CN111518808A

CN111518808A - Three ribonucleic acid sequences with anti-tumor effect and application thereof

Info

Publication number: CN111518808A
Application number: CN202010387197.4A
Authority: CN
Inventors: 侯照远; 贾浩
Original assignee: Shanghai Jiaotong University School of Medicine
Current assignee: Shanghai Jiaotong University School of Medicine
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2020-08-11
Anticipated expiration: 2040-05-09
Also published as: CN111518808B

Abstract

The application discloses three ribonucleic acid sequences and application thereof in tumor resistance, wherein the sequences are shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3, and a new method and thought are provided for tumor resistance. The sequence can be obtained by in vitro gene transcription or in vitro chemical synthesis. The three ribonucleic acid sequences can obviously inhibit the proliferation and the metastasis of tumor cells, and have strong clinical treatment value.

Description

Three ribonucleic acid sequences with anti-tumor effect and application thereof

Technical Field

The application relates to the technical field of biological medicines, in particular to ribonucleic acid which has an anti-tumor effect and can inhibit the proliferation and the metastasis of tumor cells.

Background

Cancer seriously harms human health, according to the American Cancer Society (American Cancer Society), about 1810 ten thousand new Cancer cases and 960 ten thousand death cases are found in 2018 all over the world. According to The famous medical journal "Lancet" (The Lancet), new cancer cases reach 2000 million people by 2030, wherein The 5-year survival rate of pancreatic cancer is only 4%, and The annual increase of anticancer drug expenditure is 15%. Due to the increasing morbidity and mortality, cancer has become the leading cause of death worldwide. At present, traditional cancer treatment methods such as surgery, chemotherapy and radiotherapy have been advanced to a plateau, and it is difficult to obtain more satisfactory therapeutic effects. The traditional gene therapy usually adopts a method of enhancing host anti-cancer immunity, antagonizing cancer genes, expressing suicide genes or modifying genes and cooperating with high-dose chemotherapy, and has the defects of low introduction efficiency, poor accuracy, poor controllability of exogenous gene expression in vivo and the like while obtaining a certain curative effect. With the continuous deepening of genomics and genetic engineering research and the maturation of vector technology for delivering nucleic acid drugs (DNA and RNA), new technologies such as targeted gene-virus therapy, RNAi technology and microRNA, cancer drug targeted target screening by using a CRISPR system and the like provide a new method for accurate cancer treatment.

The invention shows the sequence of nucleic acid medicine (3 pieces of ribonucleic acid), which can effectively inhibit the proliferation and metastasis of tumor cells. Provides a new method for accurately treating tumors by nucleic acid medicaments.

Disclosure of Invention

The application provides an antitumor ribonucleic acid, a vector, a protein and application thereof.

The following technical scheme is adopted in the application:

an antitumor ribonucleic acid, the sequence of which is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

A vector comprising the ribonucleic acid of claim 1.

Further, the vector is a plasmid vector or a viral vector.

The ribonucleic acid expressed protein.

The ribonucleic acid, the vector or the protein are applied to preparing antitumor medicines.

Further, the anti-tumor is to inhibit proliferation or/and metastasis of tumor cells.

Further, the tumor is a solid tumor.

Further, the solid tumor is colorectal cancer or pancreatic cancer.

The beneficial effect of this application is as follows:

the three AntiTumor-1, AntiTumor-2 and AntiTumor-3 ribonucleic acid sequences designed by the application are brand-new gene sequences and have not been reported. To date, ribonucleic acid sequences have been reported, generally micro-RNA sequences, which have stronger stability than the sequences in the present application, and in various tumors such as colorectal cancer, pancreatic cancer and the like, AntiTumor-1, AntiTumor-2 and AntiTumor-3 have been found to have antiproliferative and metastatic effects. Therefore, the invention has the advantages of 'brand new' and 'spectrum' (resisting proliferation and metastasis simultaneously), and can be applied to the field of tumor treatment.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a nucleic acid electrophoresis image of AntiTumor-1, AntiTumor-2 and AntiTumor-3.

