CN111228292A

CN111228292A - Application of human TPT1/TCTP gene in preparation of antitumor drugs

Info

Publication number: CN111228292A
Application number: CN202010166119.1A
Authority: CN
Inventors: 王巧文; 邱德辉; 谢翔峰
Original assignee: Fuzhou Carrier Biotechnology Co Ltd
Current assignee: Fuzhou Carrier Biotechnology Co Ltd
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2020-06-05
Anticipated expiration: 2040-03-11
Also published as: CN111228292B

Abstract

The invention relates to application of TPT1/TCTP gene in preparation of antitumor drugs, belonging to the field of biotechnology. The invention constructs human TPT1/TCTP gene small interfering RNA, human TPT1/TCTP gene interfering nucleic acid construct, human TPT1/TCTP gene interfering slow virus and discloses the application thereof. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the expression of human TPT1/TCTP genes, particularly the lentivirus can efficiently infect target cells, and efficiently inhibit the expression of TPT1/TCTP genes in the target cells, so that the growth of tumor cells is inhibited, the apoptosis of the tumor cells is promoted, and the siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus have important significance in tumor treatment.

Description

Application of human TPT1/TCTP gene in preparation of antitumor drugs

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to application of a human TPT1/TCTP gene in preparation of an anti-tumor drug.

Background

The tumor translation control protein (TPT 1 or TCTP) is a growth-related protein, has the functions of promoting growth and resisting apoptosis, and can inhibit the malignant phenotype of tumor cell lines by blocking the expression of the protein. No correlation report exists between TPT1/TCTP gene and other thyroid cancer and thymoma. In the prior art, human gene small interfering RNA, a human gene interfering nucleic acid construct, a human gene interfering lentivirus and the application thereof are constructed by using methods similar to genes. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus can specifically inhibit the expression of human genes, particularly the lentivirus can efficiently infect target cells, efficiently inhibit the expression of genes in the target cells, further inhibit the growth of tumor cells and promote the apoptosis of the tumor cells, and have important significance in tumor treatment.

The invention deeply researches the regulation function of TPT1/TCTP gene in tumorigenesis, thyroid cancer and thymoma cell models, and researches the effect of TPT1/TCTP in tumorigenesis and development of thyroid cancer and thymoma by taking RNAi as a means. Ribonucleic acid interference (RNAi), a gene silencing technique, can efficiently and specifically block the expression of a specific gene in vivo, thereby causing the silencing of the specific gene in vivo and causing cells to show the deletion of a certain gene phenotype. At present, the technology has shown wide prospects in the aspects of gene function research, gene therapy of tumors, research and development of new drugs and the like.

Disclosure of Invention

The invention aims to provide the application of human TPT1/TCTP gene in preparing antitumor drugs.

The following technical scheme is adopted for achieving the purpose:

the invention takes RNA interference as a means to research the effect of TPT1/TCTP gene in tumorigenesis and development, and discloses a method for inhibiting or reducing the growth, proliferation, differentiation and/or survival of tumor cells, which comprises the following steps: administering to the tumor cell a molecule capable of specifically inhibiting transcription or translation of a TPT1/TCTP gene, or capable of specifically inhibiting expression or activity of a TPT1/TCTP protein, thereby inhibiting growth, proliferation, differentiation, and/or survival of the tumor cell. The tumor cell is selected from thyroid cancer and thymoma.

The sequence of human TPT1/TCTP gene is called from Genbank; predicting a plurality of siRNA sites; synthesizing an effective siRNA sequence aiming at TPT1/TCTP gene, wherein two ends of the effective siRNA sequence contain double-stranded DNA Oligo of enzyme cutting site sticky ends; after double enzyme digestion, the lentiviral vector is connected with a double-stranded DNA Oligo to construct an RNAi plasmid for expressing the siRNA sequence of the TPT1/TCTP gene; the RNAi plasmid and auxiliary vectors required by lentivirus packaging are cotransfected with human embryonic kidney cell 293T to generate recombinant lentivirus particles, thymoma and thyroid carcinoma cell lines are selected, lentivirus is infected, qpcr is used for detecting the silencing efficiency of TPT1/TCTP genes, and lentivirus capable of efficiently silencing TPT1/TCTP genes is screened. MTT method detects the proliferation capacity of breast adenoma and thyroid carcinoma cells after TPT1/TCTP silencing.

