CN109652550A - Application of the circ-HUWE1 as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target - Google Patents

Application of the circ-HUWE1 as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target Download PDF

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CN109652550A
CN109652550A CN201910052867.4A CN201910052867A CN109652550A CN 109652550 A CN109652550 A CN 109652550A CN 201910052867 A CN201910052867 A CN 201910052867A CN 109652550 A CN109652550 A CN 109652550A
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huwe1
circ
gastric cancer
cancer
colorectal
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CN109652550B (en
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李向南
王振军
陈泓宇
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Beijing Chaoyang Hospital
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

Application the invention discloses circular rna HUWE1 (circ-HUWE1) as a kind of new potential gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target.Circ-HUWE1 (hsa_circ_0140388 | chrX:53641494-53644407- | HUWE1) it is to be formed by HUWE1 gene 20-23 exon reverse splicing, cyclization sequence has 589 bases.The present invention is prepared for the qRT-PCR primer of circ-HUWE1 and the siRNA (siRNA) of external interference circ-HUWE1.Compared with normal control, circ-HUWE1 expresses significant up-regulation in gastric cancer and Patients with Colorectal Cancer tumour and plasma sample.Experiment in vitro shows that circ-HUWE1 plays the effect of promotion sensitivity gene in gastric cancer and colorectal cancer cell.Result of study of the present invention shows that circ-HUWE1 is a kind of potential gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target newly.

