CN109576373A - Application of the circ-VAPA as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target - Google Patents
Application of the circ-VAPA as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target Download PDFInfo
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Abstract
Application the invention discloses circular rna VAPA (circ-VAPA) as a kind of new potential gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target.Circ-VAPA (hsa_circ_0006990 | chr18:9931806-9937063+ | VAPA) it is to be formed by VAPA gene 2-4 exon reverse splicing, cyclization sequence has 338 bases.The present invention is prepared for the qRT-PCR primer of circ-VAPA and the siRNA (siRNA) of external interference circ-VAPA.Compared with normal tissue, circ-VAPA expresses significant up-regulation in gastric cancer and Patients with Colorectal Cancer tumor tissues.Experiment in vitro shows that circ-VAPA plays the effect of promotion sensitivity gene in gastric cancer and colorectal cancer cell.Result of study of the present invention shows that circ-VAPA is a kind of potential gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target newly.
Description
Technical field
The present invention relates in medicine technology field, the application of tumor markers or potential treatment target spot, more particularly to
Application of the circ-VAPA as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target.
Background technique
60% the above are digestive system tumors in malignant tumour, and wherein gastric cancer, colorectal cancer are the most common digestive systems
Tumour.According to newest global cancer statistical report in 2018, in all malignant tumours, gastric cancer mortality occupied the whole world the
Three, disease incidence occupies the whole world the 5th；The colorectal cancer death rate occupies the whole world second, and disease incidence occupies global third.Research at present
The Molecular pathogenesis for showing gastric cancer includes gene mutation (P53, ARID1A, FAT4, CDH1), and it is logical that coherent signal occurs for tumour
Road is abnormal (Wnt, RTK, PI3K signal path), and chromosome instability is fixed (variation of body cell copy number, chromosome translocation), apparent to lose
It passes and learns (island mismatch repair gene CpG cytosine methylation, histone modification), microsatellite instability；The molecule of colorectal cancer is sent out
Interpretation of the cause, onset and process of an illness system includes oncogene activation (K-ras, c-myc, EGFR), and tumor suppressor gene inactivates (APC, DCC, P53), mismatch repair gene
It is mutated (hMLH1, hMSH2, hMSH6, hPMS2) and Overexpression (COX-2, CD44v) etc..However, above-mentioned point
The clinical practice that sub- target spot only has small part to be applied to gastric cancer and colorectal cancer.Therefore, it is necessary to which exploring causes gastric cancer and knot straight
Other potential pathogenesis of aspect that intestinal cancer occurs.
Circular rna (circular RNA/circRNA) is a kind of endogenic non-coding RNA.With other linear rnas point
It is sub different, circRNA no 5 ' cap ends and 3 ' tail ends, but formed after reverse splicing by Pre-mRNA.Due to
CircRNA has with the annular closed structure of Covalent bonding together, so circRNA is more stable than linear rna, it is not easy to by core
Sour excision enzyme degradation.According to current research, circRNA has following basic function: as competitive endogenous RNA
(ceRNA) microRNA is adsorbed；Regulatory transcription and alternative splicing；Act on rna binding protein；Translate into protein.It is based on
The function of the above circRNA, correlative study have confirmed that circRNA with human diseases includes that tumour is closely related.Research shows that
CircRNA is a kind of potential Tumor biomarkers and therapy target.However, most of circRNA are specific in cancer
Mechanism of action is not elucidated with yet, so mechanism of action of the further investigation circRNA in gastric cancer and colorectal cancer, for gastric cancer and
The diagnosis and treatment of colorectal cancer all have potential huge meaning.
Summary of the invention
The present invention provides circRNA VAPA (circ-VAPA) answering in terms of gastric cancer and diagnosis of colorectal carcinoma and treatment
With.The invention discloses circ-VAPA as a kind of new potential gastric cancer and diagnosis of colorectal carcinoma biomarker and treatment
Target spot: circ-VAPA expresses up-regulation in the tumor tissues of gastric cancer and Patients with Colorectal Cancer；Circ-VAPA promotes gastric cancer and knot
The proliferation of rectum cancer cell.
