CN108866061A - A kind of aptamer identifying liver cancer cells and its screening technique and purposes - Google Patents

Abstract

The invention discloses a nucleic acid aptamer for recognizing liver cancer cells and its screening method and application. The nucleotide sequence of the nucleic acid aptamer includes the characteristic sequence part shown in Seq ID No.1. The use includes the use of the above-mentioned nucleic acid aptamer in the design and preparation of anti-liver cancer drugs or detection reagents. The detection reagents include ELISA, radioimmunoassay or fluorescence method detection reagents. The present invention also includes a targeted preparation and a pharmaceutical composition, the targeted preparation includes the above-mentioned nucleic acid aptamer and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition includes the above-mentioned nucleic acid aptamer, at least one antitumor drug and a pharmaceutically acceptable carrier. Compared with the prior art, the aptamer of the present invention can specifically recognize liver cancer cells and has good application prospects.

Description

A nucleic acid aptamer for recognizing liver cancer cells and its screening method and application

technical field

The present invention relates to the field of biomedicine, in particular to a nucleic acid aptamer for recognizing liver cancer cells and its screening method and application.

Background technique

Primary liver cancer is one of the most common and most malignant tumors in the world. Therefore, research on the treatment of liver cancer is urgent. In the clinical treatment plan, although surgical treatment can cure liver cancer, it is limited to a small number of patients. For most patients, due to factors such as large lesions or high postoperative recurrence rate, they cannot be cured. Therefore, chemotherapy is still the main treatment method, but the current chemotherapy of liver cancer still lacks effective therapeutic drugs to ensure the chemotherapy effect of liver cancer, and it is urgent to further develop new therapeutic drugs or methods combined with the research on the pathogenesis of liver cancer.

The tumor stem cell hypothesis believes that there is a small number of tumor stem cells in tumor tissue, which is considered to be the root cause of tumor proliferation, invasion, metastasis and recurrence. At present, tumor stem cells have been successfully isolated from various tumors such as leukemia, breast cancer, lung cancer, brain tumor, colorectal cancer, and prostate cancer. The existence of tumor stem cells in liver cancer is closely related to its characteristics of refractory, recurrence, and drug resistance, and these characteristics also indicate that liver cancer is likely to be a disease of tumor stem cells. Medicines play an important role.

Nucleic acid aptamers are oligonucleotide (DNA/RNA) fragments screened by in vitro screening techniques that can specifically bind metal ions, polypeptides, proteins, and even whole cells. It has strict recognition ability and high affinity. However, studies have shown that nucleic acid aptamers have obvious advantages over peptide aptamers such as antibodies, such as simple and quick preparation, stable chemical properties, no immunogenicity or toxicity reported so far, Wide range of target molecules, high affinity, strong specificity, easy modification and so on.

Therefore, it is of great significance to find out a nucleic acid aptamer specific for liver cancer and apply it to the early detection of liver cancer, molecular imaging, and targeted delivery of drugs.

Contents of the invention

The object of the present invention is to provide a highly specific and stable nucleic acid aptamer for recognizing liver cancer cells and its screening method and application. The aptamer can specifically recognize liver cancer PLC8024 cell line and its derivatives.

A nucleic acid aptamer for recognizing liver cancer cells, the nucleotide sequence of the nucleic acid aptamer includes the characteristic sequence part shown in Seq ID No.1.

The beneficial effect of the present invention is that: the aptamer of the present invention has high specificity and stable performance, and the aptamer can specifically recognize the liver cancer PLC8024 cell line and its derivatives.

Further, the nucleotide sequence of the nucleic acid aptamer is:

X-GGGGGATGGAGGGTGGGTCGTATAT-Y, wherein X and Y are each independently at least one base among A, T, C, and G or at least one of X and Y does not exist.

Further, at least one of the two ends of the nucleic acid aptamer has a modification group.

