CN110129319B - siRNA of PRALR and application thereof - Google Patents

Abstract

The invention relates to siRNA of PRALR and application thereof. The invention starts from a TCONS _00013523 sequence, discovers a complete gene sequence by utilizing 5'-RACE and 3' -RACE technologies, is named as PRALR (Paclitaxel resistance-associated long-chain non-coding RNA), then constructs an expression plasmid and siRNA of the PRALR, further proves that the PRALR is the novel ovarian cancer resistance-associated lncRNA, and the proliferation, migration and invasion capacities of the knocked-down ovarian cancer Paclitaxel resistance cells are obviously reduced. The siRNA aiming at PRALR designed by the invention has very obvious effects in reversing drug resistance and inhibiting the growth of paclitaxel resistant cells of ovarian cancer, is obviously superior to other siRNA designed in the research process, and has great potential for being developed into paclitaxel resistant ovarian cancer clinical drugs.

Description

siRNA of PRALR and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to siRNA aiming at ovarian cancer drug resistance related lncRNA PRALR and application thereof in preparing a drug for treating paclitaxel-resistant ovarian cancer.

Background

lncRNA, long-chain non-coding RNA, is a generic name of RNA molecules with a length of more than 200 nucleotides (nt), which basically have no function of coding protein, but can be combined with DNA, RNA and protein, widely participate in physiological and pathological processes of organisms, and play an important role in the aspects of disease occurrence, development and regulation.

Ovarian cancer is one of the common malignant tumors of female reproductive organs, and the mortality rate of ovarian cancer is the first of all gynecological malignant tumors, thus causing serious threat to the life of women. Early symptoms are not obvious, the diagnosis is in the late stage, and the generation of high-efficiency chemotherapeutic drugs and drug resistance is the main reason.

In recent years, the research finds that lncRNA has an important role in the occurrence and development of ovarian cancer, and is expected to become a novel tumor marker and a target point for tumor treatment. By means of gene chip and high throughput sequencing method and other technology, the lncRNA related to ovarian cancer is found to have the following characteristics: LSINCT5, XIST, ZNF300P1, AB073614, HOXA11-AS, H19, HOTAIR, PVT1, ANRIL, HOST2, FAL1, MEG3, and studies have shown that LSINCT5, XIST, HOTAIR, ANRIL, etc. are expected to be molecular markers for the prognosis of ovarian cancer.

The fourth university of military medical science 2015 master paper "initial search for lncRNA screening and action mechanism in ovarian cancer drug-resistant cell lines" conducted the following studies: 1) Cisplatin-sensitive cell strain A2780 and drug-resistant cell strain CP70 are used as research objects, expression changes of lncRNA in ovarian cancer drug-resistant and non-drug-resistant cells are observed by lncRNA gene chip differential analysis, real-time fluorescence quantitative PCR and the like, and a molecular mechanism of cisplatin resistance caused by the lncRNA gene expression level changes is analyzed. 2) A2780/CP70 cells are taken as research objects, mRNA gene chip differential analysis, real-time fluorescence quantitative PCR and the like are used for observing the expression change of mRNA in drug-resistant and non-drug-resistant cells of ovarian cancer, and the correlation between the mRNA gene expression level change and cis-platinum resistance is analyzed. 3) And comparing the lncRNA gene chip with the mRNA gene chip through bioinformatics analysis, and selecting specific lncRNA. 4) And (3) verifying the correlation between the specific lncRNA and the mRNA expression level by using real-time fluorescent quantitative PCR (polymerase chain reaction), and analyzing a possible drug resistance mechanism of the lncRNA in the cis-platinum drug resistance process. 5) Cisplatin-resistant cell strain CP70 is taken as a research object, siRNA technology is applied to silence related lncRNA genes, real-time fluorescence quantitative PCR is used for observing the expression of specific genes after transfection, the MTT method is used for observing the influence of the siRNA of the specific lncRNA on the cisplatin sensitivity of ovarian cancer cells, and the internal molecular mechanism of the siRNA is analyzed. Finally, the following conclusions were drawn: the lncRNA participates in the generation of cis-platinum drug resistance, and the lncRNA-U2 plays an important role in cis-platinum drug resistance conversion after gene chip screening and RT-PCR verification; the function of lncRNA-U2 for promoting cisplatin resistance is probably realized by regulating and controlling apoptosis proteins such as Bax/Bcl-2 and the like.

Patent document CN105907858A, published 2016.08.31, discloses screening chemotherapy-resistant lncRNA in ovarian cancer tissues by lncRNA chip technology; carrying out GO and Pathway analysis on the target gene on the sample to obtain the signal path related to the functions of the target gene, tumor proliferation, apoptosis and survival and related to tumor drug resistance; and (3) screening lncRNA of the encoded protein, selecting 3 lncRNA with larger fold difference, observing the expression condition of the lncRNA, and obtaining that the expression of RP11-697E22.2 is obviously improved in all ovarian cancer cell strains, so that the lncRNA RP11-697E22.2 participates in the regulation and control of different types of ovarian cancer drug resistance and can be used as an intervention target for preventing and treating ovarian cancer.

Patent document CN107354159A, published AS 2017.11.17, discloses an application of lncRNA SMAD5-AS1 siRNA in ovarian cancer treatment, and the invention designs and synthesizes siRNA sequences for specifically targeting inhibition of SMAD5-AS1, and the siRNA sequences are transfected into ovarian cancer cell strains, so that inhibition of SMAD5-AS1 expression can significantly inhibit proliferation, migration and invasion capacity of ovarian cancer cells.

However, it is still necessary to discover a new lncRNA and its role in the development of ovarian cancer (especially drug-resistant ovarian cancer such as paclitaxel-resistant ovarian cancer), which is of great significance to the molecular mechanism research, clinical treatment and diagnosis of ovarian cancer.

Small interfering RNA (siRNA) is a double-stranded RNA of 20 to 25 nucleotides in length that has many different biological uses. It is known that siRNA is mainly involved in the phenomenon of RNA interference (RNAi) and regulates the expression of genes or RNAs in a specific manner. siRNA can be introduced into cells via a variety of different transfection techniques and produce specific knockdown effects for specific genes. Therefore, the complementarity of properly tailored siRNA can be used to target genes with known sequences, which makes siRNA an important tool for studying gene function and drug targets. The siRNA with remarkable knockdown effect designed aiming at the specific lncRNA or the gene thereof has great utilization value in the development of clinical gene therapy medicines.

