Long-chain non-coding RNA PRALR, and expression plasmid and use thereof

A carrier and polynucleotide technology, applied in the field of long-chain non-coding RNA PRALR and its expression plasmid, can solve the problems of lack of efficient chemotherapy drugs and drug resistance, early symptoms are not obvious, etc., to achieve outstanding reversal effect and reverse drug resistance Effect

Pending Publication Date: 2019-08-16
JINSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The early symptoms are not obvious, and the diagnosis is already at an advanced stage. The main

Method used

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  • Long-chain non-coding RNA PRALR, and expression plasmid and use thereof
  • Long-chain non-coding RNA PRALR, and expression plasmid and use thereof
  • Long-chain non-coding RNA PRALR, and expression plasmid and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0038] Example 1 Obtaining the full-length sequence of PRALR and construction of expression vector

[0039] 1 material

[0040] Ovarian cancer cell line OVCAR-3 (ATCC number: HTB-161);

[0041] Our self-built ovarian cancer paclitaxel-resistant cell line OV3R-PTX (disclosed in patent ZL201410708515.7);

[0042] Genomic DNA extraction reagent (Takara 9770A);

[0043] Total RNA extraction kit (Axygen AP-MN-MS-RNA-50);

[0044] Reverse transcription kit (Roche 04897030001);

[0045] High-fidelity enzyme PrimeSTAR Max DNA Polymerase (Takara R045A);

[0046] Restriction endonuclease HindⅢ (Takara 1060A), XbaⅠ (Takara 1093A);

[0047] PCR product recovery kit (Takara 9761);

[0048] DNA gel recovery kit (Takara 9762);

[0049] EasyGeno rapid recombination cloning kit (Tiangen VI201);

[0050] Competent cell DH5α (Tiangen CB101);

[0051] Small amount plasmid extraction kit (Tiangen DP103-02);

[0052] Endotoxin-free plasmid large-scale extraction kit (Tiangen DP117);

[0053] pcDNA3.1(+) / zeo(Invitrogen...

Example Embodiment

[0184] Example 2 Design and verification of PRALR-siRNA

[0185] 1 method

[0186] 1.1 Synthesis of lncRNA-siRNA

[0187] We designed 3 siRNAs against PRALR, as shown in Table 3.

[0188] Table 3PRALR-siRNA

[0189]

[0190] 1.2 Verify siRNA knockdown effect

[0191] Ovarian cancer OVCAR-3 paclitaxel-resistant cells (OV3R-PTX) were plated for 24 hours, and treated with lncRNA-siRNA and control group si-NC transfection (5μL siRNA+8μL X-treme GENE Transfection Reagent), 48 hours after transfection RNA was extracted, reverse transcribed into cDNA, and the results were tested by qRT-PCR experiment.

[0192] 2 results

[0193] The qRT-PCR test results show that PRALR-siRNA2 knockdown has the best effect ( Figure 4 ), the knockdown efficiency in OVCAR-3 paclitaxel-resistant cells is as high as 60%, and it is significantly higher than PRALR-siRNA1 and PRALR-siRNA3.

Example Embodiment

[0194] Example 3 Verification that PRALR is a PTX resistance gene

[0195] 1 method

[0196] 1.1 Cell transfection

[0197] Inoculate OVCAR-3 or OV3R-PTX cells in a 6-well plate. OVCAR-3 cells were transfected with empty vector pcDNA3.1(+)-ZEO and overexpression plasmid plncPRALR-zeo (ie plncPRALR-zeo described in Example 1); OV3R-PTX cells were transfected with NC-siRNA and PRALR-siRNA2 (ie PRALR-siRNA2 described in Example 2). The transfection steps are the same as those in Example 1. 2.2.9 1)~4).

[0198] 1.2 PTX resistance IC50 determination

[0199] The specific steps of PTX resistance IC50 determination are as follows:

[0200] 1) Step 1.1 After the cells are transfected for 24 hours, perform trypsinization, count, and inoculate the cells in 96-well plates according to the number of 10^4 cells / well;

[0201] 2) 37 degrees, 5% CO 2 After culturing for 24 hours, use 1640 medium containing 10% FBS to configure PTX drugs with concentration gradients of 0μM, 0.001μM, 0.005μM, 0.01μM, ...

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Abstract

The invention relates to a long-chain non-coding RNA PRALR, and an expression plasmid and a use thereof. Starting from TCONS-00013523 sequence, a complete gene sequence is discovered by 5'-RACE and 3'-RACE technologies and is named as PRALR (paclitaxel resistance-associated lncRNA, paclitaxel resistance-associated long-chain non-coding RNA). Then, the PRALR expression plasmid and siRNA are constructed, and the PRALR is further confirmed to be a novel ovarian cancer drug resistance-associated lncRNA; the constructed PRALR expression plasmid can significantly increase the expression quantity ofthe PRALR, and the designed siRNA can significantly reverse drug resistance of paclitaxel-resistant ovarian carcinoma cells. A powerful tool is provided for study of the mechanism of paclitaxel resistance and other chemotherapeutic drug resistance, and a reference is provided for clinical development of drugs to reverse the drug resistance of tumor to paclitaxel.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a long-chain non-coding RNA PRALR related to ovarian cancer drug resistance, its expression plasmid and its application. Background technique [0002] lncRNA, that is, long non-coding RNA, is a general term for a class of RNA molecules longer than 200 nucleotides (nt). They basically have no protein-coding function, but can combine with DNA, RNA and protein, and widely participate in the physiology of the body. It plays an important role in the development and regulation of diseases. [0003] Ovarian cancer is one of the common malignant tumors of female reproductive organs, and its mortality rate ranks first among all gynecological malignant tumors, posing a serious threat to women's lives. The early symptoms are not obvious, and the diagnosis is already at an advanced stage. The main reasons are the lack of highly effective chemotherapy drugs and the emergence of drug resistance. ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N5/10C12Q1/02A61K31/7088A61P35/00
CPCC12N15/113A61K31/7088G01N33/5011C12N2310/14C12N2510/00
Inventor 许国雄管文彩林群博
Owner JINSHAN HOSPITAL FUDAN UNIV
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