CN106520765A - SiRNA-IQGAP1 and use thereof in preparation of drugs for EBV-associated tumor - Google Patents

Abstract

The invention discloses a use of an IQGAP1 targeted siRNA in preparation of drugs for resisting EBV-associated tumor; the siRNA has a positive-sense strand sequence of 5' CGACCUGGCUUUAGAACAA dTdT 3' and an antisense strand sequence of 3' dTdT GCUGGACCGAAAUCUUGUU 5', or has a positive-sense strand sequence of 5' CAAUUGAGCAGUUCAGUUA dTdT 3 and an antisense strand sequence of 3' dTdT GUUAACUCGUCAAGUCAAU 5', or has a positive-sense strand sequence of 5' GAAAUAUACAGCAGCAAGA dTdT 3' and an antisense strand sequence of 3' dTdT CUUUAUAUGUCGUCGUUCU 5'. After the siRNA is transfected in EB virus latent infected epithelial cells and nasopharyngeal carcinoma cells, EBV latent cancer protein expression is reduced significantly along with reduction of expression of IQGAP1, so as to be conducive to suppression of EBV tumorigenesis; and the siRNA has application prospects in specific silencing IQGAP1 gene expression and EBV-associated tumor resistance.

Description

siRNA-IQGAP1 and its use in the preparation of EBV-related tumor drugs

technical field

The invention belongs to the technical field of biomedicine and relates to the application of siRNA targeting IQGAP1 gene in anti-Epstein-Barr virus-related tumor drugs.

Background technique

Epstein-Barr virus (EBV) is the first human tumorigenic virus discovered, which is associated with a variety of malignant tumors and diseases of human lymphocyte and epithelial cell origin, including nasopharyngeal carcinoma, gastric cancer, Burkitt lymphoma tumors, AIDS-related lymphoma, and post-transplantation lymphoproliferative disease (PTLD). After EBV infection, it is in a latent infection state in cells and can evade host immunity. The load of EBV in tumor tissue and blood is positively correlated with the course and prognosis of the disease. EBV maintains a certain copy number in cells, and its encoded nuclear antigen EBNA1 is a key protein for EBV replication. It replicates with cell replication and separates into daughter cells with cell division. During this process, interactions with the host cell occur to help the stable distribution of the viral genome into daughter cells. In the in vitro culture of tumor cells, it was found that EBV was not the same as in vivo, but was gradually lost with the passage of cells. Our previous studies showed that this is the cell-cell contact dependence of EBV on tumor cells. When the cell density is low, EBV is easily lost. Latent membrane protein LMP1 is the main oncoprotein of Epstein-Barr virus, and plays a role in cell transformation and tumorigenesis through cell signaling pathways such as the abnormality of NF-kB. The inventor team found that the EBV viral load is positively correlated with the expression levels of EBNA1 and LMP1, and the decrease in the expression level of oncoproteins also reduces their tumorigenic effects.

Cytoskeletal protein IQGAP1 is a 180kD molecule, which plays an important role in maintaining cell polarity, motility, cell contact inhibition, and also plays a role in cell division. The GenBank accession number corresponding to IQGAP1 is NM_003870.3, and the full length of ORF is 4974bp. IQGAP1 can interact with the key viral protein EBNA1 for Epstein-Barr virus genome replication and isolation, and through this effect, Epstein-Barr virus can be stably maintained in cells. The main function of IQGAP1 is to stably pass Epstein-Barr virus to progeny cells during cell division.

Contents of the invention

The purpose of the present invention is to provide a siRNA by targeting cellular protein IQGAP1, the siRNA-IQGAP1 sense strand sequence is: 5'CGACCUGGCUUUAGAACAA dTdT 3', as shown in SEQ ID No.1, the antisense strand sequence is: 3 'dTdT GCUGGACCGAAAUCUUGUU 5', as shown in SEQ ID No.2; or sense strand: 5'CAAUUGAGCAGUUCAGUUA dTdT 3, as shown in SEQ ID No.3, antisense strand: 3'dTdTGUUAACUCGUCAAGUCAAU 5', as in SEQ ID No.4 or sense strand: 5' GAAAUAUACAGCAGCAAGAdTdT 3' , as shown in SEQ ID No.5, and antisense strand: 3' dTdT CUUUAUAUAUGUCGUCGUUCU 5' , as shown in SEQ ID No.6.

