CN104017879A - Application of FBXO31 gene and related products in preparing gastric cancer diagnostic reagent - Google Patents

Application of FBXO31 gene and related products in preparing gastric cancer diagnostic reagent Download PDF

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CN104017879A
CN104017879A CN201410266860.XA CN201410266860A CN104017879A CN 104017879 A CN104017879 A CN 104017879A CN 201410266860 A CN201410266860 A CN 201410266860A CN 104017879 A CN104017879 A CN 104017879A
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fbxo31
gene
mir
cancer
stomach
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刘志方
张新潮
徐霞
孔叶
邹水燕
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Shandong University
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Abstract

The invention relates to application of an FBXO31 gene and related products in preparing a gastric cancer diagnostic reagent. The related products of the FBXO31 gene are one or more of spliceosome of the FBXO31 gene, antisense oligodeoxynucleotide of the FBXO31 gene, mRNA (Messenger Ribonucleic Acid) of the FBXO31 gene, a protein of the FBXO31 gene, a peptide fragment encoded by the FBXO31 gene, an antibody of the protein of the FBXO31 gene, a regulating small molecular RNA related to the FBXO31 gene or an inhibitor of the regulating small molecular RNA related to the FBXO31 gene. The invention provides a novel concept of diagnosing gastric cancer and provides a novel path for researching and developing the gastric cancer diagnostic reagent.

Description

FBXO31 gene and associated products thereof are in the application of preparing in stomach cancer diagnosis reagent
Technical field
The present invention relates to FBXO31 gene and associated products thereof in the application of preparing in stomach cancer diagnosis reagent, belong to technical field of bioengineering.
Background technology
Cancer of the stomach is one of modal malignant tumour in world wide, and its mortality ratio occupies the second of Tumor-assaciated death.According to the statistic data of 2008, cancer of the stomach incidence accounted for 8% of the total incidence of cancer, and gastric cancer mortality accounts for 10% of cancer associated death.East Asia is the highest region of incidence gastric cancer rate, particularly China.Because the commitment in cancer of the stomach is difficult to be diagnosed, cause Most patients just to reach an advanced stage or the transitivity stage in the time finding.Utilize traditional operative treatment, chemotherapy or radiotherapy do not have too large meaning improving aspect the survival rate of patients with gastric cancer.Therefore, key is to find new diagnosis or prognostic marker and treatment target, to improve the survival rate of patients with gastric cancer.
FBXO31 belongs to F-box family, the protein that contains F-box structural domain participates in the composition of Skp1-Cullin-F-box (SCF) E3 SCF-complex, the degradation pathway that mediation Ubiquitin-proteasome relies on, and then signal transduction in regulating cell, cell cycle progression, the stability of centrosome etc.F-box albumen can be divided into three extended familys according to the composition of other structural domains except F-box structural domain.FBXW contains WD40 tumor-necrosis factor glycoproteins; FBXL contains and is rich in leucine tumor-necrosis factor glycoproteins and FBXO comprises other sequences.FBXO31 belongs to FBXO group.
FBXO31 is positioned at Chromosome 16q 24.3, in neuronic growth, in the processes such as DNA damage reparation reaction and tumour generation, has played vital effect.FBXO31 is considered to a kind of candidate tumor suppressor gene at first, research finds that FBXO31 significantly reduces in breast carcinoma level, excessively express FBXO31 in breast cancer cell after, clone capable of inhibiting cell forms and cell proliferation, inducing cell aging and G1 phase cell-cycle arrest.In addition, compared with normal liver tissue, the expression level of FBXO31 in liver cancer also significantly reduces, but FBXO31 obtains contrary result in esophageal squamous cell carcinoma (squama cancer).The people such as KOGO show, FBXO31 expression level is higher, the invasive ability of tumour is stronger, clinical stages rank higher, poorer.From these results, also there is dispute in the effect of FBXO31 in tumor development.How are the expression level of current not clear FBXO31 in cancer of the stomach and function, and how it is regulated and controled.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide FBXO31 gene and associated products thereof in the application of preparing in stomach cancer diagnosis reagent.
Technical solution of the present invention is as follows:
FBXO31 gene and associated products thereof be in the application of preparing in stomach cancer diagnosis reagent, and described FBXO31 gene-correlation product is one or more the combination in the inhibition of antibody, the siRNA of FBXO31 gene or the microRNA of target FBXO31 gene of the albumen of the albumen of the mRNA of the antisense oligonucleotide of the spliced body of FBXO31 gene, FBXO31 gene, FBXO31 gene, FBXO31 genes encoding, FBXO31 genes encoding.