Disclosure of Invention

The present inventors have discovered the complete gene sequence starting from the TCONS-00013523 sequence (containing only a partial lncRNA sequence) using 5'-RACE and 3' -RACE techniques and named PRALR (Paclitaxel resistance-associated long non-coding RNA). Then, an expression plasmid and siRNA of PRALR are constructed, and the PRALR is further verified to be a novel drug-resistant relevant lncRNA of the ovarian cancer, and the proliferation, migration and invasion capacities of the knocked-down paclitaxel-resistant cells of the ovarian cancer are obviously reduced. The designed siRNA has very obvious effect in reversing drug resistance and inhibiting the growth of taxol resistant cells of ovarian cancer. The present invention has been accomplished based on this.

In a first aspect, the invention provides an siRNA molecule, wherein the sequence of the siRNA molecule is shown in SEQ ID NO. 17 and SEQ ID NO. 18.

As another preferred example, the siRNA molecule has a sequence in which the base TT is further added at the 3' end of the sequences shown in SEQ ID NO 17 and SEQ ID NO 18.

In a second aspect, the invention provides an application of the siRNA molecule in preparing a medicament for treating paclitaxel-resistant ovarian cancer.

In a third aspect, the invention provides the use of said siRNA molecule selected from the group consisting of:

a) Researching the function of protein or nucleic acid molecules interacting with PRALR, wherein the PRALR is ovarian cancer drug resistance related lncRNA, and the sequence is shown as SEQ ID NO. 1;

b) Study the mechanisms of paclitaxel resistance and other chemotherapeutic drug resistance;

c) Screening the medicine for treating the taxol-resistant ovarian cancer.

In a fourth aspect, the present invention provides a pharmaceutical composition comprising said siRNA molecule, and a pharmaceutically acceptable carrier.

As another preferred example, the pharmaceutically acceptable carrier includes excipients, diluents and adjuvants.

In a fifth aspect, the invention provides a construct comprising a gene sequence for expressing said siRNA molecule.

In a sixth aspect, the invention provides a cell line comprising said siRNA molecule, or comprising a construct as described above.

In a seventh aspect, the invention provides the use of the cell line selected from the group consisting of:

a) Researching the function of protein or nucleic acid molecules interacting with PRALR, wherein the PRALR is ovarian cancer drug resistance related lncRNA, and the sequence is shown as SEQ ID NO. 1;

b) Study the mechanisms of paclitaxel resistance and other chemotherapeutic drug resistance;

c) Screening the medicine for treating the taxol-resistant ovarian cancer.

It is to be noted that the "pharmaceutically acceptable carrier" refers to a carrier for administration of the therapeutic agent, and includes various excipients, diluents and adjuvants. The term specifically refers to agents that: they are not necessary active ingredients per se and are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. Co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.

The invention has the advantages that:

the invention starts from a TCONS _00013523 sequence (only containing a part of lncRNA sequence), and discovers a complete gene sequence by utilizing 5'-RACE and 3' -RACE technologies and is named PRALR. Then, an expression plasmid and siRNA of PRALR are constructed, and the PRALR is further proved to be a novel drug-resistant relevant lncRNA of ovarian cancer, and the proliferation, migration and invasion capacities of paclitaxel-resistant ovarian cancer cells are remarkably reduced after the PRALR is knocked down. Therefore, PRALR can be used as a molecular target for reversing paclitaxel resistance of ovarian cancer, and the inhibitor of PRALR can be used as an effective medicine for treating paclitaxel-resistant ovarian cancer. The siRNA2 aiming at PRALR designed by the invention has obvious effects in reversing drug resistance and inhibiting growth of paclitaxel resistant cells of ovarian cancer, is remarkably superior to other siRNA designed in the research process, has great potential for being developed into paclitaxel resistant ovarian cancer clinical drugs, and also has important application value in mechanism research of paclitaxel resistance and other chemotherapeutic drug resistance.

Drawings

FIG. 1.5'-RACE and 3' -RACE amplification splice the full-length sequence of PRALR.

FIG. 2.PlncPRALR-zeo plasmid map.

FIG. 3. Results of lncRNA PRALR detection by qPCR after eukaryotic cells over-express plncPRALR-zeo. Wherein A is the result of overexpressing plncPRALR-zeo in OVCAR-3 cell line and B is the result of overexpressing plncPRALR-zeo in OV3R-PTX cell line. * Represents P <0.01.

Figure 4.Sirna knockdown effect validation results. * Represents P <0.05.

Figure 5 effect of pralr on proliferative capacity of paclitaxel-resistant ovarian cancer cells. * Represents P <0.01.

Figure 6. Effect of pralr on migration ability of paclitaxel-resistant ovarian cancer cells. * Represents P <0.05.

Figure 7 effect of pralr on ability to resist invasion by paclitaxel-resistant ovarian cancer cells. * Represents P <0.05.

Figure 8. Effect of overexpression or knock-down of PRALR on PTX tolerance of cells. * Represents P <0.05; * Represents P <0.01.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

In the examples herein, the terms "PRALR", "lncPRALR" and "lncRNA PRALR" all refer to the lncRNA or its gene. The paclitaxel resistant ovarian cancer cell lines OV3R-PTX, OV3R-PTX and paclitaxel resistant ovarian cancer cells refer to the same cell line.

Example 1 acquisition of PRALR full-Length sequence and expression vector construction

1 Material

Ovarian cancer cell line OVCAR-3 (ATCC No.: HTB-161);

the ovarian cancer taxol-resistant cell line OV3R-PTX (disclosed in patent ZL 201410708515.7) self-established by the subject group;

a genomic DNA extraction reagent (Takara 9770A);

a total RNA extraction kit (Axygen AP-MN-MS-RNA-50);

reverse transcription kit (Roche 04897030001);

the high fidelity enzyme PrimeSTAR Max DNA Polymerase (Takara R045A);

restriction enzymes Hind III (Takara 1060A), xbaI (Takara 1093A);

PCR product recovery kit (Takara 9761);

DNA gel recovery kit (Takara 9762);

EasyGeno rapid recombinant cloning kit (Tiangen VI 201);

competent cell DH5 α (Tiangen CB 101);

a small plasmid extraction kit (Tiangen DP 103-02);

endotoxin-free plasmid macroextraction kit (Tiangen DP 117);

pcDNA3.1(+)/zeo(Invitrogen V86020)；

Opti-MEM I medium (Gibco, 31985-070);

X-tremeGENE siRNA Transfection Reagent(Roche 04476093001)；

FastStart Universal SYBR Green Master(ROX)(Roche 04913850001)；

RACE 5’/3’Kit(TAKARA 634858)。

2 method

2.1 identification of PRALR full Length

2.1.1 Design of 5'-RACE and 3' -RACE amplification primers

Human TCONS — 00013523 sequences were downloaded from the UCSC database and nested PCR primers were designed for 5'-RACE and 3' -RACE amplification, respectively. The amplification primers were synthesized by Jinzhi Biotechnology, inc., suzhou, and the sequences of the primers are shown in Table 1.

