WO2019222986A1

WO2019222986A1 - Long-chain non-coding rna and use thereof

Info

Publication number: WO2019222986A1
Application number: PCT/CN2018/088308
Authority: WO
Inventors: 钱政江; 杨海洋; 李燕娇; 韦命余; 李伟丽; 李翔
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2018-05-25
Filing date: 2018-05-25
Publication date: 2019-11-28

Abstract

Provided are a long-chain non-coding RNA and the use thereof, wherein the long-chain non-coding RNA is Lnc00705, located downstream of the KLF6 gene, and the transcript of which can specifically target miR-181a, thereby regulating the expression of KLF6.

Description

Long-chain non-coding RNA and application thereof

Technical field

The present application relates to the field of biomedicine, and in particular to a long-chain non-coding RNA and application thereof.

Background technique

Long non-coding RNA (LncRNA) is a non-coding RNA longer than 200 nucleotides. LncRNAs usually have an mRNA-like structure, which has a polyA tail and a promoter structure after splicing, and has dynamic expression and different splicing methods during differentiation. Most LncRNAs have distinct spatiotemporal expression specificity during tissue differentiation and development, and have characteristic expression patterns in tumors and other diseases. Studies have shown that LncRNA plays an important role in many life activities such as dose compensation effect, epigenetic regulation, cell cycle regulation and cell differentiation regulation, and has become a hotspot in genetics research. A large number of mechanisms have been reported on the role of LncRNAs, including: 1) binding to a specific protein, changing the cellular localization of that protein, 2) forming a nucleic acid protein complex with a protein as a structural component, and 3) a transcript of a gene encoding a protein Form complementary double-strands, generate endogenous siRNA under the action of Dicer enzyme, 4) form complementary double-strands with transcripts encoding protein genes, interfere with the splicing of mRNAs and form different splicing forms, 5) with small-molecule RNA (Such as miRNA, piRNA) specific binding, regulating the activity and function of small molecule RNA.

KLF6 (Kruppel-like factor 6) gene is a tumor suppressor gene. Its inactivation or abnormal expression is involved in the development of many tumors. The KLF6 gene is approximately 7 kb in length and contains four exons, 218, 574, 124, and 525 bp, respectively. Mutations often occur in

exons

2 and 3, and the mRNA is 1383 bp long. The KLF6 expression product is a 283 amino acid-containing protein with three consecutive C2H2 zinc finger structures shared by the three KLF6 families at its C-terminus. It can bind to GC-rich or CACCC-rich sequences in various gene promoter regions and interact with DNA. effect. The regions rich in acidic amino acid residues such as proline and serine are related to transcriptional activity; the regions rich in serine and threonine are related to transcription or post-translational regulatory pathways. Studies have shown that KLF6 gene is related to processes such as growth and development, cell differentiation, growth-related cell signal transduction, cell proliferation, apoptosis, angiogenesis, and injury response. Its deletion, mutation, and inactivation are related to a variety of tumors. Occurrence and development are closely related. Therefore, the KLF6 gene has great potential as a new drug target for the treatment of related diseases. Numerous studies have shown that LncRNA can regulate the expression of small molecule RNAs such as miRNA and play an important regulatory function in the occurrence of various diseases. However, there are relatively few studies on LncRNAs that regulate miR-181a. Because it has been reported that miR-181a can bind to 3′-UTR of KLF6, thereby regulating KLF6 expression at the post-transcriptional level. It has important regulatory functions in different tumors. However, it is unclear whether there is an LncRNA-specific regulation of miR-181a and then affects KLF6 expression.

Therefore, the current research progress and technology need to be further improved and developed. The mechanism that can specifically regulate the expression of miR-181a and downstream KLF6 will need to be developed, which will guide the development of targeted drugs for related tumorigenesis.

Summary of the Invention

In response to the shortcomings of the prior art and actual needs, this application provides a long-chain non-coding RNA and its application. The long-chain non-coding RNA is LncRNA00705, which is located downstream of the KLF6 gene, and uses LncRNA00705 as miR-181a and KLF6 abnormal The regulatory factors of the diseases caused by the diseases are widely used in the preparation of drugs for related diseases and have broad application prospects and market value.

To achieve this, the following technical solutions are used in this application:

In a first aspect, the present application provides a long-chain non-coding RNA. The gene of the long-chain non-coding RNA is Lnc00705. Its nucleotide sequence is shown in SEQ ID No. 1. The nucleotide sequence of its transcript is as follows. SEQ ID No.2.

SEQ ID No.1 is as follows:

SEQ ID No. 2 is as follows:

In this application, according to the UCSC online database (http://genome.ucsc.edu/), the inventor found a LncRNA named Knc6 downstream from the KLF6 gene, and further analysis found that the transcript of Lnc00705 has four miR-181a Binding sites; analysis of the binding effect of Lnc00705 to miR-181a using the luciferase reporter system; designing siRNA sequences for Lnc00705, and using gastric mucosal cell line GES-1 cells to analyze miR-181a and KLF6 expression and cell proliferation after inhibiting Lnc00705 And apoptosis; a long-chain non-coding RNA that specifically targets miR-181a and then regulates KLF6 expression was identified.