Preferred according to the present invention, the inhibition of the microRNA of described target FBXO31 gene is the miR-20a of target FBXO31 gene and the inhibition of miR-17; The inhibition nucleotide sequence of miR-20a is as shown in SEQ ID NO.1, and the inhibition nucleotide sequence of miR-17 is as shown in SEQ ID NO.2.
Preferred according to the present invention, the inhibition of above-mentioned miR-20a inhibition and miR-17 is as the effective constituent in stomach cancer diagnosis reagent.
Preferred according to the present invention, described miR-20a inhibition and the inhibition of miR-17 are synthetic.
Preferred according to the present invention, the nucleotide sequence of the siRNA of FBXO31 gene is as shown in SEQ ID NO.3 and SEQ ID NO.4.
Preferred according to the present invention, the antibody of the albumen of described FBXO31 genes encoding be for aminoacid sequence be the peptide section shown in SEQ ID NO.5.
Preferred according to the present invention, the albumen of described FBXO31 gene is that natural separation obtains or synthetic.
Preferred according to the present invention, the mRNA of described FBXO31 gene is that natural separation obtains or synthetic.
Beneficial effect
The present invention has found that FBXO31 expresses significantly reduction in stomach organization first, this phenomenon and cancer of the stomach progress are closely related, and microRNA miR-20a and the miR-17 of the expression of regulation and control FBXO31 gene are found, the small molecules miR-20a of expression and the expression of crossing of miR-17 of finding regulation and control FBXO31 gene cause that the expression of FBXO31 in cancer of the stomach reduces, thereby a kind of new approaches of diagnosis of gastric cancer are provided, for research and development stomach cancer diagnosis reagent provides new way.
Brief description of the drawings
The detected result of the expression level of Fig. 1 FBXO31 gene in stomach organization;
Figure 1A is the result figure that detects the mRNA level of FBXO31 in cancer of the stomach sample by RT-PCR method;
In figure: N: the other healthy tissues of cancer, T: stomach organization, β 2-M: internal reference.
Figure 1B is the statistical graph that detects the mrna expression level of FBXO31 in 53 pairs of cancer of the stomach samples by the method for qRT-PCR;
Fig. 1 C is the result figure that detects the protein expression level of FBXO31 in cancer of the stomach sample by Western blot method;
In figure: N: the other healthy tissues of cancer, T: stomach organization, β-actin: internal reference.
Fig. 1 D is the statistical graph that detects the protein expression level of FBXO31 in 52 pairs of cancer of the stomach samples by Western blot method;
Fig. 2 is that the organization chip of cancer of the stomach and cancer beside organism carries out immunohistochemical methods detected result photo;
Fig. 2 A is that the organization chip of cancer beside organism carries out immunohistochemical methods detected result photo;
Fig. 2 B is that the organization chip of cancer beside organism carries out immunohistochemical methods detected result photo;
Fig. 2 C is that the organization chip of stomach organization carries out immunohistochemical methods detected result photo;
Fig. 2 D is that the organization chip of stomach organization carries out immunohistochemical methods detected result photo;
Fig. 2 E is that the organization chip of stomach organization carries out immunohistochemical methods detected result photo;
Fig. 2 F is that the organization chip of stomach organization carries out immunohistochemical methods detected result photo;
Fig. 3 is the Viability statistic curve figure that FBXO31 expresses positive patient and FBXO31 and express negative patient;
The detected result of Cell clonality, cell cycle progression and cell cycle related proteins expression level after the plasmid (pCMV-myc-FBXO31) of Fig. 4 control plasmid (pCMV-myc), expression wild-type FBXO31 and mutant plasmid (pCMV-myc-FBXO31 Δ F) the difference transfection stomach cancer cell BGC-823 of F-box sequence deletion and HGC-27;
Fig. 4 A is pCMV-myc, pCMV-myc-FBXO31, the detected result of Cell clonality after pCMV-myc-FBXO31 Δ F difference transfection stomach cancer cell BGC-823 and HGC-27;
Fig. 4 B is pCMV-myc, pCMV-myc-FBXO31, the statistical graph of cell clonal formation number after pCMV-myc-FBXO31 Δ F difference transfection stomach cancer cell BGC-823 and HGC-27;
Fig. 4 C is the peak value figure of the residing cell cycle of cell of different transfections;
In figure: pCMV is the stomach cancer cell of transfection control vector, FBXO31 is the stomach cancer cell of transfection FBXO31 expression vector, and Δ FBXO31 is the stomach cancer cell of the mutational vector of F-box region disappearance in transfection FBXO31;
Fig. 4 D is the detected result of the expression level of cell cycle related proteins in different transfectional cells;
