CN111378664B - Circular RNA and application thereof - Google Patents
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Abstract
The invention belongs to the field of genetic engineering, and particularly relates to application of circular RNA circ _0058040 in preparation of a medicament for treating non-small cell lung cancer; by changing the influence of the expression of the circular RNA circ _0058040 on the proliferation, apoptosis, drug sensitivity and the like of the non-small cell lung cancer cells, the reduction of the expression of the circular RNA circ _0058040 can inhibit the proliferation of the non-small cell lung cancer cells and enhance the sensitivity of the non-small cell lung cancer cells to targeted drugs.
Description
Technical Field
The invention belongs to circular RNA, and particularly relates to application of circular RNA circ _0058040 in preparation of a medicine for treating non-small cell lung cancer.
Background
Circular RNA (circular RNA) is a new class of RNA characterized by covalent ring closure, distinct from traditional linear RNA, and is more stable and conserved than linear RNA. It was first discovered in 1976 by Nobel prize-giving Sanger in plant viruses. Within 30 years after the finding of circRNA, it is considered to be a low abundance RNA molecule formed by mis-splicing of exons, subject to experimental techniques and research methods. In recent years, a number of circrnas have been discovered in succession in different species, with the benefit of rapid development of high-throughput sequencing technologies and bioinformatics.
circRNA has become a new hotspot for tumor research due to its emerging potential role in oncogenic and tumor-suppressive pathways. In recent years, many studies have shown that certain circrnas are closely related to the corresponding human tumors. The circRNA plays an important role in the occurrence and development of tumors by regulating a series of biological functions or interfering with normal functions, including modes of transcriptional silencing, alternative splicing and the like.
According to data published by the World Health Organization (WHO), the incidence and mortality of lung cancer are on a trend of rising obviously in all countries, especially in industrially developed countries. In developed countries, lung cancer is one of the most common malignancies, the first of which in men and the second of which in women. Lung cancer has been the first death of malignant tumors by the end of the 20 th century. The morbidity and mortality of lung cancer in China are ranked first in malignant tumors and still rapidly increase every year. Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancer cases, more than 70% of patients are in middle and advanced stages, the chance of radical operative treatment is often lost, the five-year survival rate is lower than 5%, and a new and effective method for treating lung cancer needs to be found urgently. Oxitinib is a third generation tyrosine kinase inhibitor (EGFR-TKI) targeting epidermal growth factor receptors, and is a targeted drug for clinically treating lung cancer. Although the oxitinib has stronger drug effect than the first and second generations of EGFR-TKI such as gefitinib and afatinib to a certain extent, the drug resistance phenomenon is still unavoidable. With the progress of genetic engineering research, scientists have shown a great interest in developing tumor drugs by genetic engineering. The inventor finds that circ _0058040 has a good application prospect in the aspect of treating non-small cell lung cancer.
The inventor firstly finds that the circ _0058040 is a circRNA with the length of 663nt, and the circ _0058040 is knocked out by an interference sequence to have the effect of resisting the lung cancer.
Disclosure of Invention
Technical purpose
The invention aims to provide application of circ _0058040 in preparation of a medicine for treating non-small cell lung cancer.
A circular RNA circ _0058040, the nucleotide sequence of which is Seq NO. 1.
The application of the circular RNA circ _0058040 in preparing the medicine for treating the non-small cell lung cancer is characterized in that the circular RNA circ _0058040 is realized by the following interference sequences;
circ _0058040 interference sequence (5 'to 3'):
AGATGAAAGGCAATACAAGGAACGGAAdTdT, see Seq NO. 2; or
GGCAATACAAGGAACGGAAAACAGTTCTdT, see Seq No. 3.
The circRNA mainly plays a role in absorbing the ceRNA sponge, and the circRNA comprises a miRNA response element and can be combined with the miRNAs in a targeted mode to inhibit the capacity of the miRNAs for combining with the mRNA.
Advantageous effects
1. The invention discovers the influence of circ _0058040 on apoptosis, proliferation and targeted drug sensitivity of non-small cell lung cancer cells for the first time, and shows that the reduction of the expression of circ _0058040 can inhibit the proliferation of the non-small cell lung cancer cells; and has an effect on drug resistance of the oxitinib, and can increase the sensitivity of the non-small cell lung cancer cells to the targeted drugs.
2. At present, a commonly used method for interfering the circRNAs comprises the steps of preparing a circRNAs lentivirus interference vector in vitro, transfecting the circRNAs lentivirus interference vector to cells and playing a role in reducing the expression of the circRNAs in vivo. The method is simple and has high transfection efficiency.
