapidly in recent years and soon became a powerful functional genomics research. The tool can effectively and specifically inhibit the expression of human ABCB6 gene, which lays a foundation for its functional research.

 Problem solution

 Technical solution

 [0005] It is an object of the present invention to provide a small interfering RNA (siRNA) against human ABCB6 gene mRNA based on RNAi technology.

[0006] The cDNA sequence of human ABCB6 gene was obtained from GenBank. According to the basic principle of siRNA target sequence, a 19 nt siRNA was designed: siABCB6 sequence is as follows: [0007] Justice Chain: 5'- GGCAUCUGGAUCAAGUUCA -3' (SEQ ID NO: 1)

[0008] Antisense strand: 5,- UGAACUUGAUCCAGAUGCC -3, (SEQ ID NO: 2).

 Advantageous effects of the invention

 Beneficial effect

 The present invention provides siABCB6 which has the advantages of high interference efficiency, high-efficiency and specific inhibition of ABCB6 gene expression, and can be used as a powerful tool for the preparation of a medicament for treating a disease associated with abnormal expression of ABCB6 gene.

 Brief description of the drawing

 DRAWINGS

 1 is a schematic diagram showing the results of quantitative PCR detection of ABCB6 gene expression levels after transfection of A375 cells with siABCB6.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0011] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The techniques used in the following examples, including molecular techniques such as PCR amplification and detection, cell transfection, RNA extraction, and cell culture, detection techniques, and the like, are known to those skilled in the art unless otherwise specified. Conventional techniques; apparatus, reagents, cell lines, and the like, unless otherwise specified in the specification, are generally available to those skilled in the art and are available in the public.

 [0012] Example 1 Design of siRNA Oligonucleotide Sequence Targeting ABCB6 Gene

 [0013] The full sequence of ABCB6 mRNA was found in GenBank, and the AA sequence was searched from the downstream of the ABCB6 gene start codon AUG, and the adjacent 19 nt sequence at the 3' end was used as a candidate target, and the GC content was selected from 40- 50% of the siRNA sequence was confirmed by BLAST homology alignment. The secondary structure of the target mRNA sequence was evaluated by RNA structure 4.4 software. Finally, the target nucleotide sequence siABCB6 was obtained, and the sense strand sequence was SEQ ID NO. The antisense strand sequence is shown in SEQ ID NO: 2.

 Example 2 siRNA was transfected into A375 cells.

[0015] A375 cells (purchased from Biovector Plasmid Vector Culture Cell Gene Collection) were cultured in DMEM complete medium (containing 10% fetal bovine serum), and 6-well plates were plated at a ratio of 150,000 cells/well at 37°C. C, 5 % C02 was cultured for 18 h. Cell transfection was performed using the Lipofectamine 3000 Transfection Kit (Invitrogen) according to the product instructions. Transfected sputum, transfected A375 cells with 100 pmol _S iABCB6, the ratio of siRNA to liposome was 100 pmol: 10 μί.

[0016] Example 3 quantitative PCR detection of ABCB6 gene expression level

 [0017] Total RNA extraction: Total RNA from normal and transfected siABCB6 ABCB6 cells was extracted using the QIAGEN RNeasy Mini Kit.

 [0018] Reverse Transcription: Reverse transcription was performed using FastQuant RT Super Mix.

[0019] Quantitative PCR: Using SYBR Premix Ex Taq (Tli RNaseH Plus) (Dalian Bao Bio)

 Quantitative PCR was carried out, and the reaction system was 20 μί, and 1 L cDNA was added as a template for each reaction. The reaction procedure is: (1) 95 °C 30 s, (2) 95 °C 5s, (3) 60 °C 30s, (2)-(3), 40 cycles. With GAPDH as the internal reference, the results are shown in Figure 1. The quantitative PCR primers used are shown in Table 1:

 Table 1 Quantitative PCR primer sequences

 As shown in Fig. 1, the mRNA expression level of ABCB6 gene in A375 cells transfected with siABCB6 was significantly decreased compared with normal A375 cells, indicating that siABCB6 of the present invention can efficiently and specifically inhibit the expression of ABCB6 gene.

 [0022] The above is a further detailed description of the present invention in conjunction with the specific preferred embodiments. It is not intended that the specific embodiments of the invention are limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention.

 Industrial applicability

[0023] The siABCB6 provided by the invention has high interference efficiency and can efficiently and specifically inhibit the ABCB6 gene expression. The advantages of the drug can be used as a powerful tool for the preparation of drugs for the treatment of diseases related to abnormal expression of ABCB6 gene.

Claims

Claim  [Claim 1] An RNA interference fragment, wherein the sequence of the RNA interference fragment is as follows:  Justice Chain: 5,-GGCAUCUGGAUCAAGUUCA-3, (SEQIDNO: 1) Antisense Chain: 5,-UGAACUUGAUCCAGAUGCC-3, (SEQIDNO: 2) [Claim 2] The use of the RNA interference fragment of claim 1  It is characterized by the preparation of a medicament for treating a disease associated with abnormal expression of the ABCB6 gene.