CN112826826B - Application of siRNA sequence in preparation of medicine for treating ovarian cancer - Google Patents

Application of siRNA sequence in preparation of medicine for treating ovarian cancer Download PDF

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CN112826826B
CN112826826B CN202110146534.5A CN202110146534A CN112826826B CN 112826826 B CN112826826 B CN 112826826B CN 202110146534 A CN202110146534 A CN 202110146534A CN 112826826 B CN112826826 B CN 112826826B
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陈里新
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Shanghai Lantian Biomedical Technology Co ltd
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Abstract

The invention belongs to the field of biology, and particularly relates to application of a siRNA sequence in preparation of a medicine for treating ovarian cancer. The DNA sequence group for coding the siRNA sequence comprises nucleotide sequences shown as SEQ ID NO:1 and the nucleotide sequence of the forward sequence shown in SEQ ID NO:2, in reverse orientation. The siRNA can be used for preparing a medicament for treating ovarian cancer in the future, and brings good news to patients with ovarian cancer.

Description

Application of siRNA sequence in preparation of medicine for treating ovarian cancer
Technical Field
The invention belongs to the field of biology, and particularly relates to application of a siRNA sequence in preparation of a medicine for treating ovarian cancer.
Technical Field
Ovarian Cancer (OC) is characterized by high recurrence rate, high mortality rate, low survival rate, and the like, and the 5-year survival rate is only 20% -25%, because most patients are found to be in the middle and late stages due to the lack of early symptoms and signs. According to the statistics of 2018, about 295 new cases of ovarian cancer and 184799 death cases worldwide exist, and the life health of women is greatly threatened. The age of menstrual onset, spontaneous abortion, induced abortion, menstrual cycle, dysmenorrhea, pregnancy count and the like are risk factors for the onset of ovarian cancer, wherein the induced abortion, spontaneous abortion and menstrual cycle are main risk factors. The combined treatment of tumor cytoreductive surgery and platinum drug chemotherapy is recommended as the first choice for treating ovarian cancer. Platinum-based chemotherapy is effective in about 80% of ovarian cancer patients, but most patients develop platinum-resistance with increasing cycles of chemotherapy, resulting in relapse, metastasis, and death due to failure of chemotherapy. A large number of researches find that the DNA damage repair capability is enhanced, the accumulation of chemotherapeutic drugs in cells is reduced, the detoxification capability of drugs is enhanced, the apoptosis inhibition is enhanced, and the tumor microenvironment has great influence on the occurrence of platinum drug resistance. The research on the chemotherapy drug-resistant mechanism mainly based on platinum has important significance for improving the sensitivity of platinum-resistant ovarian cancer, developing and designing new drugs, and simultaneously has very important significance for improving the progression-free survival (PFS), the overall survival rate (OS) and the prognosis of ovarian cancer patients.
The small RNA is a small endogenous non-coding RNA, has the length of 20-25 nucleotides, induces the translational inhibition of a target gene by combining with a corresponding site of mRNA, inhibits the expression of cell protein, and plays a key role in the occurrence, the progression and the metastasis of malignant tumors.
However, in the prior art, whether LIM domain 2 protein (LIMD 2) is related to ovarian cancer has not been reported.
Disclosure of Invention
The invention discloses a novel siRNA sequence which can silence LIM domain 2 protein (LIMD 2) and can be used for treating ovarian cancer in the future.
Specifically, the technical scheme of the invention is as follows:
the first aspect of the invention discloses an application of siRNA sequence in preparing medicine for treating ovarian cancer, wherein the DNA sequence group for coding the siRNA sequence comprises nucleotide sequence shown as SEQ ID NO:1 and the nucleotide sequence are shown as SEQ ID NO:2, in reverse orientation. The siRNA sequences are shown below:
forward sequence: 5'-GACCCACCAACTTACCATA-3' (SEQ ID NO: 1)
Reverse sequence: 5'-AATTCTCCGAACGTGTCAC-3' (SEQ ID NO: 2)
The second aspect of the invention discloses an application of a recombinant vector in preparing a medicament for treating ovarian cancer, wherein the recombinant vector comprises the siRNA sequence.
Preferably, the method for constructing the recombinant vector comprises: the set of DNA sequences encoding the siRNA sequences described above is introduced into a lentivirus, adenovirus or adeno-associated virus.
Preferably, the method for constructing the recombinant vector comprises: the DNA sequence group encoding the siRNA sequence is cloned into a lentiviral vector pSLenti-U6-shRNA-CMV-EGFP-2A-Puro by using an AgeI restriction endonuclease and an EcoRI restriction endonuclease.
Preferably, the drug for treating ovarian cancer is a drug for inhibiting migration of ovarian cancer cells.
In a third aspect, the invention discloses a kit for treating ovarian cancer, which comprises the siRNA sequence or the recombinant vector.
In a fourth aspect, the invention discloses a medicament for treating ovarian cancer, which comprises the siRNA sequence or the recombinant vector.
In a fifth aspect of the invention, a method for treating ovarian cancer is disclosed, which comprises transferring the siRNA or the recombinant vector into a patient. Preferably, the medicament is an injection.
Compared with the prior art, the invention has at least the following beneficial effects:
the invention discloses a novel siRNA for treating ovarian cancer, which can be used for preparing a medicament for treating ovarian cancer in the future and brings gospel to patients with ovarian cancer.
Drawings
FIG. 1 is a schematic diagram of pLVshRNA-EGFP (2A) Puro vector in an embodiment of the present invention;
FIG. 2 is a schematic diagram of the pSLenti-U6-shRNA (LIMD 2) -CMV-EGFP-2A-Puro vector in an example of the present invention;
FIG. 3 is a graph showing the effect of siRNA on migration of ovarian cancer cells in an example of the present invention, wherein "cells per field at 200 × differentiation" means "number of cells per field at 200 × migration";
FIG. 4 is a schematic diagram showing the effect of siRNA on Tumor Weight and volume in nude mice in the example of the present invention, in which' "Tumor Weight" means "Tumor Weight", graph A is a schematic diagram showing the effect of siRNA on Tumor volume in nude mice, and graph B is a schematic diagram showing the effect of siRNA on Tumor Weight in nude mice;