Drawings
FIG. 1 is a diagram showing the analysis of the circular RNA expression profiles in normal lung tissue and lung cancer tissue by the gene chip (Sample A is normal lung tissue; Sample B is lung cancer tissue);
FIG. 2qRT-PCR assay of the expression level of 16HBE, a normal lung epithelial cell strain, and a non-small cell lung cancer cell strain A549 circ _0058040, with GAPDH as the internal reference;
FIG. 3 shows the results of a colony formation experiment;
FIG. 4 shows the results of cell proliferation experiments;
FIG. 5 shows the apoptosis changes of non-small cell lung cancer cell line A549 on Oxitinib before and after changing the expression of circ _0058040 by flow cytometry. (A: apoptosis graph, B: apoptosis result statistical graph);
FIG. 6 shows the change of non-small cell lung cancer cell strain A549 IC50 before and after the CCK8 method detects and changes the circ _0058040 expression;
FIG. 7 is a schematic diagram of circ _ 0058040;
FIG. 8 is a schematic diagram of lentiviral vector and shRNA inserts.
Detailed Description
The invention is further illustrated by the following examples, without restricting the invention thereto.
General description:
experimental recipes for the examples without specifying specific conditionsMethods, essentially in accordance with the Molecular Cloning A Laboratory Manual,3, all of which is published by Sambrook, J et al, Molecular Cloning, A Laboratory Manual,3rded. Huangpetang et al, science publishers, 2002.8) or according to the material suppliers suggested conditions and methods, other techniques not described in detail correspond to standard methods well known to those skilled in the art.
The material of the invention: the cell lines, lentiviral interference vectors, and culture media referred to in this application are commercially available or otherwise publicly available, and are by way of example only and not exclusive to the present invention, and may be replaced by other suitable means and biological materials, respectively.
Example 1
Detection of circ _0058040 expression in tissues and cells
(I): expression of circ _0058040 in normal lung tissue and lung cancer tissue:
1. preparing a normal lung tissue and a lung cancer tissue specimen according to the requirements of Shanghai Bohao company, and delivering the specimen to the Bohao company for chip preparation and analysis, wherein the chip comprises a plurality of probes for detecting the latest versions of the circRNAs database and tens of thousands of circRNAs. The detection principle of the chip is that according to the length and the structural characteristics of a circRNA sequence, a T7 promoter is introduced in a reverse transcription mode of a random primer, then RNA is amplified by an in vitro transcription mediated linear amplification method, and the RNA linear amplification method is used for sample marking in microarray chip analysis. And (3) carrying out reverse transcription on the linear amplification product cDNA to obtain DNA, carrying out fluorescent labeling on the DNA by Klenowfagent enzyme, finally hybridizing the fluorescent labeled DNA product with an Oligo probe on the chip, and carrying out gene expression analysis based on the chip. By using the circRNA chip detection, researchers can rapidly obtain the expression change of the circRNA related to a specific biological process or disease in high flux.
The process of detecting the sample mark by the Berhao biological circRNA chip mainly comprises the following steps:
1) 1st-strand cDNA was synthesized starting from a sample of Total RNA using the CbcScript enzyme with T7 Random Primer containing the T7 promoter sequence as Primer.
2) Second strand DNA synthesis: RNA strands in the DNA-RNA hybrid were converted into Second strand cDNA using RNaseH and DNA Polymerase, double-stranded DNA was synthesized, and DNA column purification was performed.
3) In vitro transcription synthesis of cDNA: RNA was amplified in this step by synthesizing cDNA using T7 Enzyme Mix using double-stranded DNA as a template.
4) cDNA purification: the cDNA was purified using an RNA purification column, and the cDNA was quantified by removing the reagents such as salts and enzymes in the reaction.
5) cDNA fluorescent labeling: the cDNA complementary strand of the reverse transcription was synthesized using Klenow Fragment enzyme with a template of cDNA product and Primer of Random Primer, and Cy3-dCTP or Cy5-dCTP with a fluorophore was incorporated. The labeled product was purified and quantified. These DNAs with fluorescent groups can be used for chip hybridization.
After screening the circRNAs with high specificity expression in lung cancer tissues, researchers amplify DNA by a qRT-PCR method to identify the obtained product as target DNA (circ _0058040), wherein primers of circ _0058040 are as follows: f Primer: 5'-TGCACCAGAAGGTCCTACTCA-3', see SEQ NO: 4; r Primer: 5'-ACTTCAGGTACTGCATGCTTTG-3', see SEQ NO: 5.
2. chip results experimental data from berhao corporation are shown in fig. 1 and table 1, where Ratio is the signal Ratio of Sample b/Sample a.