Among them, the target sequence corresponding to the first pair of siRNA is: CGACCTGGCTTTAGAACAA (position: 930-954, as shown in SEQ ID No.7); the target sequence corresponding to the second pair of siRNA is: CAATTGAGCAGTTCAGTTA (position: 1432-1450, as shown in SEQ ID No. ID No.8); the target sequence corresponding to the third pair of siRNAs is: GAAATATACAGCAGCAAGA (position: 4683-4701, as shown in SEQ ID No.9).

The above three pairs of siRNA are all effective for interfering with IQGAP1, and the first pair has the best effect. The three pairs of siRNAs have little difference in the effect on the transcriptional inhibition of EBV-LMP1 after interference, while the first one has the most obvious effect on the expression inhibition of EBNA1.

Through bimolecular fluorescence complementation technology (BiMC) and immunoprecipitation (IP) technology, the inventor team found that IQGAP1 directly interacts with EBNA1 (see Figure 1 and Figure 2), and QGAP1 directly binds to EBNA1 and enters the nucleus, Promote the replication and cytoplasmic separation of EBV, so that the virus genome can be stably passed along with the passage of cells. Using confocal microscopy, we also observed their interaction in the cytoplasm of dividing cells. IQGAP1 is a newly discovered oncoprotein associated with EBV that helps EBV to exert tumorigenic effects, and is a host protein necessary for the stable maintenance of the EBV genome to exert tumorigenic effects in cells. Therefore, IQGAP1 can be used as a molecular target for the treatment of EBV-related tumors.

Therefore, the present invention uses siRNA transfection technology to interfere with the expression of IQGAP1, and interferes with the expression of IQGAP1 in EBV-positive stable transfected cells, which can reduce the expression of EBV virus latent genes and viral load, and in EBV-positive nasopharyngeal carcinoma cells The same performance is significantly reduced, thereby inhibiting the stable maintenance of EBV in cells, reducing the viral load, and achieving the application of treating EBV-related tumors.

The siRNA targeting IQGAP1 has application prospects in the preparation of drugs against Epstein-Barr virus-related tumors.

Description of drawings

Figure 1 is a diagram of the experimental results of BiMC (bimolecular fluorescence complementation) of IQGAP1 and EBNA1;

Figure 2 is a diagram of the IP-WB (co-immunoprecipitation + western blot) experimental results of IQGAP1 and EBNA1;

Figure 3 is the Confocal test diagram of IQGAP1 and EBNA1. Confocal shows that endogenous IQGAP1 and EBNA1 have a strong effect when cells are isolated (the bright part in the right part of the figure);

Figure 4 is a graph showing the expression results of IQGAP1 and EBNA1 protein expression using three siRNAs in the EBV-positive nasopharyngeal carcinoma cell line 293-EBV;

Figure 5 shows the transcription level of LMP1 when three siRNAs interfere with the EBV-positive nasopharyngeal carcinoma cell line 293-EBV, where ** p<0.01; *** p<0.001;

Figure 6 is a graph showing the expression results of using the first siRNA to interfere with IQGAP1 in the EBV-positive nasopharyngeal carcinoma cell line 293-1/NL;

Figure 7 is the transcript level of EBNA1 when the first IQGAP1-siRNA interferes with the cell line 293-1/NL, where ** p<0.01; *** p<0.001;

Figure 8 is the transcript level of LMP1 when the first siRNA-IQGAP1 interferes with the cell line 293-1/NL, where ** p<0.01; *** p<0.001;

Figure 9 is a graph showing the fluorescence results of using the first siRNA to interfere with the expression of IQGAP1 in the EBV-positive nasopharyngeal carcinoma cell line C666-1;

Figure 10 is the transcript level of EBNA1 when the first siRNA-IQGAP1 interferes with the cell line C666-1, where ** p<0.01; *** p<0.001;

Figure 11 is the transcription level of LMP1 when the first siRNA-IQGAP1 interferes with the cell line C666-1, where ** p<0.01; *** p<0.001.

detailed description

Through bimolecular fluorescence complementation technology (BiMC) and immunoprecipitation (IP) technology, the inventor team found that IQGAP1 has a direct interaction with EBNA1 (see Figure 1 and Figure 2), where Figure 1 is BiMC (bimolecular fluorescence complementation) Experimental results, Figure 1 proves that IQGAP1 interacts with EBV nuclear antigen (EBNA1); Part A in Figure 1 shows the interaction between IQGAP1 and EBNA1 in the nucleus (green fluorescence), and Part B in Figure 1 is a single plasmid Negative control for transfection. Figure 2 is a diagram of the experimental results of IP-WB (co-immunoprecipitation + western blot). QGAP1 directly binds to EBNA1 and enters the nucleus to promote the replication and cytoplasmic separation of EBV, so that the virus genome can be stably passed along with the passage of cells.

