CN104198722A - Novel tumor marker GSTA1 for lung caner as well as screening method and application thereof - Google Patents
Novel tumor marker GSTA1 for lung caner as well as screening method and application thereof Download PDFInfo
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- CN104198722A CN104198722A CN201410391515.9A CN201410391515A CN104198722A CN 104198722 A CN104198722 A CN 104198722A CN 201410391515 A CN201410391515 A CN 201410391515A CN 104198722 A CN104198722 A CN 104198722A
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Abstract
The invention discloses a novel tumor marker GSTA1 for lung cancer as well as a screening method and application thereof, belonging to the field of molecular biology and medical science. The marker is characterized in that on the basis of the novel lung cancer specific binding polypeptide acquired by utilizing a bacteriophage random peptide library screening technology at the earlier stage, the polypeptide is used as a probe to select lung cancer associated antigen GSTA1 from a lung cancer cDNA library. The occurrence and development of the lung cancer have a complicated mechanism, the lung cancer involves to the alteration of a lot of lung cancer associated genes and expressions thereof in the primary stage, a plurality of adhesion molecules as well as receptors or ligands can be expressed in a patient body under the effect of a carcinogenic factor, the content of the marker released by the tumor at different development stages is also different, the variation of the specific molecular marker of the tumor cancer is detected from peripheral blood and sputum, advantages of simplicity, rapidness, little pain and easiness in reviewing can be achieved, the novel tumor marker GSTA1 is easy to receive by the patient, and an important significance on the clinical diagnosis for the lung cancer can be achieved. The molecular marker can be provided for the diagnosis and targeted therapy of the lung cancer, a candidate antigen can be provided for the research of the lung cancer tumor vaccine, and the application prospect is wide.
Description
Technical field
The present invention relates to molecular marking technique field, more specifically, relate to the new tumor markers GSTA1 of a kind of lung cancer and screening technique and application.
Background technology
Lung cancer is one of malignant tumour that the most common in global range, lethal number is maximum at present, the first cancer of Ye Shi China, and its incidence of disease and mortality ratio increase the rapidest.Due to the concealment of lung cancer onset, its diagnosis and treatment situation is far unsatisfactory, and its reason of censuring is mainly to lack means and effective methods for the treatment of of early diagnosis.When symptom appears in patient, mostly be late period, prognosis is poor, and total five year survival rate is no more than 15%, has symptom≤10%.But the patients with lung cancer of making a definite diagnosis in early days, the prognosis of operative treatment obviously improves compared with medium and advanced lung cancer, and its survival rate can reach 70%.As can be seen here, reduce the mortality ratio of lung cancer patient except reducing the incidence of disease on causal prophylaxis, the curative effect that improves early diagnosis level and drug therapy is two importances.But, the early diagnosis of lung cancer is a global problem always, diagnostic imaging (x-ray at present, CT and MRI), chemical diagnosis (cancer reaction, serology and amynologic index) and histocytology diagnosis be three large pillars of diagnosing tumor, the former has very large limitation in Patients with Subclinical diagnosis, the latter two are all using tumor markers as observation index, can be than the more Zao existence of finding tumour of diagnostic imaging, have efficient, high sensitivity, convenient, the advantage such as sample easily obtains and wound is little, therefore, in the early stage diagnosis and treatment research of lung cancer, the lung cancer tumor markers of screening and qualification high specific is the Focal point and difficult point of research always.
So-called tumor markers, refers in tumour generation and breeding, is interacted and a class material of generation by tumour cell biosynthesizing, release or tumour and host, is reflecting existence and the growth of tumour.We can be by utilizing the technology such as chemistry, immunity, molecular biology to carry out qualitative or quantitative detection to blood or secretion, by to its analysis, the characteristic that helps us to distinguish tumour from normal structure or to measure in neoplastic cell nuclei, tenuigenin and cell membrane is analyzed, using this as the mark of distinguishing tumour cell.The generation of lung cancer has very complicated mechanism with development, the initial period of its morbidity relates to the change of many lung cancer related genes and expression thereof, under the effect of carcinogenic factor, in patient body, can express various adhesion molecules and acceptor or part, its release mark thing content of different times at tumor development is also different, the variation of detection of lung cancer specificity molecular marker from peripheral blood and sputum, there is the advantages such as easy, quick, painful little, easy check, easily for patient accepts, significant to the clinical diagnosis and treatment of lung cancer.