2.1.2 5'RACE and 3' RACE amplification and sequencing

See also

Amplification was performed according to the instructions of RACE 5'/3' kit (TAKARA 634858), briefly as follows: first, the 5'-RACE and 3' -RACE full-length sequences were amplified using outer and inner primers and UPM primers. Secondly, inserting the RACE amplified fragment into a linear pRACE vector carried by the kit by adopting an In-Fusion Cloning method In the kit, then selecting 8-10 clones for sequencing identification, finally determining the sequences of 5'-RACE and 3' -RACE, and splicing a PRALR full-length sequence.

2.2 Construction of the PlncPRALR-zeo plasmid

2.2.1 primer design

The human TCONS _00013523 sequence was downloaded from the UCSC database, combined with sequences flanking the polyclonal cleavage sites Hind iii and XbaI of the eukaryotic expression plasmid pcdna3.1 (+)/zeo, and 4 pairs of primers were synthesized using the Primer Express 3.0 design for amplification of the transcribed TCONS _00013523 two exon and intron sequences. The amplification primers were synthesized by Jinzhi Biotechnology, inc., suzhou, and the sequences of the primers are shown in Table 1.

2.2.2 cell culture and genomic DNA extraction

Human ovarian cancer PTX drug-resistant cell line OV3R-PTX was seeded at 10^6 cells in T25 cell culture flasks. When the cells grow and fuse to 80% -90%, the culture medium supernatant is discarded, sterile PBS is used for washing twice, then PBS is removed as much as possible, and cell genome DNA is extracted by cracking the cells by adopting a Takara genome DNA extraction kit (Takara 9770A). The method comprises the following specific steps:

1) Adding 800 mul of lysate DNAiso into the cell surface, blowing and beating for 8-10 times by using a gun head, and transferring the lysed cells into a centrifugal tube without RNA enzyme;

2) Centrifuging at 4 deg.C at 10000g for 10min;

3) Sucking the supernatant into a new centrifugal tube without RNase, adding 300 mul of absolute ethyl alcohol, reversing and uniformly mixing for 3 minutes until flocculent precipitates are formed;

4) Carefully winding out the flocculent DNA precipitate by using a gun head, and washing the flocculent DNA precipitate once by putting the flocculent DNA precipitate into 1ml of 75% ethanol;

5) Centrifuging at 12000g for 5min, removing supernatant, and air drying for 1-2min;

6) With 200. Mu.l ddH ₂ O (Pre-heated to 65 ℃) dissolves genomic DNA.

2.2.3 PCR amplification and product purification

Two exons and introns of TCONS-00013523 were amplified using the high fidelity enzyme PrimeSTAR Max DNA Polymerase (Takara R045A). The reaction system contained 12.5. Mu.l of 2 XPrimeSTAR Max Premix, 10pmol each of the upstream and downstream primers, and 0.5. Mu.l of DNA at 50 ng/. Mu.l of the template, and the remainder was made up to 25. Mu.l with deionized water. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 2min; running 35 cycles at 95 ℃ 20s,60 ℃ 5s,72 ℃ 1min, and finally continuing to extend at 72 ℃ for 10min to obtain the target amplification product.

PCR product Purification was recovered using Takara MiniBEST DNA Fragment Purification Kit (Takara 9761). The operation is carried out according to an experimental manual of the kit, and the specific experimental steps are as follows:

1) Adding 3 times volume of Buffer DC into PCR reaction liquid, uniformly mixing, transferring into a preparation tube, centrifuging at 12000rpm for 1min, and discarding the filtrate of the collection tube;

2) Adding 650 mul Buffer WB for washing for the first time, centrifuging at 12000rpm for 1min, and collecting the filtrate;

3) Adding 650 mu l of Buffer W2 for washing for the second time, centrifuging at 12000rpm for 1min, and collecting the filtrate of the tube;

4) Centrifuging at 12000rpm for 2min to remove residual washing solution;

5) Placing the prepared tube in a clean 1.5ml centrifuge tube, adding 30 μ l deionized water (preheated to 65 deg.C) at the center of the prepared tube membrane, and standing at room temperature for 1min;

6) Centrifuging at 12000rpm for 1min to obtain purified product;

7) The concentration of the purified product was determined using nanodrop 2000.

2.2.4 plasmid vector double digestion, tapping and purification

The vector was double digested with Hind III (Takara 1060A) and XbaI (Takara 1093A). The reaction system was 10 XBuffer 6. Mu.l, hind III and XbaI 8. Mu.l each, plasmid 8. Mu.g, made up to 60. Mu.l with deionized water. Then, the mixture was digested at 37 ℃ overnight.

The cleaved product was purified by tapping using Takara MiniBEST Agarose Gel DNA Extraction Kit (Takara 9762). The method comprises the following specific steps:

1) The agarose gel containing the target DNA was cut under an ultraviolet lamp, and the gel weight was weighed and converted into a gel volume (e.g., 1mg = 1. Mu.l volume);

2) Adding Buffer GM with 5 gel volumes, mixing, heating at 37 deg.C for 10min, and intermittently mixing (every 2-3 min) until the gel block is completely melted;

3) Adding the mixed solution obtained in the step 2) into a DNA preparation tube, centrifuging for 1min at 12000g, and removing the filtrate;

4) Adding 700 mul Buffer WB,12000g, centrifuging for 30s, and removing the filtrate;

5) Washing with 700 μ l Buffer WB by the same method, centrifuging at 12000g for 1min, and discarding the waste liquid;

6) Centrifuging at 12000g for 2min to remove residual ethanol;

7) Placing the preparation tube in a clean 1.5ml centrifuge tube, adding 30 μ l deionized water (preheated to 65 deg.C) at the center of the preparation membrane, and standing at room temperature for 1min;

8) Centrifuging at 12000g for 1min to obtain purified DNA;

9) The concentration of the purified product was determined using a Narodrop 2000.