The first purpose of the invention is to provide a kind of gastric cancers and the potential diagnostic biomarkers of colorectal cancer and therapeutic target
Point is that (nucleotide sequence is such as by circ-VAPA (bsa_circ_0006990 | chr18:9931806-9937063+ | VAPA)
Shown in SEQ ID No.1).Circ-VAPA is formed by VAPA gene 2-4 exon reverse splicing, and cyclization sequence has
338 bases.
A second object of the present invention is to provide the primer pairs of specific recognition circ-VAPA, including upstream primer is under
Swim primer.The nucleotide sequence of upstream primer is as shown in SEQ ID No.2；The nucleotide sequence of downstream primer such as SEQ ID
Shown in No.3.
Third object of the present invention is expression of the open circ-VAPA in gastric cancer and Patients with Colorectal Cancer tumor tissues
Situation.
Fourth object of the present invention is function of the open circ-VAPA in gastric cancer and colorectal cancer cell.
The beneficial effects of the present invention are as follows: 1) discovery circ-VAPA can be used as gastric cancer and diagnosis of colorectal carcinoma biology mark for the first time
Will object and AD-targeted drugs；2) compared with normal tissue, circ-VAPA is in the tumor tissues of gastric cancer and Patients with Colorectal Cancer
Significant up-regulation；3) of the invention the result shows that interference circ-VAPA can inhibit the proliferation of gastric cancer and colorectal cancer cell, table
Bright circ-VAPA plays the effect of promotion sensitivity gene in the occurrence and development of gastric cancer and colorectal cancer, is clinical treatment gastric cancer and knot
The carcinoma of the rectum provides new target spot.
Detailed description of the invention
The biology synthesis and structural schematic diagram that Fig. 1 is circ-VAPA.
Fig. 2 is the result figure detected using circ-VAPA primer pair Patients with Gastric Cancer tissue expression amount.
Fig. 3 is the result figure detected using circ-VAPA primer pair Patients with Colorectal Cancer tissue expression amount.
Fig. 4 is the qRT-PCR proof diagram that drop efficiency is struck in circ-VAPA siRNA (siRNA) transfection.Si-NC represents yin
Property control group；Si-circVAPA#1, si-circVAPA#2 respectively represent two kinds of targeting circ-VAPA reverse splicing sites of transfection
SiRNA group；* represents p value < 0.01, and * * * represents p value < 0.001.
Fig. 5 is the result figure of gastric cancer and colorectal cancer cell proliferation variation after striking drop circ-VAPA.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not intended to limit the scope of the invention for illustrating the present invention.
Embodiment:
1. experimental material and method:
Clinical sample: it is connect during collecting 2016-2018 in general surgery, Beijing Chaoyang Hospital Attached to Capital Medical Univ.
By 30 gastric cancers of operative treatment and the tumor tissues and cancer side normal mucosa tissue of 60 Patients with Colorectal Cancer.
Cell line and cell culture: human gastric cancer cell line HGC-27 and colorectal cancer cell system SW480 is purchased from US mode
Culture collection warehousing (American Type Culture Collection, ATCC).Cell culture is containing 10% tire ox blood
Clearly in DMEM (Invitrogen, Carlsbad, CA, the USA) culture medium of (Gibco, NY, USA), 100U/ml penicillin is added
With 100 μ g/ml streptomysins (Gibco, NY, USA).Contain 5%CO at 37 DEG C2Environment in cultivate cell.
RNA extract and real-time fluorescence quantitative PCR (qRT-PCR): using Trizol (Invitrogen, Carlsbad, CA,
USA) extract the total serum IgE of cell and tissue: Reverse Transcriptase kit uses PrimeScriptTMRT reagent Kit (TaKaRa,
Dalian, China)；PCR kit for fluorescence quantitative uses TB GreenTM Premix Ex TaqTMLl (TaKaRa,
Dalian, China)；Using 7500 real-time fluorescence quantitative PCR instrument of ABI (Applied Biosystems, Foster City,
CA, USA) carry out PCR reaction；Using 18S rRNA as internal reference；Using 2-ΔΔCtMethod calculates RNA relative expression quantity；Primer is by giving birth to
The synthesis of work bioengineering (Shanghai) limited liability company；Primer sequence is shown in Table 1.