Fig. 5 is FBXO31 forms ability impact on the tumour in nude mouse;
Fig. 5 A is the stomach cancer cell of injection transfection control vector and the stomach cancer cell of transfection FBXO31 expression vector after 2 weeks, the graphic representation of nude mice gross tumor volume and timing relationship;
Fig. 5 B is the stomach cancer cell of injection transfection control vector and the nude mice photo of the stomach cancer cell of transfection FBXO31 expression vector after 4 weeks;
Fig. 5 C is the stomach cancer cell of injection transfection control vector and the stomach cancer cell of transfection FBXO31 expression vector after 4 weeks, the photo of transplanted tumor volume;
Fig. 5 D be the stomach cancer cell of injection transfection control vector and the stomach cancer cell of transfection FBXO31 expression vector 4 weeks afterwards transplanted tumor weight ratio compared with histogram;
Fig. 6 is the detected result that FBXO31 is subject to small molecules miR-17 and miR-20a regulation and control;
After Fig. 6 A is miR-20a and miR-17 stand-in transfectional cell, the expression level histogram of miR-17 and miR-20a;
After Fig. 6 B is miR-20a and miR-17 stand-in transfectional cell, the horizontal histogram of mrna expression of FBXO31;
After Fig. 6 C is miR-20a and miR-17 stand-in transfectional cell, the protein expression of FBXO31 detects photo;
After Fig. 6 D is miR-20a and miR-17 inhibitor transfectional cell, the expression level histogram of miR-17 and miR-20a;
After Fig. 6 E is the inhibitor transfectional cell of miR-20a and mi-17, the horizontal histogram of mrna expression of FBXO31;
After Fig. 6 F is miR-20a and miR-17 inhibitor transfectional cell, the protein expression detected result photo of FBXO31;
Fig. 6 G be miR-20a and miR-17 stand-in respectively with wild-type, binding site 1 saltant type, binding site 2 saltant types and the equal saltant type cotransfection stomach cancer cell of binding site 1,2 after, the uciferase activity histogram recording;
Fig. 7 is the detection of expression result of miR-20a and miR-17 in stomach organization and cancer beside organism;
Fig. 7 A is the expression level histogram of miR-20a in stomach organization and cancer beside organism;
Fig. 7 B is the expression level histogram of miR-17 in stomach organization and cancer beside organism;
Fig. 7 C is the statistical graph of the expression level of miR-20a in stomach organization and cancer beside organism;
Fig. 7 D is the statistical graph of the expression level of miR-17 in stomach organization and cancer beside organism;
Fig. 7 E is the statistical graph of FBXO31 and miR-20a relationship between expression in cancer of the stomach;
Fig. 7 F is the statistical graph of FBXO31 and miR-17 relationship between expression in cancer of the stomach.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described, not limitation of the present invention, all any this areas of carrying out according to content disclosed by the invention be equal to substitute, all belong to protection scope of the present invention.
Embodiment 1
RT-PCR, qRT-PCR and Western blot method detect and show that the mRNA of FBXO31 and the expression level of protein level in Human Stomach Tissue all significantly decline;
1. materials and methods
(1) patient
56 routine cancer of the stomach and surrounding tissue thereof are to come from the patient who carries out excision of tumor of stomach during 2012-2013 in Shandong Tumor Hospital, and sample is stored in-80 DEG C.The information shifting by the age to patient during operation, sex, histological type, differentiation degree, tumor size (diameter), invasion and attack situation, part and far-end is added up, and particular case is as shown in table 1:
Table 1, patient and tumour feature and the expression level in normal mucosa and stomach organization at FBXO31 albumen
1.pathology tumor-node-metastasis.
2.histology differentiation state: LDA, poorly differentiated adenocarcinoma; MDA, middle differentiation gland cancer; MucinousA, mucinous adenocarcinoma.
3.western blot analyzes the expression level of FBXO31 in cancer of the stomach and corresponding cancer beside organism, the density scan value of albumen abundance based on band compared with internal reference β-actin. relatively-: do not express
(2) RNA extracts, reverse transcription and qRT-PCR
TRIzol reagent (purchased from Invitrogen company) must RNA for extracting from tissue sample, concrete steps:
1. collecting cell, adds after 1ml Trizol fully pressure-vaccum precipitation after PBS washed cell, room temperature leaves standstill 5min.
2. add the chloroform of 0.2ml, vortex vibrator vibration 15s, room temperature leaves standstill 2min.
3. 4 DEG C, the centrifugal 15min of 12,000g.
4. upper strata water is carefully transferred to new EP pipe, adds the Virahol of 0.5ml, mix gently, room temperature leaves standstill 10min.