FIG. 2 is a schematic diagram of colorectal cancer cell proliferation (CCK-8) assay. AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0 and AntiTumor-3-pcDNA3.0 were detected in CCK-8 of colorectal cancer cell SW 1116.

FIG. 3 is a schematic diagram of pancreatic cancer cell proliferation (CCK-8) assay. The detection of AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0 and AntiTumor-3-pcDNA3.0 in CCK-8 of pancreatic cancer cells PANC-1.

FIG. 4 is a schematic diagram of colorectal cancer cell migration (Transwell) assay. 4A is AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0 and AntiTumor-3-pcDNA3.0, which are detected in colorectal cancer cells by Transwell, and 4B is a statistical chart of 4A.

FIG. 5 is a schematic diagram of pancreatic cancer cell migration (Transwell) detection. 5A is AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0 and AntiTumor-3-pcDNA3.0 detected in pancreatic cancer cells by Transwell, and 5B is a statistical chart of 5A.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The following examples do not address specific experimental procedures, generally performed according to routine experimental conditions, and may also refer to the Chinese knowledge network "role of DNA damage stress classical and non-classical (p57Kip2) pathways in tumorigenesis and therapy, doctor's graduation paper, author gianhao".

Example (b):

the three sequences are derived from human genome, three pairs of PCR primers (the primer sequences are about 20-30 bp) are respectively designed according to the sequences, and the primer sequences are respectively as follows:GAATTC:Kpn1；GAATTC:EcoR1。

AntiTumor-1-pcDNA3.0：

S：5’-GGTACcatggagacgcccatcgag(SEQ ID NO:4)-3’，

AS：5’-GAATTCgctgcatctgcgcgcccgcc(SEQ ID NO:5)-3’。

AntiTumor-2-pcDNA3.0：

S：5’-GGTACgggacatgcagcggag(SEQ ID NO:6)-3’，

AS：5’-GAATTCgcagcgcggccgtccccca(SEQ ID NO:7)-3’。

AntiTumor-3-pcDNA3.0：

S：5’-GGTACgccgtgcagggcggg(SEQ ID NO:8)-3’，

AS：5’-GAATTCcctgctcgcgctcgcgct(SEQ ID NO:9)-3’。

synthesized by Shanghai.

PCR in vitro amplification, wherein the template is human genome DNA (prepared by laboratories), polymerase and buffer solution of PCR are purchased from NEB company, and the amplification conditions are as follows: 5min at 95 ℃, three cycles (35 cycles of 30S at 95 ℃, 30S at 55 ℃ and 30S at 72 ℃) and 10min at 72 ℃. Amplification system (20 μ l): mu.g DNA, 0.2mM DNTP, 20pM of upstream and downstream primers, 2. mu.l of 10xbuffer, 1. mu.l of Taq.

The PCR product and eukaryotic expression vector (pcDNA3.0) were separately subjected to double digestion of DNA (NEB) and ligated with T4 DNA ligase at 16 ℃ overnight. The resulting product was transformed into E.coli competent cell DH5 α. After transformation, shake bacteria, and extract recombinant plasmids, namely AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0 and AntiTumor-3-pcDNA3.0 by using a DNA miniprep kit after shaking bacteria.

In vitro transcription to obtain AntiTumor-1, AntiTumor-2 and AntiTumor-3. The RNA in vitro transcription kit was purchased from Thermo FISHER. The method comprises the following specific steps: the following ingredients were added to three RNase-free plastic centrifuge tubes: AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0, AntiTumor-3-pcDNA3.050ng. Preparation of a transcription solution (20. mu.l): t7 RNA polymerase 1. mu.l, 10xbuffer 2. mu.l, NTP 4. mu.l, complement ddH₂O to 20. mu.l. Keeping the temperature at 37 ℃ for 1-2 hours. T7 RNA polymerase was inactivated by heating at 70 ℃ for 10 minutes. Taking 1-3 mul electrophoresis to detect the transcription effect. See figure 1 for specific results.

The obtained AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0 and AntiTumor-3-pcDNA3.0 are subjected to cell functional assay, 3 plasmids are respectively transfected into SW1116 (colorectal cancer) cells and PANC-1 (pancreatic cancer) cells by using LIP3000 transfection reagent, and a cell proliferation experiment (CCK-8) and a cell migration experiment (migration) are respectively carried out.