Description

Circ-HUWE1 is as gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target Application
Technical field
The invention belongs to medicine technology fields, and it is raw as gastric cancer and diagnosis of colorectal carcinoma to be related to a kind of circular rna HUWE1 The application of object marker 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, AR1D1A, 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 the non-coding RNA that a major class covalent bond forms ring structure. Circular rna found that initially it is considered as a kind of wrong montage and no research significance in 1976 in plant virus first RNA.Then research discovery circRNA has important regulating and controlling effect to gene expression in vivo it is characterized in that tool There is the cyclic structure of closure, without 5 ' cap ends and 3 ' tail ends.This cyclic structure is to hold acceptor splicing site through anti-by upstream 3 ' It holds donor splicing site to connect with downstream 5 ' to be formed to montage.It is this that circRNA is led to not with the annular closed structure of Covalent bonding together It is influenced by exonuclease, it is more stable than linear rna.Recent study finds that circRNA has following basic function: as Competitive endogenous RNA (ceRNA) adsorbs microRNA；Regulatory transcription and alternative splicing；Act on rna binding protein；It turns over It is translated into protein.Based on the function of the above circRNA, correlative study has confirmed that circRNA with human diseases includes that tumour is close Cut phase is closed.Therefore, mechanism of action of the further investigation circRNA in gastroenteric tumor, diagnosis for gastroenteric tumor and is controlled Treating has potential huge meaning.
Summary of the invention
The present invention provides circRNA HUWE1 (circ-HUWE1) in terms of gastric cancer and diagnosis of colorectal carcinoma and treatment Using.The invention discloses circ-HUWE1 as a kind of new potential gastric cancer and diagnosis of colorectal carcinoma biomarker and to control Treat target spot: circ-HUWE1 expresses significant up-regulation in the tumour and plasma sample of gastric cancer and Patients with Colorectal Cancer；Subject's work Make feature (ROC) curve and shows that circ-HUWE1 has the potential of good diagnosis of gastric cancer and colorectal cancer；Circ-HUWE1 promotees Into the proliferation of gastric cancer and colorectal 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 circ-HUWE1 (hsa_circ_0140388 | chrX:53641494-53644407- | HUWE1) (core former times acid sequence As shown in SEQ ID No.1).Circ-HUWE1 is formed by HUWE1 gene 20-23 exon reverse splicing, cyclisation Sequence has 589 bases.
A second object of the present invention is to provide the primer pairs of specific recognition circ-HUWE1, 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 table of the open circ-HUWE1 in gastric cancer and Patients with Colorectal Cancer tumor tissues Up to situation.
Fourth object of the present invention is expression feelings of the open circ-HUWE1 in gastric cancer and Patients with Colorectal Cancer blood plasma Condition, and the ROC curve of corresponding evaluation circ-HUWE1 diagnosis capability.
5th purpose of the invention is the function that open circ-HUWE1 is played in gastric cancer and colorectal cancer cell.
The beneficial effects of the present invention are as follows: 1) discovery circ-HUWE1 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 control, tumour and blood of the circ-HUWE1 in gastric cancer and Patients with Colorectal Cancer Starch significant up-regulation in sample；3) ROC curve shows the ability that circ-HUWE1 has good diagnosis of gastric cancer and colorectal cancer. 4) of the invention the result shows that interference circ-HUWE1 can inhibit the proliferation of gastric cancer and colorectal cancer cell, show circ- HUWE1 plays the effect of promotion sensitivity gene in the occurrence and development of gastric cancer and colorectal cancer, is clinical treatment gastric cancer and colorectal cancer Provide new target spot.
Detailed description of the invention
The biology synthesis and structural schematic diagram that Fig. 1 is circ-HUWE1.
Fig. 2 is the result figure detected using circ-HUWE1 primer pair Patients with Gastric Cancer tissue expression amount.* represents p value < 0.01。
Fig. 3 A is the result figure detected using circ-HUWE1 primer pair Patients with Gastric Cancer and physical examination of healthy population blood plasma expression quantity； B is the ROC curve figure for evaluating circ-HUWE1 diagnosis of gastric cancer potential.* * * represents p value < 0.0001.
Fig. 4 is the result figure detected using circ-HUWE1 primer pair Patients with Colorectal Cancer tissue expression amount.* * represents p Value < 0.001.
Fig. 5 A is the knot detected using circ-HUWE1 primer pair Patients with Colorectal Cancer and physical examination of healthy population blood plasma expression quantity Fruit figure；B is the ROC curve figure for evaluating circ-HUWE1 diagnosis colorectal cancer potential.* * * represents p value < 0.0001.
Fig. 6 is that drop effect is struck in circ-HUWE1 siRNA (siRNA) transfection in two kinds of cell lines of HGC-27 and SW480 The qRT-PCR proof diagram of rate.Si-NC represents negative control group；Si-circHUWE1#1, si-circHUWE1#2, which are respectively represented, to be turned Contaminate the siRNA group in two kinds of targeting circ-HUWE1 reverse splicing sites；* p value < 0.05 is represented, * * represents p value < 0.01, * * * P value < 0.001 is represented, * * * * represents p value < 0.0001.
Fig. 7 is the result figure of gastric cancer and colorectal cancer cell proliferation variation after striking drop circ-HUWE1.* p value < is represented 0.05, * * represents p value < 0.01, and * * * represents p value < 0.001, and * * * * represents p value < 0.0001.
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.Tissue specimen is logical Liquid nitrogen flash freezer is crossed, unloading is in -80 DEG C of refrigerators.Collect the preoperative blood of above-mentioned 30 gastric cancers and 60 Patients with Colorectal Cancer.In addition it receives Collect the blood of 45 ages and the matched physical examination of healthy population of gender as compareing.Blood plasma is obtained by centrifugation, is stored in -80 DEG C of ice Case.
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 the total serum IgE of cell and tissue) is extracted；Using TRIzolTMLS (Invitrogen, Carlsbad, CA, USA) extracts blood plasma Total serum IgE.Reverse Transcriptase kit uses PrimeScriptTMRT reagent Kit (TaKaRa, Dalian, China)；Fluorescence Quantitative PCR kit uses TB GreenTM Premix Ex TaqTMII (TaKaRa, Dalian, China)；Using AB17500 real-time fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA) carries out PCR reaction；It adopts Use 18S rRNA as internal reference；Using 2-ΔΔCtMethod calculates RNA relative expression quantity；Primer is by giving birth to work bioengineering (Shanghai) share Co., Ltd's synthesis；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-HUWE1 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 Fisher Scientific, 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-HUWE1 is formed by HUWE1 gene 20-23 exon reverse splicing.
If the qRT-PCR of Fig. 2 is the results show that compared with normal tissue, circ-HUWE1 is expressed in gastric cancer tumor tissue Significant up-regulation.* represents p value < 0.01.
If the qRT-PCR of Fig. 3 A is the results show that compared with physical examination of healthy population, circ-HUWE1 table in Patients with Gastric Cancer blood plasma Up to significant up-regulation.Area (AUC) is 0.856 under ROC curve in Fig. 3 B, shows that circ-HUWE1 has good Diagnosis of Gastric carninomatosis Person's force.* * * represents p value < 0.0001.
If the qRT-PCR of Fig. 4 is the results show that compared with normal tissue, circ-HUWE1 is in Patients with Colorectal Cancer tumor group Knit the significant up-regulation of middle expression.* * represents p value < 0.001.
If the qRT-PCR of Fig. 5 A is the results show that compared with physical examination of healthy population, circ-HUWE1 is in Patients with Colorectal Cancer blood plasma The middle significant up-regulation of expression.AUC is 0.822 in Fig. 5 B, shows that circ-HUWE1 has the latent of good diagnosis Patients with Colorectal Cancer Energy.* * * represents p value < 0.0001.
As shown in fig. 6, transfecting si-circHUWE1#1 and si- in HGC-27 gastric cancer and SW480 colorectal cancer cell system After circHUWE1#2, circ-HUWE1 expression quantity is significantly lowered, and its corresponding HUWE1 mrna expression amount then becomes without obvious Change.* p value < 0.05 is represented, * * represents p value < 0.01, and * * * represents p value < 0.001, and * * * * represents p value < 0.0001.
As shown in fig. 7, gastric cancer and colorectal cancer cell proliferative capacity are remarkably decreased after transfection si-circHUWE1#1.* generation Table p value < 0.05, * * represent p value < 0.01, and * * * represents p value < 0.001, and * * * * represents p value < 0.0001.
The above results show that circ-HUWE1 expresses up-regulation in gastric cancer and Patients with Colorectal Cancer tumor tissues and blood plasma； ROC curve shows the ability that circ-HUWE1 has good diagnosis of gastric cancer and colorectal cancer.Experiment in vitro shows circ- HUWE1 promotes the proliferation of gastric cancer and colorectal cancer cell, shows that circ-HUWE1 is played in gastric cancer and colorectal cancer and promotees cancer base The effect of cause；Circ-HUWE1 is expected to become a kind of new gastric cancer and diagnosis of colorectal carcinoma biomarker and therapy target.
Embodiment described above is exemplary, and is merely preferred embodiments of the present invention, protection of the invention Range is without being limited thereto, for those of ordinary skill in the art, under the precursor for not departing from the technology of the present invention principle, can also do Several improvements and modifications out, these improvements and modifications also should be regarded as protection scope of the present invention.

