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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 reasons are the lack of highly effective chemotherapy drugs and the emergence of drug resistance

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

Embodiment 1

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

[0039] 1 material

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

[0041] OV3R-PTX, an ovarian cancer paclitaxel-resistant cell line established by our research group (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 enzymes 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 cells DH5α (Tiangen CB101);

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

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

[0053] p...

Embodiment 2

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

[0185] 1 method

[0186] 1.1 Synthesis of lncRNA-siRNA

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

[0188] Table 3 PRALR-siRNA

[0189]

[0190] 1.2 Verify the effect of siRNA knockdown

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

[0192] 2 results

[0193] The results of qRT-PCR detection showed that PRALR-siRNA2 had the best knockdown effect ( Figure 4 ), the knockdown efficiency in OVCAR-3 paclitaxel-resistant cells was as high as 60%, and was significantly higher than that of PRALR-siRNA1 and PRALR-siRNA3.

Embodiment 3

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

[0195] 1 method

[0196] 1.1 Cell transfection

[0197] OVCAR-3 or OV3R-PTX cells were seeded in 6-well plates. OVCAR-3 cell transfection empty vector pcDNA3.1 (+)-ZEO and overexpression plasmid plncPRALR-zeo (i.e. plncPRALR-zeo described in Example 1); OV3R-PTX cell transfection NC-siRNA and PRALR-siRNA2 (i.e. PRALR-siRNA2 described in Example 2). The transfection steps are the same as 2.2.9 1)-4) in Example 1.

[0198] 1.2 Determination of PTX resistance IC50

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

[0200] 1) 24 hours after cell transfection in step 1.1, perform trypsinization, count, and inoculate cells in a 96-well plate according to 10^4 cells / well;

[0201] 2) 37 degrees, 5% CO 2 After continuing to culture for 24 hours, PTX drugs were prepared with 1640 medium containing 10% FBS, and the concentration gradients were 0 μM, 0.001 μM, 0.005 μM, 0.01 μM, 0.0...

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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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