5. 4 DEG C, the centrifugal 10min of 12,000g.
6. carefully abandon supernatant, add the ethanol of 1ml75%, washing precipitation gently.
7. 4 DEG C, the centrifugal 5min of 7,500g.
8. carefully abandon supernatant, room temperature is dried, and adds the DEPC-H of 50 μ l 2o dissolves RNA precipitation.
The PCR primer of FBXO31 is bought in Invitrogen.The primer sequence of FBXO31 is as follows:
5'-CCGGCGGGAGGCAGGAGGAGT-3'(upstream primer),
5'-GCGGCGGTA GGTCAGGCAGTTGTCG-3'(downstream primer).
Above primer is the subarea that includes across FBXO31 gene, and therefore the result of PCR product is got rid of the impact that genomic dna pollutes.
The expression (β 2-M) of B2M is used as the contrast of RNA loading and reverse transcription efficiency and amplification.
The Article 1 chain of cDNA is synthetic by random hexamers (N6) or miRNA primer and M-MLV reversed transcriptive enzyme (purchased from Ferments company).Above-mentioned random hexamers and M-MLV reversed transcriptive enzyme are all purchased from Ferments company, and miRNA primer is purchased from Guangzhou Rui Bo biotechnology company limited.
Real-time quantitative PCR is to specifications, completes with Bio-Rad CFX96TM real-time PCR system (purchased from Bio-Rad company) and the SYBR Green Kit (purchased from TaKaRa company).The mRNA level of target molecule is by the method representation of CT, does standardization by the expression of mankind β 2-M or U6.Three multiple holes have all been established in all reactions.
(3) protein imprinted analysis
RIPA lysate (purchased from green skies Bioteknologisk Institut) is used for extracting total protein from cell or tissue.
Detect the concentration of protein by BCA method (Merck).Protein carries out SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferring film.Film is hybridized with the antibody (Abcam) of FBXO31, add two of horseradish peroxidase-labeled to resist.Finally detect the expression of albumen by chemiluminescent method (ECL, Millipore).β-actin (Sigma-Aldrich) is as internal reference.Aforesaid method all can be referring to Publication about Document.
Yang?Q,Wang?B,Gao?W,Huang?S,Liu?Z,Li?W,Jia?J.SIRT1is?downregulated?in?gastric?cancer?and?leads?to?G1-phase?arrest?via?NF-κB/Cyclin?D1signaling.Mol?Cancer?Res.2013Dec；11(12):1497-507.
(3) data analysis
The mRNA of FBXO31 and the difference of protein expression are analyzed with t inspection.
2, experimental result
Detected the mRNA level of FBXO31 in 53 pairs of cancer of the stomach samples by the method for RT-PCR and qRT-PCR.Find to compare with corresponding cancer beside organism, have the mRNA level of the FBXO31 in 38 routine stomach organizations obviously to reduce (71.7%, P=0.0004), result as shown in FIG. 1A and 1B.
Detected the expression of FBXO31 protein level in 52 pairs of stomach organizations by Western blot method.Band is quantized by density scan method, find that cancer beside organism corresponding thereto compares, have the FBXO31 protein expression level in 37 routine stomach organizations to reduce significantly (71.2%, p<0.0001), result is as shown in Fig. 1 C and Fig. 1 D.This result is consistent with the result of qRT-PCR.
Above-mentioned data show compared with cancer beside organism, and in stomach organization, FBXO31 all has obvious reduction at mRNA and protein level.
Embodiment 2
Immunohistochemical methods detects the relation of the expression of FBXO31 and the clinical parameter of Patients with Gastric Cancer and postoperative survival
1, materials and methods
(1) organization chip
Paraffin-embedded mankind's stomach organization chip is purchased from Shanghai Xin Chao company limited.On a chip, contain 180 points, the sample that comprises 90 routine Patients with Gastric Cancer.Each routine patient comprises two points, and a point represents cancerous tissue, and another point represents corresponding cancer beside organism.Provide by the said firm about data such as patient's sex, age, tumor size, clinical data and life cycles.
(2) immunohistochemical methods detects
1. dewaxing and aquation: before dewaxing, 60 DEG C of tissue slicies are toasted to 2h
A. dimethylbenzene dewaxing 5mim × 3 time
B. soaked in absolute ethyl alcohol 5min × 2 time
C.95% in ethanol, soak 5min × 2 time
D.80% in ethanol, soak 5min × 2 time
2. antigen retrieval: microwave-oven-heating 0.01mol/L sodium citrate buffer (pH6.0), to boiling, is put into tissue slice, little fire heating 20min, naturally cools to room temperature.