TABLE 1 list of significant differences
3. The result analysis shows that the signal of the circ _0058040 in the lung cancer tissue (Sample B) is 8.338804, the signal of the circ _0058040 in the normal lung tissue (Sample A) is 0.197315, the Ratio (Sample B/Sample A) is 42.26142, which indicates that the circ _0058040 is highly expressed in the lung cancer tissue and is lowly expressed in the normal lung tissue, and the difference between the two is 42.26142 times, which indicates that the circ _0058040 may play a role as a cancer gene in the lung cancer.
(II) qRT-PCR analysis of the expression profile of circ _0058040 in normal lung tissue cell line 16HBE and non-small cell lung cancer cell line A549
1. The method comprises the following steps: total cellular RNA was extracted using Trizol reagent, and SYBR Green PCR Master Mix (TaKaRa, Dalian, China) and human GAPDH were used as internal control for qRT-PCR.
qRT-PCR results: as shown in fig. 2.
3. And (4) analyzing results: the circ _0058040 is low expressed in normal lung tissue cell strain 16HBE and high expressed in non-small cell lung cancer cell strain A549, which is consistent with the gene chip result.
Example 2
Construction of circ _0058040 Lentiviral interference vector transfected cells:
designing a specific interference sequence aiming at circ _0058040, wherein the sequence is (5 'to 3'): AGATGAAAGGCAATACAAGGAACGGAAdTdT, see Seq No.2, with a sequence piece that interferes with the GAPDH gene as a control and is inserted into a lentiviral vector, see FIG. 8. Infecting non-small cell lung cancer cell strain A549 cell with the packaged slow virus vector to play a role in reducing circ _0058040 expression, screening a stable transfected cell line with G418 after 48h, and naming the stable transfected cell line as A549/sh-circ _0058040 (the contrast is A549/NC),
example 3: cell proliferation assay and clone formation assay
1. Clone formation experiments:
a experimental method:
1) inoculating cells: digesting the monolayer culture cells by using 0.25% trypsin, preparing a single A549/sh-circ _0058040 (A549/NC as a control) cell suspension by using a 1640 culture solution containing 10% fetal bovine serum, and inoculating 500 cells per well into a 6-well culture plate;
2) placing into a cell culture box, changing the culture solution once every 4 days, and culturing for 2 weeks;
3) when macroscopic colonies appeared in the culture dish, the culture was terminated. Discarding the supernatant, and gently washing with PBS for 2 times;
4) adding pure methanol or 1:3 acetic acid/methanol 1ml, fixing for 15 minutes;
5) removing the methanol fixing solution, adding 1ml of 0.1% crystal violet staining solution for staining for 15 minutes, then slowly washing off the staining solution by PBS, and drying in air;
b. and (4) determining the result: as shown in fig. 3.
c. And (4) analyzing results: after A549 is transfected with the circ _0058040 lentivirus interference vector, the clone spots are reduced compared with a control group, and the fact that the reduction of the expression of the circ _0058040 can inhibit the proliferation of the non-small cell lung cancer cells is suggested.
2. Cell proliferation assay
a. A549 cell strain (A549/sh-circ _0058040) transfected by a circ _0058040 lentivirus interference vector is constructed, and an A549 cell strain (A549/NC) transfected by an empty vector is set as a control group. These cells were seeded in 96-well plates at an appropriate cell density (2000 cells/well).
b. And (4) measuring the result: after culturing for 6h, 24h, 48h, 72h and 96h, adding 20ul of CCK8 solution into each well, continuously incubating for 2h at 37 ℃, and detecting by using an enzyme-labeling instrument after terminating the culture.
c. And (4) analyzing results: the reduction of clonotypes compared with the control group after the A549 is transfected with the circ _0058040 lentivirus interference vector suggests that the reduction of the expression of circ _0058040 can inhibit the proliferation of non-small cell lung cancer cells, as shown in FIG. 4.
Example 4: apoptosis assay
1. Detecting apoptosis by a flow cytometer: a549 cell strain (A549/sh-circ _0058040) transfected by a circ _0058040 lentivirus interference vector is constructed, and an A549 cell strain (A549/NC) transfected by an empty vector is set as a control group. These cell lines were seeded in 6-well plates at 3X 105Individual cells/well. After 48h, the level of apoptosis was measured by flow cytometry.
2. And (4) determining the result: as shown in fig. 5.
3. And (4) analyzing results: compared with a control group, after transfection of the circ _0058040 lentivirus interference vector, apoptosis is increased, and the reduction of the expression of the circ _0058040 is suggested to promote apoptosis of non-small cell lung cancer cells.