2.2.5 PCR and plasmid purification product ligation

The PCR product and the large fragment of the vector are connected by an easy Geno rapid recombinant cloning kit (Tiangen VI 201). The total volume of the ligation was 10. Mu.l, and the specific procedure was as follows:

1) The insert mass was calculated according to the molar ratio of insert to vector large fragment 3:

insert (ng) = number of bases of insert × mass of vector large fragment (0.1 μ g) × 3 per base of vector large fragment. Wherein the number of bases of the insert fragment 1 is 1033bp, the number of bases of the insert fragment 2 is 1179bp, and the number of bases of the insert fragment 3 is 1810bp; the pcDNA3.1 (+)/zeo-cleaved large fragment was 4934bp.

2) Configuration of the connection System (Total 10. Mu.l)

3) Mixing, centrifuging, and standing at 50 deg.C for 1h.

2.2.6 transformation of ligation products

Transformation experiments were performed with competent cells DH5 α (Tiangen CB 101) using the following specific steps:

1) Taking out the competent cell DH5 alpha from-80 ℃ to be thawed on ice for 10min;

2) Sucking 10 μ l of the ligation product in 2.2.5, adding into 100 μ l of competent cells, mixing gently, and ice-cooling for 30min;

3) Placing the competent cell centrifuge tube after ice bath in a water bath at 42 ℃ for heating for 90s without shaking the tube;

4) Rapidly transferring the centrifuge tube to ice to cool the cells for 2min;

5) Adding 800 μ l LB liquid culture medium (the culture medium is heated to 37 deg.C in advance) into each tube, mixing, and performing shake culture at 37 deg.C and 220rpm for 45min;

6) Appropriate volumes (200. Mu.l/90 mm dish) of transformed competent cells were plated onto LB agar plates containing ampicillin, the plates were left at room temperature until the surface liquid was absorbed;

7) The plate was inverted and incubated in an incubator at 37 ℃ for 12-16h.

2.2.7 identification of plasmids

The plasmid was subjected to a generation of Sanger sequencing to verify the accuracy of the inserted sequence bases. The method comprises the following specific steps:

1) Randomly selecting 5 clones, inoculating the clones in 5ml LB-ampicillin liquid culture medium according to the plasmid resistance gene, and carrying out shaking overnight culture at 37 ℃ and 220 rpm;

2) A small amount of plasmid extraction kit (Tiangen DP 103-02) is adopted to extract the recombinant plasmid, and the steps are briefly described as follows:

a) Collecting 5ml of bacterial liquid to the bottom of a centrifugal tube, and removing supernatant as much as possible;

b) Sequentially adding the solutions P1, P2 and P3, and centrifuging to obtain a supernatant;

c) Adding the supernatant into the center of the collecting pipe, adsorbing and centrifuging;

d) Washing the collecting tube 1 times with deproteinizing solution PD, and washing with rinsing solution PW 2 times;

e) Centrifuging at high speed for 2-3min, and removing residual rinsing liquid;

f) Plasmid DNA was eluted using 50. Mu.l of deionized water (pre-heated to 65 ℃).

3) The plasmid is sent to Jinzhi biotechnology Limited company of Suzhou for a generation of Sanger sequencing, and sequencing primers are detailed in a table 1;

4) Vector NTI Advance software, version number 11.5, was used for sequencing sequence splicing;

5) Downloading genome DNA sequence from UCSC, comparing and analyzing with the spliced sequence, and observing whether base mutation exists.

2.2.8 Mass extraction of plasmids

For correctly sequenced clones, the endotoxin-free plasmid great lift kit (Tiangen DP 117) was used for the mass preparation of plasmids, and the specific steps are as follows:

1) Picking out a single colony from a flat plate, culturing in 5ml of LB culture solution containing ampicillin, and carrying out shake culture at 37 ℃ and 220rpm for 8 hours;

2) Diluting the initial culture solution by 1000 times of 150ml of LB culture solution containing ampicillin, and carrying out shaking culture at 37 ℃ and 220rpm for 12-16 hours;

3) 8000g, centrifuging at 4 ℃ for 3min, collecting the bacterial liquid at the bottom of a 50ml centrifugal tube, and completely removing supernatant;

4) Adding 8ml of RNase A-containing solution P1, and fully resuspending the bacterial liquid on a vortex device;

5) Adding 8ml of lysis solution P2 into the heavy suspension, slightly reversing and uniformly mixing for 6-8 times, and incubating for 5min at room temperature;

6) Adding 3ml of balance buffer BL into the center of the purification column, centrifuging at 8000g for 2min, and removing waste liquid;

7) Adding 8ml of neutralizing solution P4 into the heavy suspension, immediately and gently reversing and uniformly mixing for 6-8 times;

8) Passing the neutralized liquid through an endotoxin filter CS1, and collecting the filtrate at the bottom of a 50ml tube;

9) Adding isopropanol with the volume 0.3 times that of the filtered collection pipe, and uniformly mixing;

10 Adding the liquid into the CP6 column balanced in the step 6) for 2-3 times, and centrifuging for 1min at 8000 g;

11 Adding 10ml of rinsing liquid PW in the center of an adsorption column CP6, washing for 2 times, and discarding waste liquid;

12 Adding 3ml of absolute ethyl alcohol into the center of an adsorption column CP6, washing for 1 time, and discarding waste liquid;

13 8000g for 3min to remove residual ethanol;

14 Use 1ml deionized water (preheating to 65 ℃) elution plasmid DNA;

15 Concentration and purity of plasmid were determined by means of a Narodrop2000 spectrophotometry. Subpackaging and storing at-20 deg.C for use.

2.2.9 cell transfection

The plasmid Transfection is carried out by adopting X-treemeGENE siRNA Transfection Reagent (Roche 04476093001), and the specific steps are as follows:

1) Human ovarian cancer cell lines OVCAR-3 and OV3R-PTX were cultured in 6-well plates seeded at 3 x 10^5 cells the day before transfection.

2) Before the start of Transfection, the control plasmid pcDNA3.1 (+) -zeo and the overexpression plasmid plncPRALR-zeo as well as the X-tremeGENE siRNA Transfection Reagent were diluted using Opti-MEM I medium (Gibco, 31985-070):

a) Preparing 3 enzyme-free centrifuge tubes, firstly adding a serum-free culture medium into the tubes, respectively adding 150 mul of the serum-free culture medium into the tube No. 1 and the tube No. 2, and adding 300 mul of the serum-free culture medium into the tube No. 3;

b) Adding 2 mu g of control plasmid and over-expression plasmid into serum-free culture media of No. 1 tube and No. 2 tube respectively;

c) Adding 16 ul of X-tremagene siRNA Transfection Reagent into No. 3 tube serum-free medium;

d) Incubate for 5 minutes at room temperature.