At present relatively there is the lung cancer tumor markers of the meaning of representing to mainly contain following a few class: 1) embryonal antigen: taking carcinomebryonic antigen (CEA) as representative; 2) glycoprotein antigen: comprise CA19-9 (CA19-9), CA125 (CA125), CA153 (CA153), squamous cell antigen (SCC-Ag) etc.; 3) cyokeratin antigen: comprise cytokeratin 19 fragment antigen (CYFRA21-1), value of tissue polypeptide specific antigen (TPS) etc.; 4) enzyme antigen: comprise isodynamic enzyme class, as gastrin-releasing peptide precursor (ProGRP), Serum Neuron Specific Enolase (NSE), lactic dehydrogenase (LDH), matrix metalloproteinase (MMPs), tumor M 2-type pyruvate kinase (TUM2-PK) etc.; 5) tumor gene and CDKN2 protein product and antibody: as p53 and anti-p53 are anti-, c-erbB-2 and bcl-2 albumen.
Although have been found that the tumor markers that above-mentioned lung cancer is relevant, but these indexs overwhelming majority is without organ specific, they are not lung cancer specific antigen, but TSGF, not only be present in malignant tumour, be also present in benign tumour, embryonic tissue, even in normal structure, the diagnosis of lung cancer is lacked to specificity, only have reference value.On the other hand, due to the heterogeneity of the diversity of cancerous lung tissue pathology, pathology tumour cell of the same race and complicacy and the polymorphism of oncobiology behavior, cause unique identification quality testing to survey limited to the diagnostic value of lung cancer, can only reflect the variation of some side of disease, lot of domestic and foreign scholar tends to screen multiple valuable tumor markers and carries out joint-detection, to improve the positive rate of pulmonary cancer diagnosis.Detect to become to know together although joint-detection is better than individual event, not any one combination all can improve the positive rate of clinical diagnosis, and on best of breed, conclusion is quite inconsistent, even occurs contrary result.The biomarker that therefore, find a kind of high sensitivity, high specific, can be applied to lung cancer clinical diagnosis and treatment has become one of problem demanding prompt solution in lung cancer research.
Summary of the invention
The object of the present invention is to provide a kind of high sensitivity, high specific, can be applied to the biomarker of lung cancer clinical diagnosis and treatment.
To achieve these goals, the present invention is achieved by following scheme.
A kind of lung cancer tumor markers GSTA1, described mark is to utilize on the basis of new specific combined polypeptide for lung cancer of phage random peptide library triage techniques acquisition in early stage, screens from lung cancer cDNA library taking this polypeptide as probe.
Research of the present invention mainly comprises the screening of the content of four aspects: GSTA1, the lung carcinoma cell specificity identification of GSTA1, the Subcellular Localization of GSTA1, the relation of GSTA1 and lung cancer cell growth.
Concrete technical scheme is as follows:
Specific combined polypeptide for lung cancer is passed through to chemical synthesis process coupling biotin (Biotin), and immobilization is to through the ELISA reaction plate processed of Avidin (Avidin) (Biotin and Avidin can strong bonded), by immobilization polypeptide, T7 select Human Lung Cancer cDNA phage display library is carried out to 3 and take turns the screening of subtracting property, find the Lung Cancer Associated Antigen of being combined with polypeptide, a discovery wherein sequence and GSTA1 has homology.
Adopt Real-time PCR to detect mrna expression level, adopt Western blot and cellular immunofluorescence analysing protein expression, detect GSTA1 at kinds of tumor (human lung carcinoma cell LTEP-A2, SPCA1, NCI-H460, NCI-H1299, NCI-H1650, A549, human osteosarcoma cell MG-63, human liver cancer cell HepG2, Bel-7402, human cervical carcinoma cell Hela) and gene and the protein expression profile of lung normal cell MRC-5, confirm and the contrast of MRC-5 cell, the expression of the GSTA1 of cancer cell significantly increases, particularly no matter lung cancer A549 cell is in mRNA level or at protein level, its expression is far above other cells.
Adopt cellular immunofluorescence technology, simultaneously in conjunction with Hoechst33258 and DiI decoration method, utilize laser confocal scanning microscope to carry out the Subcellular Localization of GSTA1, confirm that GSTA1 mainly expresses in cytoplasm and/or cell membrane.
SiRNA according to siRNA principle of design design, chemosynthesis for new mark, by the above-mentioned lung carcinoma cell of liposome transfection, observe the impact on its growth, RT-PCR and Western Blot detect the expression of mark, MTT, srb assay detect the variation of ability of cell proliferation, Hoechst 33258 decoration methods detect Apoptosis situation, flow cytometry analysis Apoptosis situation.Confirm that reticent GSTA1 can be by impelling A549 apoptosis, thereby suppress its propagation.