The primer sequence that 1 qRT-PCR of table is used
Transfection: the specific siRNA (siRNA) and negative control (si- in targeting circ-VAPA reverse splicing site
NC) You Jima gene (Shanghai) synthesizes；It will using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA)
The siRNA of 50nM is transfected into gastric cancer and colorectal cancer cell；SiRNA sequence is shown in Table 2.
2 siRNA sequence of table
CCK-8 experiment: cell is carried out using CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan)
Proliferation experiment.About 1000 cells are inoculated in 96 orifice plates.At 0,24,48,72,96 hour, 10 μ l are added into 96 orifice plates
CCK-8 reagent.Be incubated for two hours after, using multi-function microplate reader (Thermo FisherScientific, Waltham, MA,
USA 450nm optical density (OD) value) is read.5 repetition values of every group of measurement.
Statistical analysis: for statistical analysis to result using 23.0 software of SPSS.It is soft using GraphPad Prism 7.0
Part mapping.Statistical analysis is according to circumstances carried out using paired t-test or Wilcoxon signed rank test.Data are at least three times
The form of the mean ± standard deviation of independent experiment indicates that all P values are bilateral, and P < 0.05 is considered statistically significant.
2. experimental result:
As shown in Figure 1, circ-VAPA is formed by VAPA gene 2-4 exon reverse splicing, qRT-PCR product
The reverse splicing site of Sanger sequencing result confirmation circ-VAPA.
If the qRT-PCR of Fig. 2 is the results show that compared with normal tissue, circ-VAPA expresses aobvious in gastric cancer tumor tissue
Write up-regulation (P < 0.01).
If the qRT-PCR of Fig. 3 is the results show that compared with normal tissue, circ-VAPA is in Patients with Colorectal Cancer tumor tissues
The middle significant up-regulation (P < 0.0001) of expression.
As shown in figure 4, transfecting si-circVAPA#1 and si- in HGC-27 gastric cancer and SW480 colorectal cancer cell system
After circVAPA#2, circ-VAPA expression quantity is significantly lowered, and its corresponding VAPA mrna expression amount is then without significant change.
As shown in figure 5, gastric cancer and colorectal cancer cell proliferative capacity are remarkably decreased after transfection si-circVAPA#1.
The above results show that circ-VAPA expresses up-regulation in gastric cancer and Patients with Colorectal Cancer tumor tissues；Experiment in vitro
It shows that circ-VAPA promotes the proliferation of gastric cancer and colorectal cancer cell, shows that circ-VAPA is played in gastric cancer and colorectal cancer
The effect of promotion sensitivity gene；Circ-VAPA is expected to become a kind of new gastric cancer and diagnosis of colorectal carcinoma biomarker and therapeutic target
Point.
Embodiment described above is exemplary, and is not considered as limiting the invention, for the common of this field
Technical staff can also make several improvements and modifications under the precursor for not departing from the technology of the present invention principle, these improve and become
Type also should be regarded as protection scope of the present invention.
Claims (7)
1. a kind of circular rna (circular RNA/circRNA) VAPA (circ-VAPA) is used as gastric cancer and diagnosis of colorectal carcinoma
The application of biomarker and therapy target, it is characterised in that: the circBase ID of the circular rna is hsa_circ_
0006990, No. 18 chromosome of the mankind is derived from, is cyclized and is generated by the 2 to 4th exon reverse splicing of VAPA host gene,
It is 338bp that it, which is cyclized mature sequence length, and nucleotide sequence is as shown in SEQ ID No.1.