Further, the modification groups include but are not limited to amino groups, carboxyl groups, mercapto groups, fluorescent molecules, biotin, cholesterol or polyethylene glycol groups.

Further, the nucleic acid aptamer has base modification.

Further, the base modification includes but is not limited to modification by thio, amino, locked nucleic acid, fluorine or methoxy.

The beneficial effect of the present invention is: the base of described nucleic acid aptamer is modified, makes it have more excellent stability than unmodified nucleic acid aptamer.

The present invention also includes the screening method for the above-mentioned nucleic acid aptamer, which includes the following steps: using the nucleic acid aptamer in vitro screening technology, using PLC8024 liver cancer cells as the positive screening target, and using liver immortalized cell LO2 as the negative screening target, from the Nucleic acid aptamers that specifically bind to liver cancer cell lines were screened out from the random oligomeric library.

Further, the screening method specifically includes the following steps:

S1. Synthesizing a random single-stranded library and primers: the random single-stranded library is:

ACCTTGGCTGTCGTGTTGT-25nt-AGGTCAGTGGTCAGAGCGT, said primers include 5' primers: 5'-FAM-ACCTTGGCTGTCGTGTTGT and 3' primers: 3'-Biotin-ACGCTCTGACCACTGACCT;

S2. Nucleic acid aptamer screening: incubate the random single-stranded library with liver cancer stem cells. After the incubation, collect the PLC8024 cell line and heat denature to separate the aptamer bound to the cell line, which is the nucleic acid aptamer obtained by screening. body-level library;

S3. Positive screening: incubate the supernatant of the primary nucleic acid aptamer library obtained in step S2 with the positive screening target, elute the high-affinity aptamer group bound to the surface of the target cells, and obtain enriched aptamers after amplification Positive screening aptamer library that specifically binds to target cells;

S4. Negative screening: incubate the positive screening aptamer group library with the negative screening target, collect the supernatant after cell incubation after incubation, and obtain the enriched negative screening aptamer group that specifically binds to the target cells library;

S5, analyze and identify the selectivity and affinity of each sequence of the negative screening aptamer group library obtained in said step S4, and finally obtain the nucleic acid aptamer with the characteristic sequence shown in Seq ID No.1.

Further, the screening method specifically includes the following steps:

S11. Synthesizing a random single-stranded library and primers: the random single-stranded library is:

ACCTTGGCTGTCGTGTTGT-25nt-AGGTCAGTGGTCAGAGCGT, said primers include 5' primers: 5'-FAM-ACCTTGGCTGTCGTGTTGT and 3' primers: 3'-Biotin-ACGCTCTGACCACTGACCT;

S21. Nucleic acid aptamer screening: incubate the random single-stranded library with liver cancer stem cells. After the incubation, collect the PLC8024 cell line and heat denature to separate the aptamer bound to the cell line, which is the nucleic acid aptamer obtained by screening. body-level library;

S31, PCR amplified library: performing PCR amplification on the nucleic acid aptamer primary library to obtain an amplified product;

S41. Positive screening: incubate the supernatant of the amplified product obtained in step S31 with the PLC8024 cell line to elute the high-affinity aptamer group bound to the surface of the target cell, and obtain enriched aptamers specific to the target cell after amplification. Sex-binding positive screening aptamer library;

S51. Negative screening: incubate the positive screening aptamer library obtained in step S41 with the liver immortalized cell line LO2, collect the supernatant after the incubation, and obtain the enriched negative screening that specifically binds to the target cells Aptamer library;

S61. Nucleic acid aptamer circular screening: repeat steps S11 to S51 at least once, replace the random library with the amplified product, until the affinity selectivity of the obtained aptamer library no longer increases;

S71. Sequencing identification: clone and sequence the aptamer library obtained in step S61, identify the selectivity and affinity of each sequence respectively, and finally obtain the nucleic acid aptamer with the characteristic sequence shown in Seq ID No.1.