Further, the long-chain non-coding RNA gene is located downstream of the KLF6 gene.

Further, the transcript of the long-chain non-coding RNA gene has four binding sites that bind to miR-181a.

Further, the four binding sites correspond to positions 1-7, 1696-1703, 2344-2351, and 2529-2536 of the Lnc00705 gene, respectively.

The identification method of this application is as follows:

1) According to the UCSC online database (http://genome.ucsc.edu/), an LncRNA named Lnc00705 was found downstream of the KLF6 gene, and further analysis revealed that the transcript of Lnc00705 has four binding sites of miR-181a;

2) Design siRNA sequence for Lnc00705. The luciferase reporter system was used to analyze the binding effect of Lnc00705 on miR-181a, and the gastric mucosal cell line GES-1 cells were used to analyze the effect of inhibiting Lnc00705 on miR-181a and KLF6 expression;

3) The effect of inhibiting Lnc00705 on cell proliferation and apoptosis was analyzed using GES-1 cells of gastric mucosa cell line.

In a second aspect, the present application provides the use of the long-chain non-coding RNA according to the first aspect as a miR-181a and / or KLF6 gene regulator.

In a third aspect, the present application provides a recombinant vector comprising the long-chain non-coding RNA according to the first aspect.

In a fourth aspect, the present application provides a host cell comprising the long-chain non-coding RNA according to the first aspect;

Preferably, the host cell includes any one or a combination of at least two of GES-1, MGC-803, SGC-7901, or BGC823, and is preferably a GES-1 cell.

In a fifth aspect, the present application provides an siRNA. The siRNA is designed by Lnc00705, and its nucleotide sequence is shown in SEQ ID No. 3.

SEQ ID No. 3 is as follows: CUGACCAGCAUAGAUGUUA.

In a sixth aspect, the present application provides a use of the recombinant vector according to the third aspect, the host cell according to the fourth aspect, or the siRNA according to the fifth aspect as a miR-181a regulator.

In a seventh aspect, the present application provides a use of the recombinant vector according to the third aspect, the host cell according to the fourth aspect, or the siRNA according to the fifth aspect as a KLF6 gene regulator.

In an eighth aspect, the present application provides a recombinant vector according to the third aspect, a host cell according to the fourth aspect, or an siRNA according to the fifth aspect, for preparing a medicament for treating a prostate disease, a nervous system disease, and a malignant tumor. And / or use of reagents.

Preferably, the malignant tumor includes any one or a combination of at least two of gastric cancer, prostate cancer, lung cancer or liver cancer.

Compared with the prior art, the present application has the following beneficial effects:

This application first discovered that Lnc00705 is located downstream of the KLF6 gene, and the transcription product has four binding sites of miR-181a; this application found that Lnc00705 can inhibit the expression of miR-181a, and then affect the expression of KLF6; the long-chain non-coding RNA provided by this application It can be used as a new regulator to treat diseases caused by the abnormal expression of miR-181a and KLF6. Therefore, the long-chain non-coding RNA of the present application can be used as a new regulator to prepare for the treatment of inflammatory diseases, Drugs for diseases of the prostate, nervous system and various malignancies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an analysis result of an online database of the present application, wherein FIG. 1 (A) is a position of Lnc00705 in a KLF6 gene, and FIG. 1 (B) is a schematic diagram of four binding sites of miR-181a in a transcript of Lnc00705;

FIG. 2 is an analysis result chart of a luciferase report of the present application; FIG.

Figure 3 is the expression map of miR-181a and KLF6 after inhibition of Lnc00705 in this application. Figure 3 (A) is the expression map of miR-181a, Figure 3 (B) is the expression map of KLF6 transcription level, and Figure 3 (C) KLF6 protein level expression map;

FIG. 4 is a diagram of the effect of inhibiting the expression of Lnc00705 on the proliferation and apoptosis of GES-1 cells, where FIG. 4 (A) is a line chart of cell proliferation, and FIG. 4 (B) is a flow chart of apoptosis, FIG. 4 (C) is a histogram of apoptosis.

Detailed ways

In order to further explain the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below with reference to the accompanying drawings and specific implementations, but the present application is not limited to the scope of the embodiments.

Example 1 Identification of long-chain non-coding RNA and analysis of luciferase activity

Using UCSC online genomic database analysis, it was found that there is a series of potential long-chain non-coding RNAs downstream of KLF6, as shown in Figure 1 (A), so there may be long-chain non-coding RNAs for feedback regulation of KLF6;

Based on previous reports, miR-181a has a targeted regulatory effect on KLF6; therefore, analysis of long-chain non-coding RNA transcripts revealed that one of the long-chain non-coding RNAs, the transcription product of Lnc00705, has four binding sites for miR-181a Point, see Figure 1 (B);

The nucleotide sequence of Lnc00705 is shown in SEQ ID No. 1, and the nucleotide sequence of its transcript is shown in SEQ ID No. 2.