The invention has the advantages that:

the invention discloses application of a human TPT1/TCTP gene and related medicines thereof. Also discloses the use of human TPT1/TCTP gene in tumor treatment, tumor diagnosis and medicine preparation. The invention further constructs human TPT1/TCTP gene small interfering RNA, human TPT1/TCTP gene interfering nucleic acid construct, human TPT1/TCTP gene interfering slow virus and discloses the application thereof. The siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus provided by the invention can specifically inhibit the expression of human TPT1/TCTP genes, particularly the lentivirus can efficiently infect target cells, and efficiently inhibit the expression of TPT1/TCTP genes in the target cells, so that the growth of tumor cells is inhibited, the apoptosis of the tumor cells is promoted, and the siRNA or the nucleic acid construct containing the siRNA sequence and the lentivirus have important significance in tumor treatment.

Drawings

FIG. 1 identification of TPT1/TCTP gene interference efficiency of FTC-133 human thyroid cancer cells.

FIG. 2 Thy0517 cells TPT1/TCTP gene interference efficiency identification of thymoma cells.

FIG. 3 influence of TPT1/TCTP gene interference on cell proliferation ability of human thyroid cancer cell FTC-133.

FIG. 4 the effect of TPT1/TCTP gene interference on the cell proliferation ability of Thy0517 thymoma cells.

FIG. 5 effect of TPT1/TCTP gene interference on level of human thyroid cancer cell FTC-133 apoptosis.

FIG. 6 effect of TPT1/TCTP gene interference on apoptosis level of Thy0517 thymoma cells.

Detailed Description

EXAMPLE 1 construction of TPT1 Lentiviral vector and establishment of Stable transgenic cell line

1. siRNA target design

The base sequence of human TPT1 (NM-001286272.1) was queried by the National Center for Biological Information (NCBI) of America, and 12 pairs of effective siRNA targets were designed for the coding sequence region by using DSIR software.

2. pLVX-shRNA 2-Puro-TPT 1 and negative control pLVX-shRNA2-Puro-NC plasmid construction

According to the siRNA sequence (taking SEQ ID NO: 1,2 and 3 as an example), corresponding complementary single-stranded DNA is designed, and BamHI and EcoRI cleavage sites are added at both ends.

Annealing, namely annealing the upstream and downstream segments of the single-stranded shRNA to form a double-stranded DNA segment, and then connecting the double-stranded DNA segment to the vector after enzyme digestion.

An annealing reaction system:

and (3) amplification procedure: 95 ℃ for 5 min; cooling for 1-2h at room temperature.

3. Double enzyme digestion recovery of interference vector

1) pLVX-shRNA2-Puro vector digestion system:

2) the pLVX-shRNA2-Puro vector digestion conditions are as follows: 37 ℃ for 2-3 h.

3) And (5) identifying agarose gel, cutting the agarose gel and recovering the agarose gel.

4) Connection of

Since the shRNA fragment already has the cohesive end after enzyme digestion, the shRNA fragment can be directly connected with the vector after enzyme digestion. And connecting the enzyme-digested vector with the gene. The following reagents were mixed and ligated overnight at 16 ℃. The ligation system was as follows (10. mu.l):

5) transformation of

(1) The competent cells Stbl3 were taken out from the freezer at-80 deg.C, quickly placed on ice, and left to stand for 5min to melt.

(2) Adding the connection system into the competence, slightly shaking and uniformly mixing, and placing on ice for ice bath for 30 min;

(3) placing the mixture in 42 deg.C water bath kettle, performing heat shock in water bath for 90 s, rapidly transferring to ice, timing, and standing for 3 min;

(4) adding 900 μ L of nonresistance LB liquid culture medium, setting the temperature of a constant temperature shaking table at 37 ℃, and culturing for 0.5-1 h at the rotating speed of 200 r/min;

(5) the well-spread solid LB-AMP culture dish was used, 100. mu.L of the suspended bacterial solution was spread evenly on a glass coating rod burned with an alcohol burner, and the suspension was cultured overnight in a 37 ℃ incubator by inversion.

6) And (3) identifying positive clones: selecting bacteria and carrying out PCR: sequencing and aligning the sequences. The clone with the correct sequence alignment is the successfully constructed vector.

4. Lentiviral packaging

Human embryonic kidney cell 293T cells were cultured on 10 cm-diameter culture dishes. When the cell density reaches 70% -80%, transfection is carried out by using RRE, REV and VSV/G lentivirus packaging plasmid system, 293T cells ((H) DMEM +5% FBS culture) culture dishes are taken out from the incubator, the culture medium is replaced, the plasmid mixed liquor is added into the 293T cell culture dishes according to the Lipofectamine 3000 instruction, and cell supernatants are collected at 24 hours, 48 hours and 72 hours respectively.

