CN110055253A - A kind of siRNA molecule and its application for human cystatin E B - Google Patents

Abstract

The present invention relates to a kind of siRNA molecules and its application for being directed to human cystatin E B (CSTB).The present invention devises the DNA double chain-ordering for striking and dropping effective siRNA and expression shRNA for people CSTB, and construct the slow virus carrier comprising above-mentioned DNA double chain-ordering, it is tested by ovarian cancer cell biological function, confirm that siRNA of the present invention and the construction of expression shRNA can significantly reduce CSTB mRNA and expressing quantity, human epithelial ovarian carcinoma cells proliferation is caused to inhibit, make cell block in the G2/M phase, and promote ovarian cellular apoptosis, therefore small molecule therapeutic, or the research and development reagent of research oophoroma pathomechanism be can be developed into.

Description

A kind of siRNA molecule targeting human cystatin B and its application

technical field

The present invention relates to the technical field of tumor molecular biology, in particular to a siRNA molecule targeting human cystatin B and its application.

Background technique

Ovarian cancer is the eighth most common cancer in women worldwide, but it is the most lethal gynecological cancer. The main reason is that the symptoms of ovarian cancer are not obvious in the early stage. Once diagnosed in clinic, most of them are in the advanced stage and the prognosis is poor. It is necessary to find biomarkers that can be used to diagnose and treat ovarian cancer.

The applicant's previous work has found that cysteine protease inhibitor B (Cystatin B, CSTB) is a marker of the progression of human ovarian cancer (Ovarian Cancer, OC), and found that transforming growth factor (TGF-β) 1 can regulate CSTB expression (Int J Oncol 2014, 44:1099). However, the function of CSTB in OC and the specific mechanism of its regulation by TGF-β1 are still unclear.

RNA interference (RNAi) is a gene blocking technology, which is a process in which a double-stranded RNA (dsRNA) molecule blocks the expression of specific genes or silences them at the mRNA level. Specific post-transcriptional gene silencing (Post-transcriptional gene silencing, PTGS). Using RNAi technology to study the function of genes in the process of tumor pathogenesis is an important supplement to the study of tumor pathogenesis. Moreover, RNAi has become an effective strategy for tumor gene therapy. The use of RNAi technology can inhibit the expression of proto-oncogenes, mutated tumor suppressor genes, cell cycle-related genes, and anti-apoptosis-related genes to inhibit the occurrence and development of tumors. The journal literature (Oncology Research, 2016, Vol.24, pp.487-494) disclosed the key role and possible regulatory mechanism of CSTB in gastric cancer. Human gastric cancer SGC-7901 cells were used as an in vitro model to transfect plasmids with Lipofectamine 2000 PCDNA3.1-CSTB and siRNA-CSTB, quantitative real-time PCR (qRT-PCR) and Western blotting were performed to determine the relative expression of CSTB genes and proteins, and cell proliferation, migration and apoptosis were assessed by MTT method, Transwell, and flow cytometry, respectively The results showed that compared with gastric epithelial cells, CSTB was significantly down-regulated in SGC-7901 cells. After cells were transfected with pc-CSTB and si-CSTB, CSTB was overexpressed or inhibited. Cell viability and migration were significantly decreased, and apoptosis was increased. Cells transfected with si-CSTB were significantly increased in survival and migration, and apoptosis was decreased, suggesting that CSTB can be a potential therapeutic target for gastric cancer. However, CSTB in this paper has an inhibitory effect on gastric cancer, and the knockdown effect of si-CSTB in this paper is general.

In conclusion, there is no report of CSTB in the treatment of ovarian cancer, and no siRNA molecule with significant knockdown effect against CSTB, which has the prospect of drug development.

SUMMARY OF THE INVENTION

The purpose of the present invention is to aim at the deficiencies in the prior art, and to provide siRNA molecules, related products and their uses with the development prospect of drugs and R&D reagents for human cystatin B.