The cell proliferation experiment (CCK-8) method includes inoculating 4 kinds of cells including AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0, AntiTumor-3-pcDNA3.0 and PCDNA3.0 (control group) into 96-well plate and each well has 4 kinds of cells 4 × 10³Adding 10 μ l CCK-8 solution into each well, incubating for 30min, measuring on automatic enzyme-linked immunosorbent assay, shaking for 1min, selecting 570nm wavelength, and repeating the experiment for 3 times. See fig. 2 and 3 for specific results.

Cell migration experiment (migration) method comprises re-suspending 4 groups of cells including AntiTumor-1-pcDNA3.0, AntiTumor-2-pcDNA3.0, AntiTumor-3-pcDNA3.0 and pcDNA3.0 (control group) cells in DMEM culture solution without serum, and inoculating 1 × 10 in each hole of upper chamber of transwell plate³The final volume of each well was controlled to 200. mu.l. 600. mu.l of DMEM medium containing 10% fetal bovine serum was added to the lower chamber, and the Transwell chamber was placed in CO₂Incubator, 37 ℃ C., 5% CO₂And (3) taking out the chamber after 24hrs, sucking away the culture solution in the upper chamber, wiping the uninvaved cells and Matrigel glue on the upper chamber surface of the microporous filter membrane with a cotton swab, washing with PBS for several times, fixing with 4% paraformaldehyde for 15min, dyeing with Giemsa for 10min, washing with PBS for 3 times, drying, cutting the membrane, sealing on a glass slide, and randomly selecting 5 visual fields under a 200 × light mirror for counting, wherein the specific results are shown in FIG. 4 and FIG. 5.

The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Sequence listing

<110> Shanghai college of medicine of transportation university

<120> three ribonucleic acid sequences with anti-tumor effect and application thereof

<160>9

<170>SIPOSequenceListing 1.0

<210>1

<211>200

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

cauggagacg cccaucgagc gcgaaauccg ccgcagcugc gaacgcgagg agagccugcg 60

ccggagccgg ggccugagcc cgggccgcgc aggccgugaa cucgucgagc ugcgcgugcg 120

gccggugcuc aaccugccgg guccuggccc cgcgcucccg cgcgcccugg agcgggcgcg 180

ggcgggcgcg cagaugcagc 200

<210>2

<211>200

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gggacaucga gcgggaggcc caccggcagg cggcgcuggc gcgccccgcg guccccgagc 60

cgcgcgcccg gucgccgccg cagccgcugg gcgaacucaa gcgcuucuuc gaggccgcgg 120

cggggagcgg cuccucggcg ggggcggggg acggcgcggg cccgcagagg cugccagagc 180

cugggggacg gccgcgcucg 200

<210>3

<211>100

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gccgugcagg gcgggugccg ggugcugggc agcgccccgc cgccuuucac uccgucacug 60

cuggagcagg aggugcgcgc cgugcgcgag cgcgagcagg 100

<210>4

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ggtaccatgg agacgcccat cgag 24

<210>5

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gaattcgctg catctgcgcg cccgcc 26

<210>6

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

ggtacgggac atgcagcgga g 21

<210>7

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gaattcgcag cgcggccgtc cccca 25

<210>8

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ggtacgccgt gcagggcggg 20

<210>9

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gaattccctg ctcgcgctcg cgct 24

Claims

1. An antitumor ribonucleic acid, which is characterized in that the sequence of the ribonucleic acid is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

2. A vector comprising the ribonucleic acid of claim 1.

3. The vector of claim 2, wherein the vector is a plasmid vector or a viral vector.

4. The ribonucleic acid-expressed protein of claim 1.

5. Use of the ribonucleic acid according to claim 1, the vector according to claim 2 or the protein according to claim 4 for the preparation of a medicament for the treatment of tumors.

6. The use of claim 5, wherein the anti-tumor is inhibition of proliferation or/and metastasis of tumor cells.

7. The use of claim 5, wherein the tumor is a solid tumor.

8. The use of claim 7, wherein the solid tumor is colorectal cancer or pancreatic cancer.