Example 5: cell drug resistance related experiments
CCK8 assay cell IC 50: a circ _0058040 lentiviral interference vector transfected A549 cell strain (A549/sh-circ _0058040) is constructed, and an empty vector transfected A549 cell strain (A549/NC) is set as a control group. These cell lines were seeded in 96-well plates at 2500 cells/well. And (3) treating each group with the oxitinib, detecting the cell viability by a CCK8 staining method after 3 days of administration, further calculating the IC50 of different treatment groups, and analyzing the sensitivity of the cells of the different treatment groups to the oxitinib.
2. Results the measurement is shown in FIG. 6.
3. Results analysis of the results in A549 cell line, Osciltinib-treated sh-circ-0058040 cells were administered
4. Test group IC 50: 13.05uM, control IC50 (NC): 25.87 uM. This observation suggests that decreasing the expression of circ _0058040 promotes apoptosis of non-small cell lung cancer cells and increases the sensitivity of the cells to oxitinib.
Sequence listing
<110> second subsidiary hospital of Nanjing medical university
<120> circular RNA and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 663
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 1
gaacggaaaa cagttcttcc ttcaaagcat gcagtacctg aagtaataga agactttctc 60
tgcaatttct tgatcaaaat gggaatgacc agaactcttg attgctttca gtctgaatgg 120
tatgagttaa tacagaaagg agtgactgaa cttagaactg ttgggaatgt tccagatgtc 180
tacacccaga ttatgctttt ggaaaatgag aacaaaaatt taaagaaaga tttgaagcac 240
tacaaacaag cagctgacaa agctagagaa gatttgctga aaattcagaa agaacgtgat 300
tttcatcgaa tgcatcataa gcgaatagtc caggaaaaaa acaaattaat taatgacctc 360
aaagggttga agttacatta tgcatcttat gaaccgacta taagggtgtt acatgagaaa 420
caccacactt tactgaagga gaaaatgctg acctccttgg aaagagacaa agtagttggg 480
cagatttctg gacttcaaga aacattgaag aaactgcaaa gaggacatag ttaccatggt 540
cctcaaatta aagttgatca tagtcgtgaa aaagaaaatg caccagaagg tcctactcag 600
aaaggtcttc gtgaagccag ggaacaaaac aaatgtaaaa caaagatgaa aggcaataca 660
aag 663
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 2
agatgaaagg caatacaaag gaacggaann 30
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 3
ggcaatacaa aggaacggaa aacagttcnn 30
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 4
tgcaccagaa ggtcctactc a 21
<210> 5
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<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 5
acttcaggta ctgcatgctt tg 22
Claims (1)
1. An application of an interference sequence of circular RNA circ _0058040 in preparing a medicament for treating non-small cell lung cancer is characterized in that the nucleotide sequence of RNA circ _0058040 is Seq ID NO.1, and the interference sequence is agatgaaaggcaatacaaaggaacggaadtt.
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| CN117024555B (en) * | 2023-07-18 | 2024-02-13 | 遵义医科大学珠海校区 | CircLRRFIP2-60aa and application thereof in lung squamous carcinoma treatment |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488903A (en) * | 2011-12-31 | 2012-06-13 | 南京医科大学第二附属医院 | Application of miR-224 to preparation of medicament for treating non-small cell lung cancer |
| CN103316359A (en) * | 2013-07-11 | 2013-09-25 | 南京医科大学第二附属医院 | Application of long-chain non-coding RNA in preparation of non-small cell lung cancer treatment drugs |
| CN105177005A (en) * | 2015-10-13 | 2015-12-23 | 南京医科大学第二附属医院 | Long non-coding RNA (Ribonucleic Acid) and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102488903A (en) * | 2011-12-31 | 2012-06-13 | 南京医科大学第二附属医院 | Application of miR-224 to preparation of medicament for treating non-small cell lung cancer |
| CN103316359A (en) * | 2013-07-11 | 2013-09-25 | 南京医科大学第二附属医院 | Application of long-chain non-coding RNA in preparation of non-small cell lung cancer treatment drugs |
| CN105177005A (en) * | 2015-10-13 | 2015-12-23 | 南京医科大学第二附属医院 | Long non-coding RNA (Ribonucleic Acid) and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Cell-Type Specific Features of Circular RNA Expression;Julia Salzman等;《PLOS GENETICS》;20130905;第9卷(第9期);第14页左栏第2段，表格S1 * |
| Circ 0058040 Inhibits Cadmium‐Induced Transformation of Human Bronchial Epithelial Cells by Decoying PIP5K1α;Q. Wang等;《Society of Toxicolog》;20200311;第97页第2段 * |
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