In a first aspect, the present invention provides a siRNA molecule, the siRNA molecule comprises the following sequence:

Sense strand: 5'-GGACAAACUACUUCAUCAA-3',

Antisense strand: 5'-UUGAUGAAGUAGUUUGUCC-3'.

In a second aspect, the present invention provides a DNA double-stranded sequence for knocking down the human cystatin B gene, the DNA double-stranded sequence comprising the following sequence:

Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTGATGAAGTAGTTTGTCCTTTTTTg-3',

Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTGATGAAGTAGTTTGTCC-3'.

A third aspect, the invention provides a kind of construct, described construct comprises following DNA double-stranded sequence:

Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTGATGAAGTAGTTTGTCCTTTTTTg-3';

Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTGATGAAGTAGTTTGTCC-3'.

As a preferred example, the construct is a lentiviral vector.

In a fourth aspect, the present invention provides a lentiviral particle comprising the above construct.

In a fifth aspect, the present invention provides a cell model of human cystatin B gene silencing, the cell model comprising the above lentiviral particles.

In the sixth aspect, the present invention provides the application of the siRNA molecule, the DNA double-stranded sequence, the construct, the lentiviral particle or the cell model in the preparation of a drug for the treatment of ovarian cancer .

In the seventh aspect, the present invention provides that the siRNA molecule, the DNA double-stranded sequence, the construct, the lentiviral particle or the cell model are used to inhibit the proliferation, migration or proliferation of ovarian cancer cells. Application of Invasion Reagents.

As a preferred example, the ovarian cancer cells are derived from the ovarian cancer cell line OVCAR-3.

In the eighth aspect, the present invention provides that the siRNA molecule, the DNA double-stranded sequence, the construct, the lentiviral particle or the cell model inhibit the proliferation of ovarian cancer cells for non-therapeutic purposes , migration or invasion applications.

The advantages of the present invention are:

1. Appropriate siRNA and DNA double-stranded sequences for expressing shRNA were designed for human CSTB, and a lentiviral vector containing the above DNA double-stranded sequences was constructed. Experiments show that the siRNA of the present invention and the construct expressing shRNA can significantly reduce the expression of CSTB mRNA and protein, and the knockdown effect is very obvious, which is significantly better than other siRNAs, so it can be developed as a small molecule therapeutic drug, or to study the pathological mechanism of ovarian cancer. development reagents.

2. For the first time in ovarian cancer, the therapeutic method for CSTB gene was proposed.

Description of drawings

Figure 1: si-CSTB-1, si-CSTB-2, si-CSTB-3 down-regulated CSTB mRNA and protein expression in OVCAR-3 cells.

Figure 2: Proliferation of ovarian cancer cells transfected with si-CSTB-2.

Figure 3: (A, B) CSTB protein expression; (C) proliferation of ovarian cancer cells at 72 h; (D, E, F) cell cycle detection results.

Figure 4: (A, B, C) the detection results of apoptosis of ovarian cancer cells; (D) the WB detection results of BAX protein.

Detailed ways

The specific embodiments provided by the present invention will be described in detail below with reference to the accompanying drawings.

Example 1

1. Experimental method

①Construction of CSTB-shRNA lentiviral vector

The lentiviral vector pLKO.1-TRC cloning vector (addgene product number 10878) was used for shRNA lentiviral construction. The main steps are as follows:

a) Double digestion of pLKO.1-TRC vector with AgeI and EcoRI restriction enzymes;

b) Synthesize CSTB-shRNA and NC-shRNA sequences for annealing, the sequences are as follows:

CSTB-shRNA vector insert sequence:

sense 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTTTg-3' (SEQ ID NO: 1),

antisense 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTGATGAAGTAGTTTGTCC-3' (SEQ ID NO: 2);

The insert sequence of the NC-shRNA vector is:

sense 5'-ccggTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTTg-3' (SEQ ID NO:3),

antisense 5'-aattcAAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3' (SEQ ID NO: 4).

c) Insert the annealed sequence into the linearized pLKO.1-TRC vector to construct CSTB-shRNA and NC-shRNA lentiviral vectors.