Lentivirus packaging system:

mixing cell supernatants collected three times, centrifuging at 4 deg.C and 3000 g for 25min to precipitate cell debris, filtering the supernatant with 0.45 μm microporous membrane in ultracentrifuge tube, balancing, and centrifuging 30000g for 90 min. The supernatant was decanted, the virus at the bottom of the tube was dissolved sufficiently in 200. mu.l PBS, blown up several times with a pipette tip, and quickly stored in a-80 ℃ freezer for a long period after being dispensed.

5. Qpcr detection of interference efficiency of thyroid cancer cells and thymoma cells TPT1

Establishment of stably low-expressing cell lines: human thyroid cancer cells FTC-133 cells and thymoma cells Thy0517 cells were inoculated in 6-well plates. When the cell density reaches 60%, performing formal infection experiments by using lentivirus infection MOI determined by the pre-experiments and the optimal infection conditions, after the cells are infected for 3-4 days, extracting total RNA of the cells by using a NucleoZol reagent, performing reverse transcription on 1 mu g of RNA into cDNA by using a Promega reverse transcription kit, detecting the relative expression of TPT1 according to GoTaq qPCR Master Mix instructions, and screening a stable-transformation interference cell line by using GAPDH as an internal reference. The TPT1 primers were as follows: an upstream primer: 5'-ATCCAGATGGCATGGTTGCT-3', downstream primer: 5'-TCTTTCTTTTGCATCACATTTTTCC-3', respectively; GAPDH primer: an upstream primer: 5'-GAAAGCCTGCCGGTGACTAA-3', downstream primer: 5'-GCATCACCCGGAGGAGAAAT-3' are provided.

According to 2^-ΔΔCtThe method carries out relative quantitative analysis on the data, and the calculation formula is as follows (x represents any sample): Δ Δ Ct = (C)_t.Target– C_t.GAPDH)X – (C_t.Target– C_t.GAPDH)_Control。

Among them, reverse transcription system (20. mu.L):

the first stage is as follows: denaturing and melting Total RNA

Adding the reagent according to the above, micro-centrifuging for 10 s, mixing, water bathing at 70 deg.C for 5min, and immediately ice bathing for 5 min;

and a second stage: reverse transcription reaction

Adding the reagent according to the above, micro-centrifuging for 10 s, mixing, and keeping at 25 deg.C for 5min and 42 deg.C for 60 min; 15 min at 70 ℃.

real-time PCR reaction system:

qPCR program settings:

experimental results show that compared with a negative interference group, the expression levels of TPT1 in the thyroid cancer cell FTC-133 cell and the thymoma cell Thy0517 cell are respectively reduced.

6. CCK-8 method for detecting proliferation of thyroid cancer cells and thymoma cells in normal group and interference group

Culturing human thyroid cancer cell FTC-133 cell and thymoma cell Thy0517 cell for 24 h, trypsinizing to obtain cell 4 x 10⁴The cell concentration of cells/ml was seeded in 96-well plates, 10. mu.L of 10% CCK-8 solution was added to each well at four time points of 24, 48, 72 and 96 hours, respectively, and the culture was continued for 2 hours. The absorbance of each well was measured at OD450nm using an enzyme linked immunosorbent assay.

The experimental result shows that the cell proliferation capacity of the FTC-133 cells and the Thy0517 cells of the interference group is weakened compared with that of the control group.

7. Flow cytometry was used to detect apoptosis in thyroid cancer cells and thymoma cells in both normal and interfering groups.

Digesting the FTC-133 cells and Thy0517 cells of thyroid cancer cells and thymoma cells with pancreatin, collecting the cells, washing the cells twice with PBS precooled at 4 ℃, resuspending the cells with 250 mu L of binding buffer solution, and adjusting the concentration to be 1X 10⁶Per ml; mu.L of the cell suspension was taken in a 5 ml flow tube, and 5. mu.L of Annexin V/Alexa Fluor 647 and 10. mu.L of 20. mu.g/ml propidium iodide solution were added. After being mixed uniformly inIncubating for 15 minutes at room temperature in a dark place, adding 400 mu l of PBS into a reaction tube, and detecting the apoptosis condition of the SGC-7901 cells by a flow cytometry (FACS).

The experimental result shows that the apoptosis level of FTC-133 cells and Thy0517 cells of the interference group is obviously higher than that of the control group.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fuzhou-based Biotechnology Ltd

Application of <120> human TPT1/TCTP gene in preparation of antitumor drugs

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<170>PatentIn version 3.3

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Claims

1. Application of human TPT1/TCTP gene in preparing antitumor medicine.

2. The application of human TPT1/TCTP gene in preparing thyroid cancer and thymoma resisting medicine.