Using confocal microscopy, we also observed their interaction in the cytoplasm of dividing cells. Figure 3 is the Confocal test diagram of IQGAP1 and EBNA1. Confocal shows that endogenous IQGAP1 and EBNA1 have a strong effect when cells are isolated (the bright part in the right part of Figure 3). IQGAP1 is a newly discovered EBV-related and help EBV The oncoprotein that plays a tumorigenic role is a host protein that is necessary for the stable maintenance of the Epstein-Barr virus genome to play a tumorigenic role in cells. Therefore, IQGAP1 can be used as a molecular target for the treatment of EBV-related tumors.

Example 1: Design of siRNA targeting gene IQGAP1 (siRNA-IQGAP1 for short)

Search NCBI GeneBank to get the full sequence and RNA sequence of IQGAP1, use the existing online software to analyze the biology of IQGAP1, and select the coding region as the sequence for siRNA design. According to the principles of siRNA design, preliminary design and screening were carried out on relevant siRNA design software. Synthesize according to the selected siRNA sequence. siRNA design principles: (1) Use the mRNA sequence of IQGAP1, start from the start codon AUG, search for the "AA" double-linked sequence, and record the base sequence at its 3' end as a potential siRNA target site; (2) The GC content of the designed siRNA sequence is between 30% and 50%; (3) avoid secondary structure repetition; (4) the 3' stability of the sence strand is lower than that of the 5' end; (5) align the potential sequence with the corresponding genome Databases were searched for homologous sequences, excluding sequences homologous to other coding sequences or ESTs. Exclude potential siRNAs with 8 consecutive clips at the 5' end of the aitisence chain paired with other genes; exclude any potential siRNA with 14 consecutive bases paired with other genes.

Finally, three pairs of IQGAP-1-specific siRNA (siRNA-IQGAP1 for short) were obtained, and the sequences were:

The sequences of the sense strand and antisense strand of No. 1 siRNA-IQGAP1 are respectively shown in SEQ ID No.1 and SEQ ID No.2, that is, the sense strand sequence is: 5'CGACCUGGCUUUAGAACAA dTdT 3', and the antisense strand sequence is: 3' dTdTGCUGGACCGAAAUCUUGUU 5';

The sequences of No. 2 siRNA-IQGAP1 sense strand and antisense strand are respectively shown in SEQ ID No.3 and SEQ ID No.4, that is, sense strand: 5' CAAUUGAGCAGUUCAGUUA dTdT 3, antisense strand: 3' dTdT GUUAACUCGUCAAGUCAAU 5';

The sequences of the sense strand and antisense strand of No. 3 siRNA-IQGAP1 are shown in SEQ ID No.5 and SEQ ID No.6 respectively, that is, the sense strand: 5' GAAAUAUACAGCAGCAAGA dTdT 3', the antisense strand: 3' dTdT CUUUAUAUGUCGUCGUUCU 5' .

Example 2: Cell Transfection with IQGAP-1 Specific siRNA (siRNA-IQGAP1)

Three pairs of siRNA-IQGAP1 were synthesized. The synthesized powdered siRNA-IQGAP1 reagent was diluted into a 20uM solution with sterile enzyme-free water, and the final concentration used was 5nM.

Cells C2089, 293-EBV, C22, and 293-1/NL were planted in six-well plates, about 1.5x10 ⁵ -6x10 ⁵ of each cell, and cultured in a cell culture incubator (37°C, 5% CO ₂ ). Take the transfection of one well in a six-well plate as an example. The next day, 5ul of the diluted siRNA-IQGAP1 solution was added to a sterile enzyme-free EP tube filled with 200ul of serum-free DMEM medium (the final concentration of siRNA-IQGAP1 added to the cells was 5nM). Add 6ul of HiPerFect transfection reagent (QIGEN, product number: 301705), mix well, and place at room temperature (15-25°C) for 5-10 minutes. Discard the cell culture medium in the six-well plate and replace it with fresh DMEM medium containing 10% serum, 1.8ml per well. Add the mixture kept at room temperature in the third step above to the prepared cells, and gently shake the six-well plate to dissolve it evenly in the medium. Put them in a cell culture incubator (37°C, 5% CO2) to continue culturing. Cellular protein and total RNA were collected at 36 hours.

Example 3: Real-time fluorescent quantitative PCR (real-time PCR) and western-blotting (western-blot) detection of the expression level of IQGAP1 protein in four cell lines and the effect of interference on the mRNA level

For the cell lines C22 and 293-1/NL with EBV copy number, interference was carried out according to the above steps, and the total protein and total RNA of the cells were collected after 36 hours. Then use western-blot and real-time PCR to detect the post-interference effect of protein level and mRNA level respectively.