SEQ ID No.1 is as follows:

SEQ ID No. 2 is as follows:

It can be known from FIG. 1 (B) that the four binding sites correspond to positions 1-7, 1696-1703, 2344-2351, and 2529-2536 of the Lnc00705 gene, respectively.

HEK293A cells (purchased from ATCC, Manassas, VA) were cultured in DMEN medium containing 10% fetal bovine serum. When the cell density reached 70%, the cells were transfected by the calcium phosphate method and co-transfected with KLF6 gene 3 ' -UTR dual luciferase reporter vector, Lnc00705 overexpression vector, miR-181a binding site mutated vector, miR-181a analogue and corresponding control. Two days after transfection, the luciferase reporter activity test was performed, and the results are shown in the figure 2 shown;

As can be seen from FIG. 2, compared with the control, luciferase activity was reduced after transfection of miR-181a, and luciferase activity was restored after co-transfection of miR-181a and Lnc00705; The mutation partially restored luciferase activity, and Lnc00705 was able to inhibit the targeted regulation of KLF6 by miR-181a.

Example 2 Long-chain non-coding RNA targeting miR-181a inhibits KLF6 expression

The gastric mucosal cell line GES-1 was used for routine culture. When the cell density reached 70%, Lnc00705 siRNA was transfected with Lipofectamine 2000 (Invitrogen). After 6 hours of transfection, the medium was changed and the culture was continued for 48 hours for experiments;

The nucleotide sequence of Lnc00705 siRNA is shown in SEQ ID No. 3:

Detection of mRNA and protein expression levels were performed using qRT-PCR and western blot respectively, and the results are shown in Figure 3 (A) -Figure 3 (C);

From Fig. 3 (A) to Fig. 3 (C), it can be seen that after inhibiting the expression of Lnc00705, miR-181a expression is up-regulated, while KLF6 expression is down-regulated at both mRNA and protein levels.

Example 3 Effect of long-chain non-coding RNA on GES-1 cell proliferation and apoptosis

Using MTT method and flow cytometry to analyze the effects of GES-1 cell proliferation and apoptosis after inhibiting Lnc00705, the results are shown in Fig. 4 (A)-Fig. 4 (C);

As can be seen from Figs. 4 (A) to 4 (C), compared with the control, inhibition of Lnc00705 promoted the proliferation of GES-1 cells and inhibited apoptosis.

In summary, the present application provides a long-chain non-coding RNA and its application. The long-chain non-coding RNA is LncRNA00705, which is located downstream of the KLF6 gene. LncRNA00705 is used as a regulator of diseases caused by miR-181a and KLF6 abnormalities. , Used to prepare drugs for related diseases, has broad application prospects and market value.

The applicant states that this application uses the foregoing embodiments to describe the detailed method of the application, but the application is not limited to the detailed method, which does not mean that the application must rely on the detailed method to be implemented. Those skilled in the art should be aware that any improvement to this application, equivalent replacement of each raw material of the product of this application, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of this application.

Claims

A long-chain non-coding RNA. The gene of the long-chain non-coding RNA is Lnc00705. The nucleotide sequence of the Lnc00705 is shown in SEQ ID No. 1. The nucleotide sequence of the transcript of the Lnc00705 is shown in SEQ. ID No.2.
The long-chain non-coding RNA according to claim 1, wherein the gene of the long-chain non-coding RNA is located downstream of the KLF6 gene.
The long-chain non-coding RNA according to claim 1 or 2, wherein the transcript of the long-chain non-coding RNA gene has four binding sites that bind to miR-181a.
The long-chain non-coding RNA according to claim 3, wherein the four binding sites correspond to positions 1-7, 1696-1703, 2344-2351, and 2529-2536 of the Lnc00705 gene, respectively Bit.
Use of the long-chain non-coding RNA according to any one of claims 1 to 4 as a regulator of miR-181a and / or KLF6 genes.
A recombinant vector comprising the long-chain non-coding RNA according to any one of claims 1-4.
A host cell comprising the long-chain non-coding RNA according to any one of claims 1-4.
The host cell according to claim 7, wherein the host cell comprises any one or a combination of at least two of GES-1, MGC-803, SGC-7901 or BGC823, preferably a GES-1 cell.
An siRNA is designed by Lnc00705, and its nucleotide sequence is shown in SEQ ID No. 3.
Use of a recombinant vector according to claim 6, a host cell according to claim 7 or 8 or an siRNA according to claim 9 as a miR-181a regulator.
Use of a recombinant vector according to claim 6, a host cell according to claim 7 or 8 or an siRNA according to claim 9 as a KLF6 gene regulator.
A long-chain non-coding RNA according to any one of claims 1-4, a recombinant vector according to claim 6, a host cell according to claim 7 or 8, or an siRNA according to claim 9 for Use of preparation of medicines and / or reagents for treating prostate diseases, neurological diseases and malignant tumors.