3) Mixing and incubation of transfection complexes:

a) Sucking 150 mul of transfection reagent diluent out of the No. 3 tube, adding the transfection reagent diluent into the No. 1 or No. 2 tube, and gently mixing the transfection reagent diluent and the No. 2 tube;

b) Incubate for 15 minutes at room temperature.

4) The complex is added to the cells and the plate or flask is gently shaken or rotated to ensure that the culture is evenly distributed over the surface of the plate.

5) 37 degree, 5% CO ₂ And continuing to culture for 48h, and then extracting RNA.

2.2.10 Total RNA extraction

Cell total RNA is obtained by cell lysis and extraction by using an Axygen total RNA extraction kit (Axygen AP-MN-MS-RNA-50). The method comprises the following specific steps:

1) Adding 600 mul of lysate buffer R-I to the cell surface, blowing and beating for 8-10 times by using a gun head, and transferring the lysed cells into a centrifuge tube without RNA enzyme;

2) Pumping 8-10 times by using a syringe with a 21-25-gauge needle, fully cracking cells, adding 220 mu l of buffer R-II, shaking and uniformly mixing for 30s; centrifuging at 12000g at 4 ℃ for 10min;

3) Sucking the supernatant into a new centrifugal tube without RNA enzyme, adding 400 mul of isopropanol, and fully and uniformly mixing;

4) Adsorbing RNA with column, and centrifuging at 6000g for 1min;

5) Adding 500 μ l of buffer W1A solution, centrifuging at 12000g for 1min, and washing once;

6) Adding 700 μ l AW2 solution, centrifuging at 12000g for 1min, and washing for 2 times;

7) After the waste liquid of the collecting pipe is discarded, 12000g of the waste liquid is centrifuged for 2min to remove the residual ethanol;

8) Total RNA was obtained by elution with 100. Mu.l buffer TE (pre-heated to 65 ℃).

2.2.11 reverse transcription to obtain cDNA and qPCR to detect the expression of lncRNA

Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis kit (Roche 04897030001). First, 50mM olig (dT) 18.5. Mu.l and 600mM Random Primer 1. Mu.l were added to 500ng of total RNA, and the volume was made up to 6.5. Mu.l with DEPC water. The mixture was heated at 65 ℃ for 10min and then immediately placed on ice for 2min. Then, 2. Mu.l of 5 XTranscriptor Reaction buffer, 1. Mu.l of 1mM dNTP, 1U of RNase inhibitor and 5U of reverse Transcriptase were added to the Reaction system to carry out reverse transcription. Reverse transcription conditions were 25 deg.C, 10min,55 deg.C, 40min,85 deg.C, 5min. Finally, the cDNA is obtained and frozen at-20 ℃ for later use.

qPCR was performed using FastStart Universal SYBR Green Master (ROX) (Roche 04913850001). The reaction system included 2 XFastStart Universal SYBR Green M buffer 7.5. Mu.l, primers LncPRALR-F/R of 10. Mu.M each 0.5. Mu.L, cDNA 1.5. Mu.L, DEPC stream water make-up to 15. Mu.l, and qPCR was performed. PCR was carried out at 95 ℃ for 10min for 1 cycle, 95 ℃ for 10s,60 ℃ for 30s (fluorescence collected) for 40 cycles, and a melt curve was added.

3 results

3.1 primer design

The sequences of the primer pairs for amplification of the full length of the exons and introns of PRALR and for amplification of 5'-RACE and 3' -RACE are shown in Table 1, using seamless cloning ligation techniques, with the lower case letters being the sequences on the vector and the upper case letters being the amplification sequences corresponding to the genome of PRALR.

TABLE 1 amplification of full Length PRALR exons and introns and amplification of 5'-RACE and 3' -RACE primer pairs sequences

3.2 PRALR full-Length sequence

The full-length sequence of PRALR (SEQ ID NO: 1) was further spliced by amplification using 5'-RACE and 3' -RACE, and the gene sequence of PRALR (containing NO intron) is shown in FIG. 1 and SEQ ID NO: 2.

3.3 plncPRALR-zeo plasmid map

The Vector NTI software (version 11.5) was used to construct a recombinant plasmid map, the details of which are shown in FIG. 2. Wherein the PRALR gene sequence (including intron) inserted into the plasmid is shown as SEQ ID NO. 3.

3.4 sequencing results

The insert sequences of 1 recombinant plasmid were selected and aligned in the genbank database, and were found to be identical to the genomic sequence NC _018918.2 (table 2).

TABLE 2 alignment of the insert sequences of the recombinant plasmids with the genomic DNA in the genbank database

3.5 Results of overexpression of plncPRALR-zeo in OVCAR-3 and OV3R-PTX

The pncPRALR-zeo plasmid was transfected into OVCAR-3 and OV3R-PTX cell lines, and qPCR detection showed significant up-regulation of 130648-fold expression of lncPRALR in OVCAR-3 (A in FIG. 3) and 170-fold expression of lncPRALR in OV3R-PTX (B in FIG. 3) compared with the plnPRALR-zeo transfected group and blank and pcDNA3.1 (+) -zeo empty vector transfected group. Therefore, the PRALR expression plasmid constructed by the invention can obviously improve the PRALR expression quantity in OV3R-PTX cells, and is very beneficial to researching the functions of protein or nucleic acid molecules interacting with PRALR and the mechanisms of paclitaxel resistance and other chemotherapeutic drugs resistance.

Example 2 Effect of PRALR on the biological behavior of paclitaxel-resistant ovarian cancer cells

1 method

1.1 Synthesis of lncRNA-siRNA

We designed 3 sirnas against PRALR as shown in table 3.

TABLE 3 PRALR-siRNA

1.2 verification of siRNA knockdown Effect

Ovarian cancer OVCAR-3 paclitaxel resistant cells (OV 3R-PTX) are plated for 24 hours, treated lncRNA-siRNA and control si-NC are transfected (5 muL siRNA +8 muL X-treme GENE Transfection Reagent), RNA is extracted 48 hours after Transfection, and results are detected through qRT-PCR experiments after reverse transcription into cDNA.