3. immunohistochemical staining:
A. take out section, in distilled water, soak 5min.PBS soaking flushing 5min × 3 time.Then drip 3%H2O2 room temperature treatment 10min, blocking-up endogenous peroxidase activity, continues to keep the moistening of section.PBS soaking flushing 5min × 3 time.
B. add primary antibodie FBXO31 (1:200 dilution), 4 DEG C, wet box spends the night.PBS soaking flushing 5min × 3 time.
C. drip biotin labeled two and resist, hatch 30min for 37 DEG C.PBS rinses 5min × 3 time.
D. drip HRP-Streptavidin, hatch 30min for 37 DEG C.
The e.DAB 5-20min that develops the color, by the time obviously positive, background is clear, and both difference contrast gradients are large.Color development stopping, tap water rinses 2min, slowly sweeps away from section upper end.Hematorylin is redyed 2min.Tap water rinses 10-15min.1% hydrochloride alcohol differentiation.1% ammoniacal liquor returns indigo plant.Dehydration, transparent, mounting, microscopy.
(3) data analysis
Relation between FBXO31 protein expression level and different clinical parameter is analyzed (seeing document below) with x2 and is evaluated.Cox proportional hazards regression models carries out single factor and multiplicity, estimates the impact on patient's survival of clinicopathologic features and FBXO31 protein expression level.Survival curve is drawn by Kaplan – Meier method.X2 analytical procedure can be referring to:
Wang?DD,Chen?YB,Pan?K,Wang?W,Chen?SP,Chen?JG,Zhao?JJ,Lv?L,Pan?QZ,Li?YQ,Wang?QJ,Huang?LX,Ke?ML,He?J,Xia?JC.Decreased?expression?of?the?ARID1A?gene?is?associated?with?poor?prognosis?in?primary?gastric?cancer.PLoS?One.2012；7(7):e40364.doi:10.1371/journal.pone.0040364.Epub2012Jul13.
2, experimental result
Carry out immunohistochemical methods detection with the organization chip that contains 90 pairs of cancer of the stomach and cancer beside organism, find that FBXO31 has obvious dyeing in all cancer beside organisms, result as shown in Figure 2 A and 2 B.Be negative and there is 52.2% dyeing in stomach organization, result is as shown in Fig. 2 E and Fig. 2 F.Having 38.9% dyeing situation is the weak positive, and result is as shown in Fig. 2 C and 2D.
On the whole, compared with cancer beside organism, the reduction that has 84.4% stomach organization to show FBXO31 protein expression level.This result has further been verified the conclusion that FBXO31 reduces in expression in gastric carcinoma level.
By analyzing contacting of FBXO31 expression and patients clinical index, result shows: the expression of FBXO31 albumen and tumor size (P=0.022), infiltration degree (P=0.024) and tumor grade (P=0.012) have obvious relation.And and the age, sex, it doesn't matter for metastases in local lymph node, as shown in table 2:
Table 2.FBXO31 expresses the dependency with various clinical case parameters
acase number in each grouping
*statistical significance (P<0.05).
Single factor Cox regression analysis shows tumor size (P=0.016), infiltration degree (P=0.044), metastases in local lymph node (P<0.001) and FBXO31 express (P=0.004) to be had significantly and contacts with patient's survival.Analyzed by Cox Model shows, metastases in local lymph node (P=0.003) and FBXO31 expression (P=0.026) are the independent predictor of Patients with Gastric Cancer survival rate, as shown in table 3:
Single argument and the multivariate analysis of table 3 Patients with Gastric Cancer Viability
HR, risk ratio; CI, fiducial interval; Case number in the each grouping of a; *statistical significance (P<0.05).
FBXO31 expresses compared with positive patient expresses negative patient with FBXO31, and Viability significantly raises (P=0.002, log-rank test), and result as shown in Figure 3.
Embodiment 3
Colony formation and the impact of Flow cytometry FBXO31 on proliferation of human gastric cancer cell ability and cell cycle progression
1. materials and methods
(1) experiment material and cell cultures
Mankind's gastric carcinoma cell lines BGC-823, HGC-27, buys in Chinese Academy of Sciences's Shanghai biological chemistry and Institute of Cell Biology.
Cell cultures is at 37 DEG C, 95% air, 5%CO 2constant temperature incubator.
Substratum is 1640 substratum (purchased from Invitrogen company, Carlsbad, CA) of 10% foetal calf serum (FBS), 100U/mL penicillin and 2mmol/L L-glutaminate for containing volume percent.
Mankind miR-20a stand-in, miR-17 stand-in, blank stand-in, miR-20a inhibition, miR-17 inhibition, blank inhibition is all bought from Rui Bo biotech firm (GuangZhou, China).