2. the application of gastric cancer according to claim 1 and diagnosis of colorectal carcinoma biomarker comprising detection comes from can
Circ-VAPA expression quantity in the sample of gastric cancer or colorectal cancer object can be suffered from, wherein the circ-VAPA of higher expression quantity and institute
It states a possibility that object suffers from gastric cancer or colorectal cancer and bad prognosis and increases correlation.
3. gastric cancer according to claim 1 and diagnosis of colorectal carcinoma biomarker circ-VAPA are in preparation gastric cancer and knot
Application in carcinoma of the rectum diagnostic products.
4. application according to claim 3, it is characterised in that: the product is selected from preparation, chip or kit.
5. application according to claim 3, it is characterised in that: the product includes drawing for specific recognition circ-VAPA
Object pair.
6. application according to claim 5, it is characterised in that: the primer pair includes upstream primer and downstream primer, institute
The nucleotide sequence of upstream primer is stated as shown in SEQ ID No.2；The nucleotide sequence of the downstream primer such as SEQ ID No.3
It is shown.
7. gastric cancer according to claim 1 and treatment of colorectal cancer target spot circ-VAPA are in preparation gastric cancer and colorectal cancer
Application in treatment product.
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Cited By (5)
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|---|---|---|---|---|
| CN112011620A (en) * | 2020-09-23 | 2020-12-01 | 山东大学第二医院 | Application of circ-SLC38A1 as target in medicine for inhibiting bladder cancer cells |
| CN112159848A (en) * | 2020-09-30 | 2021-01-01 | 长治医学院 | Application of cyclic RNA as gastric cancer diagnosis biomarker and prognosis evaluation reagent |
| CN114317539A (en) * | 2022-01-12 | 2022-04-12 | 上海卡序生物医药科技有限公司 | hsa _ circ _0001137 circular RNA and application thereof in cancer diagnosis and treatment |
| CN114705859A (en) * | 2022-04-26 | 2022-07-05 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Biomarker for diagnosis, treatment and prognosis of liver cancer bone metastasis and application thereof |
| CN114790457A (en) * | 2022-04-01 | 2022-07-26 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Application of circB3GALNT2 in colorectal cancer metastasis prediction and treatment |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011620A (en) * | 2020-09-23 | 2020-12-01 | 山东大学第二医院 | Application of circ-SLC38A1 as target in medicine for inhibiting bladder cancer cells |
| CN112011620B (en) * | 2020-09-23 | 2021-04-27 | 山东大学第二医院 | Application of circ-SLC38A1 as target in medicine for inhibiting bladder cancer cells |
| CN112159848A (en) * | 2020-09-30 | 2021-01-01 | 长治医学院 | Application of cyclic RNA as gastric cancer diagnosis biomarker and prognosis evaluation reagent |
| CN114317539A (en) * | 2022-01-12 | 2022-04-12 | 上海卡序生物医药科技有限公司 | hsa _ circ _0001137 circular RNA and application thereof in cancer diagnosis and treatment |
| CN114317539B (en) * | 2022-01-12 | 2023-05-23 | 上海卡序生物医药科技有限公司 | hsa_circ_0001137 circular RNA and application thereof in cancer diagnosis and treatment |
| CN114790457A (en) * | 2022-04-01 | 2022-07-26 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Application of circB3GALNT2 in colorectal cancer metastasis prediction and treatment |
| CN114790457B (en) * | 2022-04-01 | 2022-12-06 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Application of circB3GALNT2 in colorectal cancer metastasis prediction and treatment product |
| CN114705859A (en) * | 2022-04-26 | 2022-07-05 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Biomarker for diagnosis, treatment and prognosis of liver cancer bone metastasis and application thereof |
| CN114705859B (en) * | 2022-04-26 | 2023-02-24 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Biomarker for diagnosis, treatment and prognosis of liver cancer bone metastasis and application thereof |
| WO2023207072A1 (en) * | 2022-04-26 | 2023-11-02 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Biomarker for diagnosis, treatment and prognosis of liver cancer bone metastases and use thereof |
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