1.3 Effect of PRALR on proliferative Capacity of ovarian cancer paclitaxel-resistant cells

WST-1 kit assay: OVCAR-3 paclitaxel-resistant cells were plated on 96-well plates at 5000 density per well (37 ℃,5% CO) in a constant temperature incubator ₂ ) And (5) incubating for 24h. Transfection experiments: the control groups of si-NC and PRALR-siRNA2 and the transfection reagent are respectively dissolved in the opti-MEM culture medium and are stood for 5 minutes at room temperature, then the opti-MEM culture medium containing the transfection reagent is respectively added into the si-NC group and the siRNA2 group and is stood for 15 minutes at room temperature, and finally the mixed reagent is respectively added into the corresponding 96-well plate in equal amount. After 6h of culture, the medium is replaced by a fresh FBS-containing medium and placed into a constant-temperature incubator for incubation. At three time points of 0h, 24h and 48h, 100 mu L of fresh complete culture medium and 10 mu L of CCK-8 reagent are added into each small hole, the mixture is uniformly mixed, a 96-hole culture plate is incubated in an incubator for 2 hours, the 96-hole culture plate is covered and is operated in a dark place, the absorbance is measured at 450nm by using an enzyme labeling instrument, and the result is subjected to statistical analysis. The experiment was repeated three times.

1.4 Effect of PRALR on migration Capacity of drug-resistant cells of ovarian cancer paclitaxel

Transwell migration experiment: the OVCAR-3 paclitaxel resistant cells are inoculated in a 6-well plate, trypsinized after 24h of transfection, resuspended and mixed evenly by serum-free medium, and the cell count is 8 ten thousand per 100 mu L. Approximately 8 million cells/well were added to the upper Transwell chamber, the cell suspension volume was 100. Mu.L, and medium containing 10% serum was added to the lower Transwell chamber at 700. Mu.L/well. Placing at 5% of CO ₂ Incubators were incubated at 37 ℃ for 48h. After the incubation time was completed, the upper chamber medium was discarded and washed twice with PBS solution, and the upper chamber cotton swab was removed to gently wipe the cells in the upper chamber that did not penetrate the membrane. Fixing with 4% paraformaldehyde for 15 minutes, dyeing with crystal violet stain for 20 minutes, washing with PBS solution for 1 time, drying for 5 minutes, observing the cell penetrating the membrane under a microscope (200X), randomly selecting three fields, photographing, counting, and performing statistical analysis on the result. The experiment was repeated three times.

1.5 Effect of PRALR on the invasive potential of paclitaxel-resistant cells in ovarian cancer

Transwell migration experiment: aseptically spreading the diluted Matrigel in the upper chamber of a Transwell chamber, about 100. Mu.L/well, placing in 5% CO ₂ The incubator is placed at 37 ℃ for 4 hours, OVCAR-3 paclitaxel resistant cells are inoculated in a 6-well plate, after transfection for 24 hours, trypsinized, resuspended and mixed evenly by serum-free medium, and the number of cells is 8 ten thousand per 100 mu L. About 8 million cells/well were added to the upper Transwell chamber to which Matrigel had been added, the cell suspension volume was 100. Mu.L, and the lower Transwell chamber was 700. Mu.L/well of medium containing 10% serum. Placing at 5% of CO ₂ Incubators were incubated at 37 ℃ for 48h. After the incubation period ended, the upper chamber medium was discarded and washed twice with PBS solution, and the cells that did not penetrate the membrane in the upper chamber were gently wiped off with a cotton swab from the upper chamber. Fixing with 4% paraformaldehyde for 15 minutes, dyeing with crystal violet stain for 20 minutes, washing with PBS solution for 1 time, drying for 5 minutes, observing the cell penetrating the membrane under a microscope (200X), randomly selecting three fields, photographing, counting, and performing statistical analysis on the result. The experiment was repeated three times.

2 results of

2.1 Verification of siRNA knockdown effect

The qRT-PCR detection result shows that the PRALR-siRNA2 has the best knockdown effect (figure 4), and the knockdown efficiency in OVCAR-3 paclitaxel resistant cells is as high as more than 60 percent and is obviously higher than PRALR-siRNA1 and PRALR-siRNA3.

2.2 Effect of PRALR on proliferative capacity of paclitaxel-resistant ovarian cancer cells

The WST-1 experiment result shows that after the IncRNA PRALR is knocked down by siRNA-2, the proliferation capacity of the ovarian cancer paclitaxel resistant cells is inhibited (figure 5).

2.3 Effect of PRALR on migration Capacity of drug-resistant cells of ovarian cancer paclitaxel

The results of Transwell migration experiments showed that the migration ability of paclitaxel-resistant ovarian cancer cells was inhibited after lncRNA PRALR knock-down by siRNA-2 (FIG. 6).

2.4 Effect of PRALR on the invasive potential of paclitaxel-resistant cells in ovarian cancer

The results of the Transwell invasion experiment show that after the IncRNA PRALR is knocked down by siRNA-2, the invasion capacity of the ovarian cancer paclitaxel resistant cells is inhibited (FIG. 7).

Example 3 verification that PRALR is a PTX drug resistance Gene

1 method

1.1 transfection of cells

OVCAR-3 or OV3R-PTX cells were seeded in 6-well plates. OVCAR-3 cells were transfected with the empty vector pcDNA3.1 (+) -ZEO and the overexpression plasmid plncPRALR-ZEO (i.e., plncPRALR-ZEO as described in example 1); OV3R-PTX cells were transfected with NC-siRNA and PRALR-siRNA2 (i.e., PRALR-siRNA2 as described in example 2). The transfection procedure was as in example 1, 2.2.9) to 4).

1.2 PTX drug resistance IC50 assay

The specific procedure for the determination of PTX resistance IC50 is as follows:

1) Step 1.1, after cell transfection is carried out for 24 hours, pancreatin digestion is carried out, counting is carried out, and cells are inoculated in a 96-well plate according to the number of 10^4 cells/well;

2) 37 degree, 5% CO ₂ After further incubation for 24h, the PTX drug was prepared in a 10% FBS-containing 1640 medium at a concentration gradient of 0. Mu.M, 0.001. Mu.M, 0.005. Mu.M, 0.01. Mu.M, 0.05. Mu.M, 0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 4. Mu.M, 5. Mu.M, and each concentration was added to cells of different transfection groups in 3-well 96-well platesThe preparation method comprises the following steps of (1) performing;

3) 37 degree, 5% CO ₂ After further culturing for 48 hours, the cells are replaced with 100. Mu.L of fresh 10-percent FBS 1640 medium per well, followed by 10. Mu.L of CCK8 reagent, 37 degrees, 5% ₂ After incubation for 2h, detecting the absorbance value of each well at the wavelength of 450 nm;

4) IC50 values for PTX resistance of each transfection group were statistically analyzed using Graphpad Prism 5 software.

