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Modified LNA oligonucleotide for targeting lncRNAs to resist AR-related tumor castration-recurrent

A technology of oligonucleotides and nucleotides, applied in the direction of anti-tumor drugs, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effect of not easy to degrade, prolong the action time, and high stability

Inactive Publication Date: 2015-04-29
SHANGHAI TONGJI HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report about further inhibiting hormone castration resistance and recurrence and metastasis of AR-related tumors by targeting lncRNA PCGEM1 to block the expression of this specific target gene

Method used

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  • Modified LNA oligonucleotide for targeting lncRNAs to resist AR-related tumor castration-recurrent
  • Modified LNA oligonucleotide for targeting lncRNAs to resist AR-related tumor castration-recurrent
  • Modified LNA oligonucleotide for targeting lncRNAs to resist AR-related tumor castration-recurrent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Synthesis of lncRNA PCGEM1-LNA oligonucleotides

[0048] The effect of an LNA oligonucleotide on the expression of a target nucleic acid can be detected in any of a variety of cell types, provided that the target nucleic acid is present at measurable levels, the target is expressed endogenously, or is encoded by the Transient or stable transfection expression of the nucleic acid of the nucleic acid.

[0049] The synthetic lncRNA PCGEM1-LNA oligonucleotide sequence is as follows:

[0050] PCGEM1-LNA-1: T+T+T+TCCAAAGGG+T+CCGCTGTCCCTG+G+A+G (SEQ ID NO. 5);

[0051] PCGEM1-LNA-2: A+T+T+CCCCTCAGA+A+ATCTCAGGGCTT+G+T+C (SEQ ID NO. 6).

[0052] Wherein + is selected from any one of β-D-oxygen-LNA nucleotide analogues.

[0053] The sequences of four GAS5-LNA oligonucleotides are as follows:

[0054] a) T G T G T A TCCAAAGGG T T A CCGCTGTCCCTG T G T A A G (SEQ ID NO. 1);

[0055] b) T G T G T A TCCAAAGGG A T T CCGCTGTCCCTG T G T A A G (SEQ ID NO. 2)...

Embodiment 2

[0060] Example 2 Verification of lncRNA PCGEM1 participating in castration resistance of AR-related tumors (attached figure 1 , 2 、3)

[0061] 1. The expression of lncRNA PCGEM1 in prostate malignant or metastatic tumors, high-grade malignant tumors, low-grade malignant tumors and benign tumors was detected by nucleic acid fluorescence in situ hybridization. The specific steps of nucleic acid fluorescence in situ hybridization are:

[0062] (1) Dewaxing of paraffin-embedded tissue sections of clinical prostate cancer:

[0063] 1) Xylene dewaxing 3 times, 5min each time;

[0064] 2) 100% alcohol twice, 2 minutes each time;

[0065] 3) Remove the alcohol, place the slice obliquely, mark the end section down, and air dry.

[0066] (2) Protease treatment:

[0067] 1) 40ml of proteinase K digestion solution for each staining jar, the preparation method is as follows: 40ml of 2×SSC is poured into a Facal tube, and preheated in a water bath. Add the digestive enzyme solution i...

Embodiment 3

[0118] Example 3 In vitro analysis: lncRNA PCGEM1-LNA oligonucleotide antisense inhibition of tumor cell PCGEM1 expression (attached Figure 4 )

[0119] LNCaP or 22RV1 cells were treated with 0.3×10 6 Cell seeding. at 37°C, 5% CO 2 cultivated under conditions. The next day after inoculation, use RNA transfection reagent (2.5μg / ml) to transfect cells with two different lncRNA PCGEM1-LNA oligonucleotides SEQ ID NO.1 and SEQ ID NO.3 (10μg / ml) . Briefly, cells were incubated with Lipofectamine in OptiMEM for 10 min, followed by the addition of lncRNA PCGEM1-LNA oligonucleotides SEQ ID NO.1 or SEQ ID NO.3. After 12 hours, the transfection mixture was removed, cells were washed, and plated Into an appropriate growth medium, grow at 37°C for about 48 hours, and then perform fluorescence quantitative PCR analysis.

[0120] Results: For the detection results in 22RV1 cells, see Figure 4 . The detection results in LNCaP are: compared with the experimental group transfected wit...

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Abstract

The invention relates to a modified LNA oligonucleotide for targeting lncRNAs to resist AR-related tumor castration-recurrent. Researches show that the expression of PCGEM1 is highly correlated with malignancy as well as recurrence and metastasis of prostate cancers, and is highly positive correlated with the expression of an AR isoform, such as AR3 (AR-V7), and researches also prove that the PCGEM1 modifies and regulates the expression of the AR isoform, such as AR3, through alternative transcription. Based on the research results, the LNA oligonucleotide can directly target the target gene PCGEM1, can efficiently and accurately block the expression of the PCGEM1, and blocks a PCGEM1-mediated androgen receptor isoform AR3 through joint targeting, so that the castration resistance as well as recurrence and metastasis possibility of AR-related tumors subjected to hormone castration treatment can be effectively reduced. The modified LNA oligonucleotide is good in specificity and high in stability; animal experiments show that the modified LNA oligonucleotide can suppress tumor growth, and the effect is very significant.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a modified LNA oligonucleotide targeting lncRNAs for resisting castration recurrence of AR-related tumors. Background technique [0002] Cancer accounts for nearly a quarter of human deaths. Tumor recurrence and metastasis have remained a major obstacle to improving overall survival over the past few decades, which may be largely attributed to the still lack of a comprehensive understanding of cancer biology. For example, the latest progress in functional genomics research shows that most of the genes involved in human gene transcription are non-coding RNAs. However, little is known about long non-coding RNAs (lncRNAs) in regulating cancer gene expression and mechanisms (Xie C, Yuan J, Li H, Li M, Zhao G, Bu D, et al. NONCODEv4: exploring the world of long non-coding RNA genes. Nucleic acids research. 2013; doi: 10.1093 / nar / gkt1222.). [0003] Recent studies have show...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61P35/00C12Q1/68
Inventor 邱忠民张子强Y·莫吕寒静朱竹先
Owner SHANGHAI TONGJI HOSPITAL
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