Brief description of the drawings
Fig. 1. under inverted fluorescence microscope, observe the expression (200 ×) of different cell GSTA1; A is A549 cell; B is Bel-7402 cell; C is NCI-H1299 cell; D is NCI-H1650; E is Hela cell; F is LTEP-A2 cell; G is MG-63 cell; H is SPCA1; I is MRC-5 cell.
Fig. 2. the difference of GSTA1 expression in different carcinoma cell and lung normal cell MRC-5; A is the relative expression quantity of the mRNA of GSTA1 in different cells; B is the relative expression quantity of the albumen of GSTA1 in different cells; * p<0.05; * p<0.01, cancer cell group vs.MRC-5 group.
Fig. 3. under laser confocal microscope, observe the expression of A549 cell and MRC-5 cell GSTA1, A is A549 cell, and B is MRC-5 cell.
Fig. 4. the impact on A549 cell proliferation after GSTA1 silence; * p<0.05, * * p<0.01, experimental group vs.lipo group.
Fig. 5. nucleus apoptosis situation after A549 cell GSTA1 silence.
Embodiment
Further describe the present invention below in conjunction with Figure of description and specific embodiment.Unless stated otherwise, reagent, the equipment that the present invention adopts is the conventional reagent of the art and equipment.Certainly the employing of embodiment Instrumental and material is not limited to enumerating of the present embodiment, but can solve technical matters of the present invention, and to realize corresponding technique effect be foundation.In addition, in embodiment, unspecified molecular biology method is the method for this area routine, and concrete operations can be referring to molecular biosciences guide or product description.
embodiment 1
The lung cancer specificity checking of GSTA1
By cell A549, SPCA1, NCI-H1650, NCI-H1299, LTEP-A2, Bel-7402, Hela, MG-63 and lung normal cell MRC-5 with 1 × 10
4individual cells/well is inoculated in 96 orifice plates, after 24h, adopt 4% paraformaldehyde room temperature to fix 15min, 0.5% tween is changed 10min thoroughly, 0.4% 2 anti-source serum room temperature sealing 1h, primary antibodie 4 is spent overnight incubation, after PBS washing fully, the two anti-1h of hatching, omnidistance black out operation, after PBS washing, adopts under inverted fluorescence microscope and observes.
Result as shown in Figure 1, visible by microscopic examination coloration result, and the green fluorescence of different carcinoma cell is better than than normal cell MRC-5, illustrates that thus the expression of GSTA1 in cancer cell is far above lung normal cell.
embodiment 2
According to the ORF sequence distribution design of mark and synthetic upstream and downstream primer, with the mRNA in Trizol extraction different carcinoma cell, adopt the expression of Real-time pcr analysis lung cancer marker GSTA1 in different carcinoma cell; Extract the total protein of above-mentioned cell, carry out Western blot analyze lung cancer marker GSTA1 in different cells protein expression situation.
Result as shown in Figure 2, the GSTA1 of different carcinoma cell (LTEP-A2, SPCA1, NCI-H460, A549, HepG2) at mrna expression level and protein expression level far above lung normal cell MRC-5, particularly A549 cell, its expression is far above other cells.
embodiment 3
The Subcellular Localization of GSTA1
By A549 cell and MRC-5 cell with 1 × 10
5individual cells/well is inoculated in laser co-focusing special culture dish, cultivates after 24h, and 4% paraformaldehyde room temperature is fixed 15min, and 0.5% tween is changed 10min thoroughly, and after 0.4% donkey serum room temperature sealing 1h, GSTA1 primary antibodie 4 is spent overnight incubation, and 1 × PBS washes 4 times, each 7 minutes; Two anti-incubated at room 1h, 1 × PBS washes 4 times, each 7 minutes; Hoechst33258 incubated at room 15min, 1 × PBS washes 4 times, each 5 minutes; DiI incubated at room 15min, PBS fully washs rear confocal laser scanning microscope.
Result as shown in Figure 3, Hoechst33258 carries out nucleus dyeing, DiI is cell membrane red fluorescence probe, GSTA1 green fluorescence is mainly expressed at endochylema, and the expression of A549 cell GSTA1 is higher than MRC-5 cell, GSTA1 green fluorescence and cell membrane red fluorescence are superimposed, can illustrate that GSTA1 expresses at endochylema and/or after birth.
embodiment 4
Employing liposome method is by siRNA (SiRNA-1) transfection to A549 cell, and wherein SiRNA-NC is the negative control of GSTA1 sequence without homology.RT-PCR and Western blotting verify after its reticent effect, by the A549 cell of above-mentioned GSTA1 silence with 1 × 10
4individual cells/well is inoculated in 96 orifice plates, then adds 200ulOpti-MEM nutrient culture media, and GSTA1 is disturbed one sky respectively, and 2 days, 3 days, 4 days, within 5 days, add afterwards the MTT of the 5mg/ml of 20ul, cultivate 4h, under 570nm wavelength, measure the absorbance in each hole by microplate reader.Taking lipo group as blank, calculate cell survival rate: cell survival rate (%)=(OD processing-OD blank)/(OD contrast-OD blank) × 100%
As shown in Figure 4, the survival rate of GSTA1 cell after reticent 5 days only has 51% to result, and its A549 cell survival rate reduces along with the increase of time, and has the dependence of time.
