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SiRNA of PRALR and use thereof

A use and molecular technology, applied in the application field of drugs, can solve the problems of lack of efficient chemotherapeutic drugs, drug resistance, and inconspicuous early symptoms.

Active 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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  • SiRNA of PRALR and use thereof
  • SiRNA of PRALR and use thereof
  • SiRNA of PRALR and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining the full-length sequence of PRALR and constructing the expression vector

[0040] 1 material

[0041] Ovarian cancer cell line OVCAR-3 (ATCC number: HTB-161);

[0042] OV3R-PTX, an ovarian cancer paclitaxel-resistant cell line established by our research group (disclosed in patent ZL201410708515.7);

[0043] Genomic DNA extraction reagent (Takara 9770A);

[0044] Total RNA extraction kit (Axygen AP-MN-MS-RNA-50);

[0045] Reverse transcription kit (Roche 04897030001);

[0046] High-fidelity enzyme PrimeSTAR Max DNA Polymerase (Takara R045A);

[0047] Restriction enzymes HindⅢ (Takara 1060A), XbaI (Takara 1093A);

[0048] PCR product recovery kit (Takara 9761);

[0049] DNA gel recovery kit (Takara 9762);

[0050] EasyGeno Rapid Recombination Cloning Kit (Tiangen VI201);

[0051] Competent cells DH5α (Tiangen CB101);

[0052] Small plasmid extraction kit (Tiangen DP103-02);

[0053] Endotoxin-free plasmid extraction kit (Tiangen DP117);

[...

Embodiment 2

[0186] Example 2 Effect of PRALR on Biological Behavior of Paclitaxel-resistant Ovarian Cancer Cells

[0187] 1 method

[0188] 1.1 Synthesis of lncRNA-siRNA

[0189] We designed three siRNAs targeting PRALR, as shown in Table 3.

[0190] Table 3 PRALR-siRNA

[0191]

[0192] 1.2 Verify the effect of siRNA knockdown

[0193] 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.

[0194] 1.3 Effect of PRALR on the proliferation of paclitaxel-resistant ovarian cancer cells

[0195] WST-1 kit detection method: OVCAR-3 paclitaxel-resistant cells were inoculated on a 96-well plate at a density of 5000 cells / well, and kept in a constant temperature incubator (37°C, ...

Embodiment 3

[0209] Example 3 verifies that PRALR is a PTX drug resistance gene

[0210] 1 method

[0211] 1.1 Cell transfection

[0212] 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.

[0213] 1.2 Determination of PTX resistance IC50

[0214] The specific steps of PTX resistance IC50 determination are as follows:

[0215] 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;

[0216] 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 an siRNA of PRALR and a use thereof. Starting from a TCONS-00013523 sequence, a complete gene sequence is discovered by 5'-RACE and 3'-RACE technologies and named as PRALR (paclitaxel resistance-associated lncRNA, paclitaxel resistance-associated long-chain non-coding RNA), an expression plasmid and siRNA of the PRALR are constructed, the PRALR is further confirmed to be anovel ovarian cancer drug resistance-associated lncRNA, and the proliferation, migration and invasiveness abilities of ovarian cancer paclitaxel-resistant cells decrease significantly after knockdown. The siRNA designed for the PRALR plays an obvious role in reversing drug resistance and inhibiting the growth of paclitaxel-resistant ovarian cancer cells, is significantly superior to other siRNAsdesigned in the research process, and has great potential for developing paclitaxel-resistant ovarian cancer clinical drugs.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a siRNA targeting lncRNA PRALR associated with ovarian cancer drug resistance and its application in the preparation of drugs for treating paclitaxel-resistant ovarian cancer. 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 effect...

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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