The constructed lentiviral vector, pMD2.G (addgene product number 12259) and pCMV-dR8.2 (addgene product number 8455) were co-transfected into HEK293T to package the lentivirus.

②The effect of CSTB on the proliferation of ovarian cancer cells

a) Select ovarian cancer cell line OVCAR-3, use lentiviral technology to knock down CSTB, construct stable knockdown CSTB cell line OV-CSTB-shRNA, and lentiviral NC-shRNA construct cell line OV-NC-shRNA as control. The CSTB knockout efficiency was verified by Immunoblotting.

b) CCK-8 method: OV-NC-shRNA and OV-CSTB-shRNA were respectively counted (4000 cells/well), inoculated in a 96-well plate and incubated for 24h, 48h and 72h. CCK8 detection reagent was added at each time point and OD absorbance was measured at 450 nm after 2 hours.

③The effect of CSTB on ovarian cancer cell cycle

a) Flow Cytometry: OV-NC-shRNA and OV-CSTB-shRNA cells were plated in 6-well plates (300,000 cells/well), and when the cell confluence reached 85% to 90%, trypsin digestion was completed. The digestion medium was terminated, 1000 rpm, 5 min; washed once with 2 ml of PBS; 1000 rpm, 5 min; suspended cells with 300 μl PBS, added 700 μl pre-cooled absolute ethanol, fixed at 4°C for more than 3 h; 1000 rpm, 5 min; discarded the supernatant, washed with PBS, 1000 rpm, 5 min; discard the supernatant, add 500 μl of PI dye (BD, cat550825) to each tube, protect from light at room temperature for 15 min, analyze the cell cycle by flow cytometry, and repeat the experiment three times.

④The effect of CSTB on apoptosis of ovarian cancer cells

a) Flow Cytometry: OV-NC-shRNA and OV-CSTB-shRNA cells were plated in 6-well plates (300,000 cells/well), and the cell culture medium was collected when the cell confluence reached 85% to 90%. Digest with EDTA-free trypsin, terminate the digestion in complete medium, 1000rpm, 5min; wash twice with 2ml PBS; 1000rpm, 5min; discard the supernatant, add 100μl 1×Binding Buffer, add 5μl PI and 1μl APC- AnnixV (protect from light during this process); after staining at 4 degrees for 15 minutes, add 500 μl of 1×Binding Buffer to each well, protect from light, and mix gently; for apoptosis analysis by flow cytometry, the experiment was repeated 3 times.

b) Western blotting experiment: detection of pro-apoptotic protein Bax.

2. Experimental results

①CSTB affects the proliferation of ovarian cancer cells

A variety of siRNAs were designed, and the information of si-CSTB-1, si-CSTB-2, si-CSTB-3 is shown in Table 1. Compared with the control, the above three siRNAs can down-regulate CSTB mRNA and protein expression in OVCAR-3 cells, but si-CSTB-2 has the most obvious knockdown effect, up to more than 90%, which is significantly higher than that of si-CSTB-1 and si-CSTB-2. -CSTB-3 and other siRNAs (P<0.05) (Fig. 1).

Table 1. siRNA Design

As shown in Figure 2, the OVCAR-3 ovarian cancer cell line transfected with si-CSTB-2 exhibited significant proliferation inhibition.

As shown in Figure 3, the sh-RNA lentivirus constructed with si-CSTB-2 knocked down the CSTB protein (A, B), compared with the sh-NC group, the ovarian cancer cells could significantly inhibit the proliferation at 72h (C). ); analysis of cell cycle results showed that knockdown of CSTB protein could cause cells to arrest in G2/M phase (D, E), and three replicate experiments were statistically significant (F), and the experiments were repeated three times. *P<0.05, **P<0.01.