For high EBV copy number cell lines C2089 cells and 293-EBV cell lines, two transfections are required to see the effect. After 24 hours of transfection, the cells were subcultured and plated again, and the above siRNA transfection step was performed again, and then the cell protein and total RNA were collected. Then use western-blot and real-time PCR to detect the post-interference effect of protein level and mRNA level respectively.

Wherein, the specific steps of fluorescent quantitative PCR are:

(1) RNA extraction from tissue samples (TRIZol):

Collect si-IQGAP1 cells and control cells transfected for 36 hours, add 1ml RNA TRIZol; place at room temperature for 5-10 minutes, add 0.2ml chloroform to EP tube, place on ice for 5 minutes, centrifuge at 4°C, 12000rpm, 15 minutes; distinguish carefully Put the upper aqueous phase in a new EP tube, repeat the chloroform extraction step; add pre-cooled 0.5ml isopropanol, mix well, place on ice for 15 minutes, 4 ° C, 12000rpm centrifuge, after 10 minutes, discard the supernatant solution; add 75% ethanol, centrifuge at 4°C, 7600rpm, for 5 minutes; after drying at room temperature for 5-10 minutes, add 20ul-50ulDEPC water. In the instrument NanoDrop 2000, the RNA concentration was measured, and the OD260/280 ratio was between 1.9-2.0, and the RNA quality was detected by EB gel electrophoresis, and stored at -80°C.

(2) reverse transcription

Reverse transcription was performed on the total RNA extracted above, and the reverse transcription reaction system was configured according to the instructions of Thermo Scientific Revertaidfirst strand cDNAsynthesis Kit (20ul):

Total RNA 2ug

dNTP(10mM) 2ul

RNasin 1ul

5x Buffer 4ul

M-MuLV Reversr Transcriptase (200U/uL) 1ul

Oligo (dT18) primer 1ul

Water, nuclease-free make up to 20ul total system

Program: 42°C, 60 minutes; 70°C, 5 minutes; the reaction ends.

(3) Realtime-PCR

After diluting the reverse transcript five times, use the reverse transcription product as a template, PCR reaction system (25ul):

LMP1 primer:

Forward: 5'-TGAACACCACCACGATGACT-3'

Reverse: 5'-GTGCGCCTAGGTTTTGAGAG-3'

The primer sequence of the internal reference gene β-actin used is:

Forward: 5'-TCACCAACTGGGACGACATG-3'

Reverse: 5'-GTCACCGGAGTCCATCACGAT-3'

The Realtime PCR reaction system was configured according to the instructions of Thermo Scientific Maxima SYBR Green qPCR MasterMix (2x) (25ul):

2×SYBR Green qPCR Master Mix(2x) 12.5ul

Upstream and downstream primers 2ul

Template cDNA 2ul

Water, nuclease-free make up to 25ul total system.

PCR program: 95°C, 10 minutes; ① 95°C, 30 seconds; ② 58°C, 30 seconds; ③ 72°C, 30 seconds; ①②③ for 45 cycles to terminate the reaction.

The specific steps of Western Blot are as follows:

(1) Total protein extraction: Collect the cells in the logarithmic phase, wash them twice with PBS, add an appropriate amount of protein lysate (containing 1% PMSF), shake and mix on the shaker and place on ice for 20-45 minutes, 12000rpm , centrifuged for 25 minutes, and the supernatant was transferred to a clean EP tube for later use. The concentration of the extracted protein was determined according to the BCA method. Take 30-50 ug of total protein, add an appropriate amount of SDS-PAGE protein loading buffer, and place in a boiling water bath at 100 degrees for 10 minutes.

(2) SDS-polyacrylamide gel electrophoresis

The concentration of the separation gel is selected according to the molecular mass of the protein to be detected. After the separation gel is prepared, cover the top of the separation gel with water. Prepare the integrating gel after the separating gel is completely solidified, and insert the sample loading comb immediately. After it solidifies, pull out the comb and pour it into the electrophoresis solution to prepare for electrophoresis and sample loading. Add the prepared protein samples with the same amount of protein into the sample wells in an appropriate order, and add the pre-stained protein markers at the appropriate positions. The initial voltage is 70V for 30 minutes; after the bromophenol blue enters the separation gel, the voltage is 80V for about 2 hours, and the electrophoresis is stopped when the bromophenol blue is close to the bottom of the separation gel.