PCMV-myc-FBXO31 carrier and F-box sequence deletion mutational vector pCMV-myc-FBXO31 Δ F are all according to building with the method in Publication about Document.
1.Kumar?R,Neilsen?PM,Crawford?J,McKirdy?R,Lee?J,Powell?JA,Saif?Z,Martin?JM,Lombaerts?M,Cornelisse?CJ,Cleton-Jansen?AM,Callen?DF.FBXO31?is?the?chromosome16q24.3senescence?gene,a?candidate?breast?tumor?suppressor,and?a?component?of?an?SCF?complex.Cancer?Res.2005Dec15；65(24):11304-13.
(2) colony formation
The cell of transfection different carriers after 48 hours moves in 6 orifice plates, and 300, every hole cell, hatches 10 days.Then in orifice plate, add methyl alcohol, fixed cell, then adds Viola crystallina to cell dyeing.The clone who is greater than 50 cell count is write down.This experimental design three multiple holes and in triplicate.
(3) the Flow cytometry cell cycle
Cell after transfection is collected, and under 4 DEG C of conditions, the ethanolic soln that is 70% containing volume percent of preparing with PBS damping fluid is fixed 4 hours.Then dye 30 minutes at the dark place of 37 DEG C with the propidium iodide that contains RNase A.Cell cycle divides and selects flow cytometer (BD Biosciences).Each experiment has repeated three times, and data are analyzed with FCS Express V3.0612 software.
(4) data analysis
The difference that clone forms is to be analyzed by the T inspection of two tail non-matchings.P<0.05 has been considered to statistical significance.
2. experimental result
Biological function by further research FBXO31 in stomach cancer cell, adopt control plasmid (pCMV-myc), express the plasmid (pCMV-myc-FBXO31) of wild-type FBXO31 and the mutant plasmid (pCMV-myc-FBXO31 Δ F) of F-box sequence deletion transfection stomach cancer cell BGC-823 and HGC-27 respectively.Then the cell after transfection is carried out to the detection of clonality, find through contrast, the cell of transfection wild-type FBXO31 forms number than the clone of transfection control vector and obviously reduces.Do not affect but the plasmid of F-box sequence deletion forms number to clone, result as shown in Figure 4 A and 4 B shown in FIG..Therefore the restraining effect that FBXO31 forms clone depends on its F-box sequence.
In tumour, because the stagnation of cell cycle has important effect to suppressing cell proliferation.Therefore whether we are further because the impact of its cell cycle causes by the restraining effect that flow cytometry has been studied FBXO31 on cell proliferation.Find by research, FBXO31 crosses expression can increase cell in the stagnation of G1 phase, and reduces the ratio of G2-M.But plasmid FBXO31 (the FBXO31 Δ F) cell cycle that has lacked F-box sequence does not affect, and result as shown in Figure 4 C.Therefore F-box sequence suppresses proliferation of human gastric cancer cell for FBXO31 important effect.
Embodiment 4
Western blot detects the impact that FBXO31 cell cycle albumen cyclinD1 expresses
1. materials and methods
(1) cell cultures and cell transfecting are shown in embodiment 3;
(2) Western blot is shown in embodiment 1, and CyclinE1 antibody is purchased from Cell Signaling Technology, and P21 antibody is purchased from Epitomics, and CyclinD1 antibody is purchased from Epitomics.
2. experimental result
In order further to inquire into the molecular mechanism that FBXO31 causes cell-cycle arrest, we have studied the expression of a series of cyclins in stomach cancer cell, comprise P21, cyclinD1 and CyclinE1.Our result shows, the expression that has significantly suppressed cyclinD1 is expressed in crossing of wild-type FBXO31, and the mutant FBXO31 Δ F of F-box structural domain disappearance on the expression of cyclinD1 without impact (Fig. 4 D).In addition, FBXO31 crosses to express and regulates albumen to comprise Cyclin E1 and CDK inhibition (CDKI to other; P21) the not significant impact of expression level.These data show, FBXO31 blocked by the induced expression stomach cancer cell G1 phase of suppressing cyclinD1 albumen.
Embodiment 5
Nude mice by subcutaneous becomes knurl experiment to detect the impact of FBXO31 on nude mice in-vivo tumour formation ability
1. materials and methods
Nude Mouse Model
The nude mice of athymic BALB/c is bought from Peking University (China, Beijing) in the time of 5-6 week size.Then in special bioclean environment, raise.Transfection the BGC-823 stomach cancer cell of pCMV-myc or pCMV-myc-FBXO31 be injected at the subcutaneous of nude mice both sides.Each side injection the volume of 100ul, density is in 0.1mL 5 × 10 5individual visible cell.After one week, the size of subcutaneous tumour is carried out one-shot measurement with each week of vernier calliper master.The volume of tumour is with (length) × (wide 2the formula of)/2 calculates.After surrounding, these mouse are condemned to death.The tumour of dissecting is out weighed.