②Knockdown of CSTB induces apoptosis of ovarian cancer cells

As shown in Figure 4, after knockdown of CSTB protein by sh-CSTB lentivirus constructed with si-CSTB-2, compared with sh-NC group, 48h can induce an increase in early apoptotic cells in ovarian cancer cells (A, B, C) . 48 hours WB detection, after knockdown of CSTB protein, BAX protein was up-regulated relative to sh-NC group (D). The experiments were repeated three times. *P<0.05.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as It is the protection scope of the present invention.

SEQUENCE LISTING

<110> Jinshan Hospital Affiliated to Fudan University

<120> A kind of siRNA molecule against human cystatin B and its application

<130>/

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 55

<212> DNA

<213> Artificial sequences

<400> 1

ccggggacaa actacttcat caactcgagt tgatgaagta gtttgtcctt ttttg 55

<210> 2

<211> 55

<212> DNA

<213> Artificial sequences

<400> 2

aattcaaaaa aggacaaact acttcatcaa ctcgagttga tgaagtagtt tgtcc 55

<210> 3

<211> 55

<212> DNA

<213> Artificial sequences

<400> 3

ccggttctcc gaacgtgtca cgtctcgaga cgtgacacgt tcggagaatt ttttg 55

<210> 4

<211> 55

<212> DNA

<213> Artificial sequences

<400> 4

aattcaaaaa attctccgaa cgtgtcacgt ctcgagacgt gacacgttcg gagaa 55

<210> 5

<211> 19

<212> RNA

<213> Artificial sequences

<400> 5

guccicagcuu gaagagaaa 19

<210> 6

<211> 19

<212> RNA

<213> Artificial sequences

<400> 6

uuucucuuca agcugggac 19

<210> 7

<211> 19

<212> RNA

<213> Artificial sequences

<400> 7

ggacaaacua cuucaucaa 19

<210> 8

<211> 19

<212> RNA

<213> Artificial sequences

<400> 8

uugaugaagu aguuugucc 19

<210> 9

<211> 19

<212> RNA

<213> Artificial sequences

<400> 9

cccuugaccu uaucuaacu 19

<210> 10

<211> 19

<212> RNA

<213> Artificial sequences

<400> 10

aguuagauaa ggucaaggg 19

Claims (10)

1. a kind of siRNA molecule, which is characterized in that the siRNA molecule includes following sequence:

Positive-sense strand: 5'-GGACAAACUACUUCAUCAA-3',

Antisense strand: 5'-UUGAUGAAGUAGUUUGUCC-3'.

2. a kind of for striking the DNA double chain-ordering of drop human cystatin E 1 B gene, which is characterized in that described DNA double chain-ordering includes following sequence:

Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTT Tg-3',

Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGT CC-3'.

3. a kind of construction, which is characterized in that the construction includes following DNA double chain-ordering:

Upstream chain: 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTT Tg-3'；

Downstream chain: 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGT CC-3'.

4. construction according to claim 3, which is characterized in that the construction is slow virus carrier.

5. a kind of lentiviral particle, which is characterized in that the lentiviral particle includes construction as claimed in claim 3.

6. a kind of cell model of human cystatin E 1 B gene silencing, which is characterized in that the cell model Include lentiviral particle as claimed in claim 5.

7. siRNA molecule described in claim 1, DNA double chain-ordering as claimed in claim 2, building as claimed in claim 3 Lentiviral particle described in object, claim 5 or cell model as claimed in claim 6 are in the drug of preparation treatment oophoroma Application.

8. siRNA molecule described in claim 1, DNA double chain-ordering as claimed in claim 2, building as claimed in claim 3 Lentiviral particle described in object, claim 5 or cell model as claimed in claim 6 increase in preparation inhibition ovarian cancer cell The application in reagent grown, migrate or invaded.

9. application according to claim 8, which is characterized in that the ovarian cancer cell is from Ovarian Cancer Cells OVCAR-3。

10. siRNA molecule described in claim 1, DNA double chain-ordering as claimed in claim 2, structure as claimed in claim 3 Lentiviral particle described in object, claim 5 or cell model as claimed in claim 6 are built in the inhibition ovary of non-treatment purpose Application in cancer cell multiplication, migration or invasion.