2 results of

The results of the PTX drug resistance IC50 assay for the different groups of transfectants are shown in FIG. 8. The lncPRALR over-expressed group (transfected plncPRALR-zeo) in OVCAR-3 cell line increased 4.6-fold over the PTX resistance coefficient IC50 of the control group (transfected empty vector group), P <0.01 (see fig. 8, a); knock-down of lncPRALR (lncPRALR-siRNA 2 transfected group) in OV3R-PTX cell line was 2.1 fold lower than the PTX resistance coefficient IC50 of control group (NC-siRNA transfected group), P <0.05 (see B in fig. 8). And researches find that the drug resistance coefficient IC50 of the OV3R-PTX cell line with the siRNA2 knocked down is obviously lower than that of other OV3R-PTX cell lines with the siRNA knocked down.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Jinshan Hospital affiliated to Fudan university

<120> siRNA of PRALR and application thereof

<130> /

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 579

<212> RNA

<213> Artificial sequence

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gagguugcag agaaaaggga acacuuauac acuguuggug ggaguguaaa uuaguucaac 60

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agcaauccca uuacugggua uauacucaga ggaauauaag ucacucuacu auaaacacac 180

acgcaugcaa auuuucauug uagcacuauu uacaauagca aagacaugga aucaaccuaa 240

augcucauca augacagauc ggauaaagaa aauaugcauc uucaaccuuu cuggacuaca 300

gcaccugcca acugaccccc acaucauggc uccaacgcug gcuucaucac auaucuucuc 360

ucucaguccc uuuggcucuc agggugcaau agcuuaacaa aauugcugau cuaggugccu 420

cagcauuguu ucuuugucau agucucugua uuaucuuauu uugagccauu cuuuuccugc 480

aaagauccua acugaaugag auaacuacuu cauaagcuau uuguaagguu uaaauaaauu 540

aaugcauauu uaauacauga aaaaaaaaaa aaaaaaaaa 579

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<212> DNA

<213> Artificial sequence

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gaggttgcag agaaaaggga acacttatac actgttggtg ggagtgtaaa ttagttcaac 60