embodiment 5
The A549 cell of GSTA1 silence is inoculated in 48 orifice plates to 37 degree, 5%CO
2cultivate respectively 1 day, 2 days, 3 days, 4 days, after 5 days, with liquid-solid fixed 20 min of paraformaldehyde that add 4% after PBS washing 2 times, exhaust after immobile liquid, add immediately 200ul Hoechst 33258(5 mg L
-1) dyeing liquor, lucifuge reaction 10min, PBS washes 3 times, each 7min, natural air drying, is placed in and causes observing and random picture with 340nm exciting light under fluorescent microscope, and experiment repeats 3 times.
Result as shown in Figure 5, is observed under inverted fluorescence microscope, does not have the cell karyon of GSTA1 silence large, and fluorescent dye is shallow, and brightness homogeneous, is light blue.And the reticent group of GSTA1 visible cell form presents variation, karyopyknosis, color depth and form sapphirine, the visible dense block fluorescence of fine and close particle that dyes, and along with the increase of time, change more obvious.As can be seen here, GSTA1 down-regulated expression can promote Apoptosis.
embodiment 6
By the A549 cell of GSTA1 silence with 9 × 10
6individual cells/well is inoculated in the double dish of 60mm, 37 degree, 5%CO
2cultivate after 72h, collect suspension cell and attached cell, centrifugal (2000rpm, 5min), abandon supernatant, wash the Binging Buffer suspension cell that adds 500ul after 2 times with PBS, add the Annexin V-FITC of 5ul, after mixing, add the Propidium Iodide(PI of 5ul), mix, lucifuge reaction 5min, in flow cytometer sample detection, and carry out data processing with software that instrument is joined, calculate apoptosis rate.
Flow cytometry A549-SiRNA, total apoptosis rate of A549-NC and A549 group cell is respectively: (9.42 ± 0.22) %, (1.2 ± 0.1) %, (0.02 ± 0.03) %, P<0.5 VS A549 groups of cells, total apoptosis rate of the reticent group of GSTA1 is higher than control group and NC group, and GSTA1 down-regulated expression can promote Apoptosis.
Claims (4)
1.GSTA1 is in the application of the tumor markers as pulmonary cancer diagnosis and magnetic target therapy.
2. a tumor markers for diagnosis and magnetic target therapy lung cancer, is characterized in that, described mark is GSTA1 albumen.
3. the tumor markers of diagnosis according to claim 2 and magnetic target therapy lung cancer, is characterized in that, GSTA1 expresses at endochylema and/or after birth.
4.GSTA1 down-regulated expression is in the application promoting in Apoptosis.
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| CN110579611A (en) * | 2019-09-18 | 2019-12-17 | 郑州大学 | Combined detection serum marker, kit and detection method for early screening and diagnosis of lung cancer |
| CN112011833A (en) * | 2019-05-30 | 2020-12-01 | 上海桀蒙生物技术有限公司 | Method for screening and isolating tumor neoantigens |
| CN112250733A (en) * | 2020-10-14 | 2021-01-22 | 山西诺恩精准医疗器械有限责任公司 | Fluorescent molecular probe for targeted labeling of non-small cell lung cancer and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108624664A (en) * | 2018-01-16 | 2018-10-09 | 新开源汇诚（武汉）医疗科技有限公司 | A kind of rapid amplifying method of the early diagnosis marker of acute liver damage |
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| CN112011833B (en) * | 2019-05-30 | 2024-04-26 | 上海桀蒙生物技术有限公司 | Method for screening and isolating tumor neoantigens |
| CN110579611A (en) * | 2019-09-18 | 2019-12-17 | 郑州大学 | Combined detection serum marker, kit and detection method for early screening and diagnosis of lung cancer |
| CN112250733A (en) * | 2020-10-14 | 2021-01-22 | 山西诺恩精准医疗器械有限责任公司 | Fluorescent molecular probe for targeted labeling of non-small cell lung cancer and application thereof |
| CN112250733B (en) * | 2020-10-14 | 2023-05-02 | 山西诺恩精准医疗器械有限责任公司 | Fluorescent molecular probe for targeted labeling of non-small cell lung cancer and application thereof |
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