FIG. 5 is a graph showing the effect of siRNA on body weight of nude mice in the examples of the present invention.
Detailed Description
The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.
The instruments, equipment, reagents used in the examples are available from various sources, for example, purchased, or may be prepared.
Example 1
The embodiment discloses a preparation method of a recombinant vector, which comprises the following steps:
firstly, a DNA sequence group (a forward sequence: 5'-GACCCACCAACTTACCATA-3' (SEQ ID NO: 1); a reverse sequence: 5'-AATTCTCCGAACGTGTCAC-3' (SEQ ID NO: 2)) for coding an siRNA sequence is cloned into a lentiviral vector pLVshRNA-EGFP (2A) Puro vector by using an AgeI restriction endonuclease and an EcoRI restriction endonuclease to obtain the pSLenti-U6-shRNA (LIMD 2) -CMV-EGFP-2A-Puro vector. The lentivirus is then packaged with a helper plasmid. Next, the packaged lentivirus was used to infect ovarian cancer cells A2780, and 24 hours later, the cells were screened with DMEM medium containing puromycin at 5. Mu.g/ml for 48 hours, and the resulting transfected ovarian cancer cells were subjected to cell migration and subcutaneous tumorigenicity experiments.
The schematic diagram of the pLVshRNA-EGFP (2A) Puro vector and the schematic diagram of the pSLenti-U6-shRNA (LIMD 2) -CMV-EGFP-2A-Puro vector are respectively shown in a figure 1 and a figure 2.
Example 2
Cell migration is an important index of tumor metastasis, and the movement capacity of tumor cells can be researched by applying Transwell.
The Transwell chamber has a permeable polycarbonate membrane on the bottom, the upper chamber is serum-free cell suspension, the lower chamber is serum-containing medium, and certain components in serum, such as some cytokines, are essential for cell growth, so that the cells have tropism for the factors, and the cell tropism is used for promoting cell transfer to the lower chamber, and different cells show different movement transfer capacities.
The transwell chamber of the Corning transfer assay kit consisted of a 24-well tissue culture plate and a 12-well cell culture insert containing a polycarbonate membrane with a pore size of 8 μm, and the transfer cells migrated and adhered to the bottom of the polycarbonate membrane.
This example studies the effect of siRNA on ovarian cancer cell migration, including the following steps:
(1) The kit was removed, the desired number of chambers were placed in a new 24-well plate, 100. Mu.L serum-free medium was added to the upper chamber, and the plate was placed in an incubator at 37 ℃ for 1h.
(2) Serum-free cell suspensions obtained in example 1 were prepared and counted, and the number of cells was adjusted according to the preliminary experiment, typically 105/well (24-well plate).
(3) Carefully remove the medium in the upper chamber and add 100. Mu.L of cell suspension, and 600. Mu.L of 30% FBS medium in the lower chamber. At the same time, the cell suspension was used to spread an MTS 96 well plate, approximately 5000 cells were seeded per well, and OD570 was measured after seeding as a transfer reference.
(4) The incubation was carried out in a 37 ℃ incubator for a period of time (the specific time was adjusted according to the preliminary experiment).
(5) And reversely buckling the chamber on absorbent paper to remove the culture medium, lightly removing non-transferred cells in the chamber by using a cotton swab, dripping 2-3 drops of Giemsa staining solution to the lower surface of the membrane to stain the transferred cells for 3-5min, soaking and washing the chamber for several times, and airing in the air.
(6) Taking a picture by a microscope: for each transwell cell, fields of view were randomly selected and 100 × 4 photographs and 200 × 9 photographs were taken.
(7) Counting by 200 × photos, analyzing data, comparing the difference of cell transfer capacity of experimental group and control group, calculating the number of transferred cells (Migratory cells per field) of each group, obtaining p value by T-Test analysis, judging whether there is significant difference (p is less than 0.05, there is significant difference, otherwise there is no significant difference), and the result is shown in FIG. 3.
Example 3
The embodiment relates to a nude mouse tumorigenicity experiment, which specifically comprises the following steps:
6-week-old female mice were injected subcutaneously with 200. Mu.l (3X 10) of the solution obtained in example 1 in the bilateral plateau region 6 ) Cell mixture (100. Mu.l cell PBS + 100. Mu.l Matrigel). Tumor size was measured every 3 days. Tumor volume was calculated according to the following formula: v = a × b 2 /2. After 3 weeks, nude mice were euthanized by intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg) and tumor body weighing was performed. Fixing a part of the tissues by 10% formaldehyde for subsequent histological examination; the remaining tissue was quickly frozen in liquid nitrogen and stored in a-70 ℃ freezer for subsequent molecular biological testing. Density of injected cellsIs 3X 10 7 Per milliliter.
Experimental results show that the nude mice inoculated with the ovarian cancer cell strain have the advantages that the cell growth after RNA interference is inhibited, and the volume and the weight of the nude mice are lower than those of tumor cells without interference. The results of the experiment are shown in FIG. 4.
The experimental result shows that the weight of the nude mice inoculated with the ovarian cancer cell strain is reduced by more than 10% basically after 7 weeks, and is concentrated on about 15%, while the weight of the nude mice subjected to RNA fragment intervention is reduced by about 2%. The RNA fragment can be used as a limited drug for treating ovarian cancer, as shown in FIG. 5, wherein in FIG. 5, groups 79, 83 and 84 are SiControl groups, groups 80, 81 and 82 are Si-LIMD2 experimental groups, and the only difference between the SiControl group and the Si-LIMD2 experimental group is that: the SiControl group was introduced with an empty vector, and the Si-LIMD2 experimental group was introduced with a recombinant vector comprising a group of DNA sequences encoding siRNA sequences.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Shanghai lan Tian biomedical science and technology Co., ltd
Application of siRNA sequence in preparation of medicine for treating ovarian cancer
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gacccaccaa cttaccata 19
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aattctccga acgtgtcac 19