The beneficial effect of the present invention is that: the scheme of the present invention adopts an in vitro screening technique known as the ligand system evolution (Systematic evolution of ligand by exponential enrichment, SELEX) technology, and the nucleic acid aptamer obtained by screening has a higher molecular weight than protein antibodies. High affinity and specificity without immunogenicity, can be chemically synthesized in vitro, small molecular weight, can modify and replace different parts, and the sequence is stable, easy to store and easy to label, and the secondary antibody does not need to be labeled.

The present invention also includes the use of the above-mentioned nucleic acid aptamer in the design and preparation of anti-liver cancer drugs or detection reagents.

Further, the detection reagents include but not limited to ELISA, radioimmunoassay and fluorescence method detection reagents.

The present invention also includes a targeting agent, including the above-mentioned nucleic acid aptamer.

Further, the targeting formulation also includes a carrier or excipient selected from pharmaceutically acceptable.

Further, the carrier is selected from any one of nano drug carriers, tumor imaging agents or dye molecules.

The present invention also includes a pharmaceutical composition, which includes the above-mentioned nucleic acid aptamer, at least one antitumor drug and a pharmaceutically acceptable carrier.

The beneficial effects of the present invention are: when using the nucleic acid aptamer of the present invention to detect liver cancer cells, the operation is simpler and faster, and the synthesis cost of the nucleic acid aptamer of the present invention is lower than that of antibody preparation, and the cycle is short and reproducible. Good performance; the characteristic sequence of the nucleic acid aptamer of the present invention can be used as a molecular probe or drug target against liver cancer, which will provide strong support for molecular diagnosis and targeted therapy of liver cancer, and has important clinical value.

Description of drawings

Fig. 1 is the specific binding diagram of the aptamer (left figure) and control aptamer (right figure) of the present invention and PLC8024 liver cancer cell line;

Fig. 2 is a graph showing the specific binding of the aptamer according to the embodiment of the present invention to the paraffin blocks of tumor tissue (left figure) and paracancerous tissue (right figure).

Detailed ways

In order to describe the technical content, structural features, achieved goals and effects of the present invention in detail, the following will be described in detail in conjunction with the embodiments and accompanying drawings.

An embodiment of the present invention is: a nucleic acid aptamer for recognizing liver cancer cells and a screening method thereof, wherein the nucleotide sequence of the nucleic acid aptamer includes AGGAGGTTAGGGGTGGGTGGGTGGT, and the screening method comprises the following steps:

1. Initial DNA library (i.e. primary library) preparation:

Design and synthesize the two ends to contain 19 nucleotides (primers), and the middle includes the nucleic acid sequence random nucleic acid library and the amplification primer of 25 nucleotide random sequences, specifically as follows:

ACC TTG GCT GTC GTG TTG T (as shown in Seq ID No.2)-25nt-A GGT CAG TGG TCAGAG CGT (as shown in Seq ID No.3)

5' primer: ACC TTG GCT GTC GTG TTG T (as shown in Seq ID No.4)

3' primer: ACG CTC TGA CCA CTG ACC T' (as shown in Seq ID No.5)

2. Target Cell Preparation

2.1 Use the trypan blue staining experiment to determine the viability of liver cancer cells, and at the same time determine the number of cells used for incubation.

2.2 The library was incubated with liver cancer cells, and candidate aptamers were screened. Use the binding buffer to dissolve the random nucleic acid library above, the binding buffer solution is prepared with DPBS buffer, containing 5mM magnesium chloride, 4.5g/L glucose, 0.1mg/mL yeast tRNA and 1mg/mL bovine serum albumin (bovine serum albumin, BSA) . Shake at a constant temperature of 95°C for 5 minutes, put it into ice quickly, and then incubate with the cultured and pretreated PLC8024 cell line on ice for 1 hour.