Claims (7)

1. a kind of circular rna (circular RNA/circRNA) HUWE1 (circ-HUWE1) is examined as gastric cancer and colorectal cancer The application of disconnected biomarker and therapy target, it is characterised in that: the circBase ID of the circular rna is hsa_circ_ 0140388, mankind's X chromosome is derived from, is cyclized and is generated by the 20 to 23rd exon reverse splicing of HUWE1 host gene, Being cyclized mature sequence length is 589bp, 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 Can with circ-HUWE1 expression quantity in the sample of gastric cancer or colorectal cancer object, wherein the circ-HUWE1 of higher expression quantity with It is related that the object suffers from the increase of a possibility that gastric cancer or colorectal cancer and bad prognosis.
3. gastric cancer according to claim 1 and diagnosis of colorectal carcinoma biomarker circ-HUWE1 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-HUWE1 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-HUWE1 are in preparation gastric cancer and colorectal cancer Application in treatment product.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103502473A (en) * 2011-03-01 2014-01-08 耶鲁大学 Predicting gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs)
US20150141273A1 (en) * 2012-04-26 2015-05-21 Stichting Vu-Vumc Biomarkers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103502473A (en) * 2011-03-01 2014-01-08 耶鲁大学 Predicting gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs)
US20150141273A1 (en) * 2012-04-26 2015-05-21 Stichting Vu-Vumc Biomarkers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIANG-NAN LI等: "RNA sequencing reveals the expression profiles of circRNA and indicates that circDDX17 acts as a tumor suppressor in colorectal cancer", 《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》 *

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