tattgtgtaa agcaatatgt caattcctca aagagctaaa aaagaactac cattggactc 120

agcaatccca ttactgggta tatactcaga ggaatataag tcactctact ataaacacac 180

acgcatgcaa attttcattg tagcactatt tacaatagca aagacatgga atcaacctaa 240

atgctcatca atgacagatc ggataaagaa aatatgcatc ttcaaccttt ctggactaca 300

gcacctgcca actgaccccc acatcatggc tccaacgctg gcttcatcac atatcttctc 360

tctcagtccc tttggctctc agggtgcaat agcttaacaa aattgctgat ctaggtgcct 420

cagcattgtt tctttgtcat agtctctgta ttatcttatt ttgagccatt cttttcctgc 480

aaagatccta actgaatgag ataactactt cataagctat ttgtaaggtt taaataaatt 540

aatgcatatt taatacatga a 561

<210> 3

<211> 3951

<212> DNA

<213> Artificial sequence

<400> 3

cttcaagcat aattgcctgg gaataatagt agatttaaat tcaaatttta ttttgtgaag 60

gataacattt aaattaaatt tttttttgtg aaagcaacta tcttaactat ctgtgctaac 120

gacagctcta tgatagaagg aactttgttt attatgctta ctatgcagat gcagaaacac 180

agaaaggaag aggattagca actttcccag atctgagttg agctgagatt tgagtcaagg 240

caatattgct tctggggctg taacaactac attttattgt ctcctgaaca gatacttcct 300

tttcttactt aataagaata gaccttggag gtctggacct ggacatgaac gtacactttt 360

ctaaagaaga tatacacatg gccaacaagc atgtgaaaaa aagctcaata tcactgatca 420

ttagagaaat gcaaatgaaa atcacaatga gagaccatct cacaccagtc agaatggcta 480

ttattaaaaa ctcaaaaaat aacagatgct ggcgaggttg cagagaaaag ggaacactta 540

tacactgttg gtgggagtgt aaattagttc aactattgtg taaagcaata tgtcaattcc 600

tcaaagagct aaaaaagaac taccattgga ctcagcaatc ccattactgg gtatatactc 660

agaggaatat aagtcactct actataaaca cacacgcatg caaattttca ttgtagcact 720

atttacaata gcaaagacat ggaatcaacc taaatgctca tcaatgacag atcggataaa 780

gaaaatatgg tacgtataca ccatgcaata ctgtgcagcc ataaaaaaga atgagatatt 840

ttgtgggaac atggatggag ctggagacca tcatcctttg caaactaaca aaggaacaga 900

aaagcaaata gtgcatgttt tcacttataa gtgggaacta aatgatgaga atttatgaac 960

acaaagtaac aacagacact gtggcctact tgagggtgga aggtaggagg agggaaagga 1020

tcagaaaaaa taactgttgg gtaattggct tatgtattag tcccttctca tactgctata 1080

aagacatgcc tgagactggg taatttacag agaaaagagg tttaattgac tcacacttct 1140

gcaggctgta catgtacagg aggcatggct ggggagacct caggaatctt aaaatcatgg 1200

cagaagggct aggggaagca agcaagtctt cacgtggcag caggagaaag agagagcaaa 1260

acaaagggga aagtgctaca cactttcaaa caaccagatc tcatgagaac tcactatcac 1320

aaaaacaaca aggggaaact gtccgcatga tccaatcacg tcccaccaga tccttccccc 1380

aacactgagg attacaattc aacatgaaat ttaccggggg gacaaagagt caaacggtat 1440

cagcttagta cctgggtgag gaaataatct gtacaacaaa acctcatgac aaacatttac 1500

ctatataaca aacctgcaca tgtacctaaa atctaaagta agagttaaaa aaaattatac 1560

aggagtatac ttagaaggca tagtttctga attctaaatc attattcata aaaattttac 1620

ccaatatttt tcattgcata tttcctataa aaaataatag atcaaagact actaattgta 1680

aaaagaaaaa aaaagcaagc tgctaatatt tattgtgcct ttactctttg ccaggcctta 1740

ttaaacgctt tacctttgtt acactgttta actctccaaa taactctaaa atgtagttac 1800

agttattgtt tctgagaaag gatggaactg aggctctata agttcaagtc actatagact 1860

actggtctag gccagtagtt ctatttttaa cacgtttatg aaacaccttg aaaagttatt 1920

aaaagagttc cctggtttca acaccagaag atctaattca gcccaaaaat ttgctgatgc 1980

ttctggtcag aggaccacac tttgaaaact actgctctcc taatggtgtt agcaattgtc 2040

tgaatcctga gactttaacc aacatatgaa cacacaaaac gtgatgttca ttccgtagat 2100

attgattgat gctgaatcca aacctcatca aactaaatta ttcatgtacc atgacagaca 2160

ggaatcctaa aggcattttt ataaaagaca tttgcctcta atccttaagg acagacactt 2220

aagtagcaca ttttggtaca tagaaattag atttcagtac taaactctac tcacttttct 2280

caaagctatt aaatcagcaa atagttggta tcaagcatca ttgtaaatga atgaaaattg 2340

ttgttttaaa taataaactt taaaagtgag agattaatag attcccaaat tgataaagcc 2400

agttagtgcc agattctacc acaccctatt ttcagtttcc ttgaatgagt agtatgtcaa 2460

cagctcccct tggtcctcca gtataatttc tccccagatt tcatttaggt acatgactac 2520

tgataattag caatggaatt taaattcctc aggagctctt gcatctaggc atgggaccaa 2580

ggctaagttc tagtcaatgg gatgtaagca gaaatgatgt gcgtaacatc aagaatcagt 2640

ttttcagaga gataatttgc ttttcttttc tccttcttgt ctttgatagc atcttcaacc 2700

tttctggact acagcacctg ccaactgacc cccacatcat ggctccaacg ctggcttcat 2760

cacatatctt ctctctcagt ccctttggct ctcagggtgc aatagcttaa caaaattgct 2820

gatctaggtg cctcagcatt gtttctttgt catagtctct gtattatctt attttgagcc 2880

attcttttcc tgcaaagatc ctaactgaat gagataacta cttcataagc tatttgtaag 2940

gtttaaataa attaatgcat atttaataca tgaagctgtg cctagtttat agtaattact 3000

caaaaatgtt acttatttct ccatccaaaa catacaaaca atagtatatc cttcatgagg 3060

acttcttgtg gactaaaaag gcagtatctt taaagtgttt tgcagtctct gacaaaacca 3120

cactattacc cttactcaag gttttaattg attgtatgta cacaaggtta gtctatatga 3180

gctttggaaa gatgggaaat tcaaagtaga acagttaagg aaggaaagtg tatgttccac 3240

taataaaata tatattagtt cctgtaatca gattgtgagt gacaatggaa tccagtggac 3300

ccattttgcc ctcagacaga tggctgagaa attgctggtg tggcaagcaa gcaactctga 3360

ctgcaaaatg gcagactcat ttgcttgggg gacaggaatg agggatggtt cacatgggaa 3420

gcacaaagtc acccttgatg aaagttcaga gaagaaagag atcatcaaac ttactctaat 3480

taccaaaagg tgatgacatt agcagaagaa ttagagtttg aaatagtact tttggtagag 3540

aggactgatt tctaagaaag cccatactca tgatacaaag gcagttggtg ggccccaaag 3600

cagaatagct gcctcaggtg ggcaagtagc taaagttcac caagcactgt gtcccaaaac 3660

tcacatacac gctataccta cctgcatccc atgatgccag gagagccaca tttctgtggc 3720

aagggacagt gaacagagag gagccaacat gggaatgaga aacagagtta taaacaaaaa 3780

gcaagagagg aaccctcagg gtaacagaat cagtcactct tgaatatgtg acagagtaga 3840

aggtaattac ctcttatatc cggatattga aattccaatt ctagtctctg gaccatactt 3900

ctttgaaggt tagaaatatc agtagctcag ttggatctca gattggagct g 3951

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<211> 44

<212> DNA

<213> Artificial sequence

<400> 4

ctagcgttta aacttaagct tcttcaagca taattgcctg ggaa 44

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<400> 5

ctcctcctac cttccaccct caag 24

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence

<400> 6

cttgagggtg gaaggtagga ggag 24

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence

<400> 7

ggattcctgt ctgtcatggt acatg 25

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<400> 8

catgtaccat gacagacagg aatcc 25

<210> 9

<211> 43

<212> DNA

<213> Artificial sequence

<400> 9

ggtttaaacg ggccctctag acagctccaa tctgagatcc aac 43

<210> 10

<211> 40

<212> DNA

<213> Artificial sequence

<400> 10

gattacgcca agcttgaact accattggac tcagcaatcc 40

<210> 11

<211> 41

<212> DNA

<213> Artificial sequence

<400> 11

gattacgcca agcttgtcac tctactataa acacacacgc a 41

<210> 12

<211> 42

<212> DNA

<213> Artificial sequence

<400> 12

gattacgcca agcttccgat ctgtcattga tgagcattta gg 42

<210> 13

<211> 40

<212> DNA

<213> Artificial sequence

<400> 13

gattacgcca agcttggttg attccatgtc tttgctattg 40

<210> 14

<211> 45

<212> DNA

<213> Artificial sequence

<400> 14

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 15

<211> 19

<212> RNA

<213> Artificial sequence

<400> 15

cuacaggugu gaagcaaga 19

<210> 16

<211> 19

<212> RNA

<213> Artificial sequence

<400> 16

ucuugcuuca caccuguag 19

<210> 17

<211> 19

<212> RNA

<213> Artificial sequence

<400> 17

gggugcaaua gcuuaacaa 19

<210> 18

<211> 19

<212> RNA

<213> Artificial sequence

<400> 18

uuguuaagcu auugcaccc 19

<210> 19

<211> 19

<212> RNA

<213> Artificial sequence

<400> 19

gauccuaacu gaaugagau 19

<210> 20

<211> 19

<212> RNA

<213> Artificial sequence

<400> 20

aucucauuca guuaggauc 19

Claims (6)

1. The siRNA molecule is characterized in that the sequence of the siRNA molecule is shown as SEQ ID NO. 17 and SEQ ID NO. 18.

2. An siRNA molecule according to claim 1, characterized in that said siRNA molecule has a sequence further comprising base TT at the 3' end of the sequences shown in SEQ ID NO 17 and SEQ ID NO 18.

3. A pharmaceutical composition comprising the siRNA molecule of claim 1 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier comprises excipients, diluents and adjuvants.

5. A construct comprising a gene sequence that expresses the siRNA molecule of claim 1 or 2.

6. A cell line comprising an siRNA molecule according to claim 1 or 2 or comprising a construct according to claim 5.

Images