2.3 Dissociate and elute the bound sequence. After the incubation, pour out the liquid in the incubation flask, wash and incubate with washing buffer (the washing buffer is prepared with DPBS buffer, containing 5mM magnesium chloride and 4.5g/L glucose) Cells in culture flasks. Scrape the cell pellet with DNase-free water, heat the cell and DNA mixture at 95°C for 10 minutes, heat denature and separate the aptamers bound to the cell surface, centrifuge at 13,000 rpm for 5 minutes, and collect the supernatant as the specific nucleic acid aptamer for liver cancer cells obtained by screening library.

3. Library amplification: Perform PCR amplification on the library, take 100 μL of PLC8024 cell-specific DNA aptamers obtained from the above screening, and pre-amplify all the obtained one-cell specific nucleic acid aptamer libraries after the first round of screening After 16 cycles, the amplification in this step is carried out again to obtain the amplification product.

4. Digest the double-stranded DNA obtained by PCR amplification into a single-stranded DNA library.

5. Obtain candidate aptamers through multiple rounds of positive and negative screening processes, wherein the operation steps of the positive screening process are as follows: incubate the supernatant of the amplified product in the step with the PLC8024 cell line, elute the aptamer bound to The high-affinity aptamer group on the surface of the target cell is amplified to obtain an enriched positive screening aptamer library that specifically binds to the target cell;

The operation steps of the negative screening process are as follows: incubate the above-mentioned positive screening aptamer library with the liver immortalized cell line LO2, and collect the supernatant after the incubation of the cells to obtain the enriched aptamer specificity for the target cells. Combined aptamer library.

6. After completing the screening process, the final product was sequenced after TOPO-TA amplification, and the sequencing results were analyzed to obtain a highly enriched aptamer:

5'-GGGGGATGGAGGGTGGGTCGTATAT-3'

7. Identification of aptamer specificity and clinical significance

7.1 Use the original DNA library sequence as a negative sequence control to synthesize aptamers with 5'FAM fluorescent molecules and control aptamers.

7.2 Incubate 3×10 ⁵ PLC8024 cells with 250 nM fluorescent aptamer and control library in 200 μL binding buffer at 4 °C for 1 h.

7.3 After incubation, wash 3 times with 500 μL elution buffer, 5 min each time, and finally resuspend the cell pellet with 500 μL elution buffer, and measure the fluorescence intensity of the cells by flow cytometry.

7.4 Observe the binding specificity of the aptamer with a fluorescence microscope. Plant 1.5×10 ⁵ cells in a 35mm culture dish and grow for 24 hours. After the residual medium is eluted with the elution buffer, dissolve the fluorescently labeled aptamer in 1 mL of the binding buffer. The final concentration of the antibody and the control library reached 250nM, incubated at 4°C for 1 hour, and the observation results were shown in Figure 1. It can be seen from Figure 1 that the nucleic acid aptamer of the sequence of the present invention has a strong binding to PLC8024 cells binding force, while the binding force of the control aptamer was weaker.

7.5 Immunohistochemistry

Take the paraffin blocks of the tumor and paracancerous tissues of the liver cancer patients, dewax the sections with xylene, and then hydrate them with graded alcohols (100%, 95%, 90%, 80% and 70%) for 5 min at each concentration, and then After washing with DPBS solution for 5 min, the sample was treated with 0.01 mol/L, pH 6.0 citrate solution at 95°C for 20 min to restore the antigen, and left at room temperature until natural cooling. The blocking solution was prepared with binding buffer (adding 20% fetal bovine serum (FBS), 0.1 mg/mL silicon fish essence DNA) for blocking at room temperature for 1 h, and then in 200 μL of binding buffer, 250 nM FAM-labeled appropriate The ligand or control library was incubated on ice in the dark for 30 minutes, and the fluorescence was observed with a fluorescence microscope. The observed results are shown in Figure 2. Through observation, it was found that the aptamer of the sequence of the present invention has a good ability to recognize and detect liver cancer tissues.