2, experimental result
Detect crossing of FBXO31 and express the tumour formation ability of stomach cancer cell in nude mouse that whether suppress.
The BGC-823 cell of the stomach cancer cell BGC-823 of transfection FBXO31 expression vector and transfection control vector is injected respectively to nude mice by subcutaneous, detect its tumorigenesis ability.Inject after 1 week, subcutaneous start to have visible tumour form, measure its gross tumor volume with vernier callipers, later every 7 days, measure and calculate gross tumor volume.Result shown in Fig. 5 A shows: the stomach cancer cell of injection transfection control vector is after 2 weeks, and in nude mouse, gross tumor volume sharply increases, but after the stomach cancer cell of injection transfection FBXO31, the increase not obvious (Fig. 5) of gross tumor volume in nude mouse.After surrounding, put to death mouse.
Result: compared with transfection control vector group, the transplanted tumor volume of transfection FBXO31 group obviously reduces (Fig. 5 B and 5C).Statistical analysis shows: the tumor weight of transfection control vector group is apparently higher than the tumor weight (P=0.0161 of transfection FBXO31 group; Fig. 5 D).Particularly, after injection transfection FBXO31 cell, in two Mice Bodies, do not have tumour to form.Therefore, FBXO31 has brought into play and has suppressed a swollen neoplastic very important role.
Embodiment 6
QRT-PCR, Western blot and uciferase activity are measured and are detected miR-17 and the regulation and control of 20A to FBXO31
1. materials and methods
(1) cell cultures, cell transfecting are shown in embodiment 3
(2) QRT-PCR and Western blot detect and see that embodiment 1, miR-20a, the primer of miR-17 and U6 buy in sharp rich biology (GuangZhou, China).
(3) structure of report carrier and luciferase assays
In mankind FBXO31 gene, in the sequence of 3 '-UTR of 217bp length, contain miR-20a and miR-17 binding sequence.This section of sequence is inserted in the SpeI/Hind III restriction enzyme site of pMIR-REPORT luciferase carrier (called after pMIR-FBXO31/wt).In 3 ' of FBXO31-UTR, there is the fully-complementary sequence of two miR-20a and miR-17 gCACTTT.Using pMIR-FBXO31/wt as template, land used point mutation test kit, these two sequences by single or together sudden change fall.Primer sequence is as shown in table 4, has recorded normal carrier pMIR-FBXO31/wt and two mutant pMIR-FBXO31/mut1 and the pMIR-FBXO31/mut2 needed primer sequence of the 3 '-UTR that builds FBXO31 in this table.These mutant are called after pMIR-FBXO31/mut1 respectively, pMIR-FBXO31/mut2 and pMIR-FBXO31/mut1,2.Stomach cancer cell is cultivated in 24 orifice plates, suitable report carrier and miRNA is temporarily proceeded to Lipofectamine2000.After 48 hours, cell is collected and cracking.Uciferase activity detects by Dual-Luciferase Reporter Assay system (Promega, Madison, WI, USA).Sea cucumber fluorescent value is used for doing standardization.For each plasmid structure, transfection experiment has all been established three multiple holes.
Table 4
2. experimental result
Because the expression level of FBXO31 obviously declines in cancer of the stomach, and block and suppresses in the induction G1 phase and also play very important regulating effect aspect the formation of in-vivo tumour, study by experiment the molecular mechanism that causes FBXO31 downward in cancer of the stomach.Because miRNAs is the short non-coding RNA that a class is made up of 19-25 Nucleotide, can be by being combined with the complementary sequence of 3 ' of said target mrna-non-translational region (UTR), thus suppress the translation of said target mrna or cause the degraded of said target mrna.Therefore whether be subject to the negative regulation of miRNAs by detecting FBXO31, use different databases, as TargetScan, the miRNA of PicTar and miRanda prediction target FBXO31, finds that two conservative sites generation parts of miR-20a and miR-17 and FBXO313 ' UTR are complementary.
Therefore,, to stand-in or the inhibition of stomach cancer cell BGC-823 and HGC-27 transfection miR-20a or miR-17, detect the expression of FBXO31 with quantitative PCR and Western blot.Find by research, miR-20a or miR-17mimics have significantly reduced the expression level of FBXO31, and miR-20a or miR-17 inhibition have significantly increased FBXO31mRNA and protein level, and result is as shown in Fig. 6 A-Fig. 6 F.