(3) Membrane transfer: Wet a PVDF membrane of appropriate size with methanol for 3-5 minutes. Cover the electrophoresis gel with PVDF membrane, and cover the non-contact surface of PVDF membrane and gel with filter paper and sponge, and place it in an electrophoresis tank filled with pre-cooled transfer buffer. Film transfer conditions: voltage 80V, about 90 minutes.

(4) Blocking: Put the transferred PVDF membrane into the ready-made blocking solution (PBST containing 5% skimmed milk), with the membrane face up, place on a shaker at room temperature for 1-2 hours.

(5) Antibody incubation: After blocking, incubate with the primary antibody prepared in blocking solution (PBST containing 5% skimmed milk), overnight at 4°C. The PVDF membrane incubated with the primary antibody overnight was washed with PBST on a shaking plate for 3 times, 20 minutes each time, and incubated with the secondary antibody at 37°C for 1 hour. Then rinse with PBST 3 times, 10-20 minutes each time.

(6) Development: Configure the luminescence solution according to the chemiluminescence ECL kit, image on the BIO-RAD chemiluminescence imaging instrument, and save the result picture.

In EBV stably transfected cell lines, RNA interfered with the expression of IQGAP1, and the expression of EBV latent protein decreased (see Figure 4 to Figure 8). Figure 4 and Figure 5 In the EBV-positive cell 293-EBV, three siRNAs interfered with the expression of IQGAP1. Figure 4 shows the interference effect and the decrease of EBNA1 protein expression, and Figure 5 shows that the transcription level of LMP1 decreased significantly. Figure 6, Figure 7 and Figure 8 are the results of using No. 1 siRNA to interfere with the expression of IQGAP1 in EBV-positive cells 293-1/NL, where Figure 6 shows the interference effect, and it can be seen from Figure 7 that the transcription level of EBNA1 has decreased significantly , it can be seen from Figure 8 that the transcript level of LMP1 decreased significantly.

Example 4: Effect of siRNA-IQGAP1 in EBV-positive nasopharyngeal carcinoma cell line C666-1

Using the method described in Example 3, the effect of siRNA-IQGAP1 in the EBV-positive nasopharyngeal carcinoma cell line C666-1 was detected. The siRNA used in this example is No. 1 siRNA-IQGAP1, and its sequence is: sense strand: 5'CGACCUGGCUUUAGAACAA dTdT 3', antisense strand: 3'dTdT GCUGGACCGAAAUCUUGUU 5'. The test results are shown in Figures 9 to 11.

As shown in Figures 9 to 11, siRNA-IQGAP1 interfered with the expression of IQGAP1 in the EBV-positive nasopharyngeal carcinoma cell line C666-1, and the expression of EBV latent protein decreased. Figure 9 shows the interference effect; Figure 10 shows that the transcription level of EBNA1 significantly decreases after interference; Figure 11 shows that the transcription level of LMP1 significantly decreases after interference.

SEQUENCE LISTING

<110> Central South University

<120> siRNA-IQGAP1 and its use in the preparation of EBV-related tumor drugs

<130> CSU20161-3003

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Claims (8)

1. the positive-sense strand and antisense strand sequence of a kind of interference siRNA of targeting proteins IQGAP1, the siRNA is respectively such as SEQ ID Shown in No.1 and SEQ ID No.2.

2. the positive-sense strand and antisense strand sequence of a kind of interference siRNA of targeting proteins IQGAP1, the siRNA is respectively such as SEQ ID Shown in No.3 and SEQ ID No.4.

3. the positive-sense strand and antisense strand sequence of a kind of interference siRNA of targeting proteins IQGAP1, the siRNA is respectively such as SEQ ID Shown in No.5 and SEQ ID No.6.

4. the Nasopharyngeal Carcinoma Cell Line of siRNA is disturbed in a kind of transfection just like the targeting proteins IQGAP1 one of described in claim 1 ~ 3 293-EBV。

5. the Nasopharyngeal Carcinoma Cell Line of siRNA is disturbed in a kind of transfection just like the targeting proteins IQGAP1 one of described in claim 1 ~ 3 293-1/NL。

6. the Nasopharyngeal Carcinoma Cell Line of siRNA is disturbed in a kind of transfection just like the targeting proteins IQGAP1 one of described in claim 1 ~ 3 C666-1。

7. applications of the siRNA one of described in claim 1 ~ 3 in anti-EBV associated tumors medicine is prepared.

8. applications of the siRNA one of described in claim 1 ~ 3 in anti-EBV correlations medicine for nasopharyngeal is prepared.