The experimental methods in the present invention are conventional methods unless otherwise specified, and the materials used in the experiment are purchased from conventional biochemical reagent stores unless otherwise specified; the source of PLC8024 liver cancer cells used in the present invention All cells used in this experiment are from tumors in South China State Key Laboratory of Science, other cells come from American Tissue CultureCollection.

The above is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the description of the present invention and the contents of the accompanying drawings, or directly or indirectly used in other related technologies fields, all of which are equally included in the scope of patent protection of the present invention.

sequence listing

<110> The Fifth Affiliated Hospital of Sun Yat-sen University

<120> A nucleic acid aptamer for recognizing liver cancer cells and its screening method and application

<160> 5

<170> SIPO Sequence Listing 1.0

<210> 1

<211> 25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gggggatgga gggtgggtcg tatat 25

<210> 2

<211> 19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

accttggctg tcgtgttgt 19

<210> 3

<211> 19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aggtcagtgg tcagagcgt 19

<210> 4

<211> 19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

accttggctg tcgtgttgt 19

<210> 5

<211> 19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

acgctctgac cactgacct 19

Claims (9)

1. A nucleic acid aptamer for recognizing liver cancer cells, characterized in that: the nucleotide sequence of the nucleic acid aptamer includes the characteristic sequence part shown in Seq ID No.1. 2. The nucleic acid aptamer for recognizing liver cancer cells according to claim 1, wherein at least one of the two ends of the nucleic acid aptamer has a modification group. 3. The nucleic acid aptamer for recognizing liver cancer cells according to claim 1, characterized in that: the nucleic acid aptamer has base modifications. 4. A screening method for a nucleic acid aptamer according to any one of claims 1-3, characterized in that: comprising the following steps: using a nucleic acid aptamer in vitro screening technique, using PLC8024 liver cancer cells as a positive screening target, Using liver immortalized cell LO2 as negative screening target, nucleic acid aptamers that specifically bind to liver cancer cell lines were screened from a random oligomeric library synthesized in vitro. 5. screening method according to claim 4, is characterized in that: specifically comprise the following steps: S1. Synthesizing a random single-stranded library and primers: the random single-stranded library is: ACCTTGGCTGTCGTGTTGT-25nt-AGGTCAGTGGTCAGAGCGT, the primers include 5' primer: 5'-FAM-ACCTTGGCTGTCGTGTTGT and 3' primer: 3'-Biotin-ACGCTCTGACCACTGACCT; S2. Nucleic acid aptamer screening: incubate the random single-stranded library with the positive screening target. After the incubation is completed, collect the PLC8024 cell line and heat denature to separate the aptamer bound to the cell line, which is the nucleic acid aptamer obtained from the screening. Ligand primary library; S3. Positive screening: incubate the supernatant of the primary nucleic acid aptamer library obtained in step S2 with the positive screening target to elute the high-affinity aptamers bound to the surface of the target cells, and obtain enriched aptamers after amplification. Positive screening aptamer library for target cell specific binding; S4. Negative screening: incubate the positive screening aptamer group library with the negative screening target, collect the supernatant after cell incubation after incubation, and obtain the enriched negative screening aptamer group that specifically binds to the target cells library; S5. Analyze and identify the selectivity and affinity of each sequence of the negative screening aptamer library obtained in step S4, and finally obtain the nucleic acid aptamer with the characteristic sequence shown in Seq ID No.1. 6. Use of the nucleic acid aptamer according to any one of claims 1-3 in designing and preparing anti-liver cancer drugs or detection reagents. 7. The use according to claim 6, characterized in that: the detection reagents include enzyme-linked immunoreaction, radioimmunoassay or fluorescence method detection reagents. 8. A targeting preparation, characterized in that it comprises the nucleic acid aptamer according to any one of claims 1-3. 9. A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises the nucleic acid aptamer according to any one of claims 1-3, at least one antitumor drug and a pharmaceutically acceptable carrier.