Claims (7)

1. The application of an siRNA sequence in the preparation of a medicament for treating ovarian cancer is characterized in that a DNA sequence group for coding the siRNA sequence comprises a nucleotide sequence shown as SEQ ID NO:1 and the nucleotide sequence of the forward sequence shown in SEQ ID NO:2 in reverse order;
forward sequence: 5'-GACCCACCAACTTACCATA-3' (SEQ ID NO: 1)
Reverse sequence: 5'-AATTCTCCGAACGTGTCAC-3' (SEQ ID NO: 2).
2. Use of a recombinant vector in the manufacture of a medicament for treating ovarian cancer, wherein the recombinant vector comprises a set of DNA sequences encoding the siRNA sequence of claim 1.
3. The use according to claim 2, wherein the recombinant vector is constructed by a method comprising: introducing a set of DNA sequences encoding the siRNA sequence of claim 1 into a lentivirus, adenovirus or adeno-associated virus.
4. The use of claim 3, wherein the recombinant vector is constructed by a method comprising: cloning a DNA sequence group encoding the siRNA sequence of claim 1 into a lentiviral vector pLVshRNA-EGFP (2A) Puro by using an AgeI restriction endonuclease and an EcoRI restriction endonuclease to obtain pSLenti-U6-shRNA-CMV-EGFP-2A-Puro.
5. The use of any one of claims 1-4, wherein the agent for treating ovarian cancer is an agent that inhibits migration of ovarian cancer cells.
6. The use of claim 5, wherein the medicament comprises the siRNA sequence of claim 1 or the recombinant vector of claims 2-4.
7. The use of claim 5, wherein the medicament is an injection.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN107354159A (en) * 2017-08-03 2017-11-17 哈尔滨医科大学 Applications of the long-chain non-coding RNA SMAD5 AS1 siRNA in treatment of ovarian cancer

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US20090232893A1 (en) * 2007-05-22 2009-09-17 Bader Andreas G miR-143 REGULATED GENES AND PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION
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CN107354159A (en) * 2017-08-03 2017-11-17 哈尔滨医科大学 Applications of the long-chain non-coding RNA SMAD5 AS1 siRNA in treatment of ovarian cancer

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