Further determine whether that FBXO31 is the direct target of miR-20a or miR-17, by building a luciferase reporting carrier pMIR-FBX who contains FBXO313 ' UTR region, the impact of research miR-20a or the uciferase activity of miR-17 on pMIR-FBX.
Find that by research miR-20a and 17 significantly reduces the uciferase activity of pMIR-FBX, result is as shown in Fig. 6 g.Because the 3 ' UTR of FBXO31 found two with the region of miR-20a and 17 complementations, by building three mutant, pMIR-FBX/mut1 (site 1 suddenlys change), pMIR-FBX/mut2 (site 2 suddenlys change) and pMIR-FBX/mut1,2 (site 1,2 all suddenlys change) are verified.Luciferase reporter gene detected result shows: pMIR-FBX/mut1 shows has significant restraining effect to uciferase activity, and pMIR-FBX/mut2 is not obvious to the restraining effect of uciferase activity, and pMIR-FBX/mut1,2 have lost the restraining effect (Fig. 6 G) to uciferase activity completely.Therefore, the Two Areas of 3 ' UTR of FBXO31 is all having certain effect aspect miR-20a or miR-17, but Two Areas is prior.
Embodiment 7
QRT-PCR detects expression in stomach organization of miR-20 and miR-17 and analyzes miR-20a or the dependency of miR-17 and FBXO31 expression.
1. materials and methods
(1) patient: with embodiment 2;
(2) QRT-PCR detects and sees embodiment 1.MiR-20a, the primer of miR-17 and U6 is bought in sharp rich biology (GuangZhou, China).
(3) statistical analysis
The dependency that FBXO31 and miR-20a (17) express in cancer of the stomach is to be analyzed by linear regression.All data analyses are all that this software of 17.0 versions (SPSS Inc., Chicago, IL, USA) carries out by Statistical Package for the Social Sciences.P<0.05 has been considered to statistical significance.
2. experimental result
Finally, detect whether overexpression of miR-17, miR-20A in Primary Gastric cancerous tissue, by the expression level with miR-20a and miR-17 in real-time quantitative PCR method 56 pairs of cancer of the stomach of mensuration and corresponding non-cancer normal mucosa tissue, find by research, compared with surrounding normal mucous membrane, the expression level of miR-20a is high expression level in 66.1% (37/56) stomach organization, the expression level of miR-17 shows high expression level in 38/56 (67.9%) clinical cancer of the stomach sample, and result is as shown in Fig. 7 A and 7B.Can be found out by Fig. 7 C and Fig. 7 D, in tumor tissues, the expression of miR-20a and miR-17 is apparently higher than surrounding normal mucous membrane.
Statistical analysis shows, in tumor tissues, the expression of miR-20a and miR-17 is apparently higher than surrounding normal mucous membrane.In 52 pairs of cancer of the stomach and cancer beside organism's sample, there are 26 pairs to show that FBXO31 expresses decline in cancer, and the trend that miR-20a raises, there are 27 pairs of samples to show FBXO31 and in cancer, express decline, and miR-17 expresses the trend raising, statistical study shows, FBXO31 and miR-20a or miR-17 are high negative correlation (P < 0.0001) in cancer of the stomach, and result is as shown in Fig. 7 E and Fig. 7 F.

Claims (5)

1.FBXO31 gene and associated products thereof be in the application of preparing in stomach cancer diagnosis reagent, and described FBXO31 gene-correlation product is one or more the combination in the inhibition of antibody, the siRNA of FBXO31 gene or the microRNA of target FBXO31 gene of the albumen of the albumen of the mRNA of the antisense oligonucleotide of the spliced body of FBXO31 gene, FBXO31 gene, FBXO31 gene, FBXO31 genes encoding, FBXO31 genes encoding.
2. application as claimed in claim 1, is characterized in that, the inhibition of the microRNA of described target FBXO31 gene is the miR-20a of target FBXO31 gene and the inhibition of miR-17; The inhibition nucleotide sequence of miR-20a is as shown in SEQ ID NO.1, and the inhibition nucleotide sequence of miR-17 is as shown in SEQ ID NO.2.
3. application as claimed in claim 2, is characterized in that, the inhibition of miR-20a inhibition and miR-17 is as the effective constituent in stomach cancer diagnosis reagent.
4. application as claimed in claim 1, is characterized in that, the nucleotide sequence of the siRNA of described FBXO31 gene is as shown in SEQ ID NO.3 and SEQ ID NO.4.
5. application as claimed in claim 1, is characterized in that, the antibody of the albumen of described FBXO31 genes encoding be for aminoacid sequence be the peptide section shown in SEQ ID NO.5.
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