CN103157116A - Novel cancer suppressor gene UNC5D and new applications of encoded protein thereof - Google Patents
Novel cancer suppressor gene UNC5D and new applications of encoded protein thereof Download PDFInfo
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Abstract
The invention provides a novel cancer suppressor gene UNC5D gene and new applications of encoded protein, and particularly relates to the application of the UNC5D gene and/or the encoded protein in the drugs preparation for treating cancers, the application of testing UNC5D gene and encoded protein reagent in the preparation of compositions used for the auxiliary diagnosis of cancers and/or prognostic judgment, the application of the UNC5D gene and/or the encoded protein in preparation with effects of inhibiting the growth, the migration, the tumorigenicity of tumor cells, and/or inducing apoptosis. Researches find that the UNC5D is the new potential cancer suppressor gene, and especially plays an important role in the occurrence and development process of a kidney cancer.
Description
Technical field
The present invention relates to the new application of a new potential antioncogene and encoding proteins thereof, specifically, the present invention relates to UNC5D as a new potential antioncogene and the application of encoding proteins thereof.
Background technology
UNC5D belongs to UNC5 receptor family member.The UNC5 receptor family was the receptor of nerve growth factor netrin-1 by reporting for work in 1997, and in this family, except UNC5D, other member also comprises UNC5A, UNC5B, UNC5C.And UNC5D finds the latest in UNC5 family.Netrin-1 and receptor thereof are to be found in nerve to play an important role in phylogeny at first, because it can regulate and control the directional migration of nerve fiber.In recent years, the effect in tumor occurs of Netrin-1 and receptor thereof more and more comes into one's own.UNC5A, UNC5B and the effect of UNC5C in tumor development in Netrin-1 and UNC5 family have all had report in succession.Oncobiology function about the UNC5D gene does not have any report yet at present.
Summary of the invention
One object of the present invention be to study UNC5D tumor particularly mechanism, the research UNC5D of the expression characteristic in renal carcinoma and its expression regulation new function and the new application of UNC5D is provided.
In the present invention, described UNC5D comprises UNC5D gene or UNC5D albumen etc.Described UNC5D albumen is the albumen that aminoacid sequence forms as shown in SEQ ID No.2, or in aminoacid sequence shown in SEQ ID No.2 through replacing, lack or adding one or several aminoacid and have the derived protein of identical function with the albumen that aminoacid sequence shown in SEQ ID No.2 forms.Described UNC5D gene is the polynucleotide sequence of coding UNC5D albumen of the present invention, be preferably the polynucleotide sequence of aminoacid sequence shown in coding SEQ ID NO:2, perhaps except the coded sequence of above-mentioned aminoacid sequence, can also comprise non-coding sequence, such as intron, coded sequence 5 ' or the non-coding sequence of 3 ' end etc.Wherein preferred UNC5D gene is the polynucleotide sequence shown in SEQ ID No.1, and it is the gene of NM 080872.2 for the registration number at GenBank.Perhaps, preferred UNC5D gene is a kind of nucleotide sequence of separation, and it comprises coding UNC5D protein sequence, for example coded sequence shown in SEQ ID No.1: the 329th～3190 nucleotide sequence.Those of ordinary skills are known, and the nucleotide sequence of UNC5D gene of the present invention can be entirely identical to the coded sequence as shown in SEQ ID NO:1, also can due to the degeneracy of genetic code, not exclusively be equal to the coded sequence of above-mentioned nucleotide.
The present invention has studied the expression of UNC5D in the multiple normal structure of the mankind, reach tumor cell line, the renal carcinoma of pairing and the expression characteristic in cancer beside organism in the Various Tissues source, find UNC5D down-regulated expression or disappearance in the tumor cell line in Various Tissues source, its expression in renal carcinoma tissue is starkly lower than cancer beside organism.The present invention also further by immunohistochemical experiment, confirms UNC5D albumen down-regulated expression in renal carcinoma tissue.
The present invention is by analyzing the clinical information data, find UNC5D in renal carcinoma tissue the down-regulated expression degree and the Fuhrman classification of renal carcinoma be dependency.
The present invention has further disclosed the mechanism of UNC5D down-regulated expression, finds that loss of heterozygosity (LOH) and the methylating of UNC5D gene promoter area CpG island on chromosome is the important mechanisms that causes UNC5D down-regulated expression in kinds of tumor cells system and renal carcinoma tissue.
The present invention confirms by experiment: recover the expression of UNC5D in the renal carcinoma cell line (786-O and A498) of UNC5D down-regulated expression or disappearance, but the cloning efficiency of inhibition tumor cell propagation, cell migration and tumor cell.Express the variation of cell cycle before and after UNC5D by analyzing renal carcinoma cell line 786-O, find that it is to realize by the release that slows down G2/M that UNC5D suppresses cell proliferation.
The present invention is by crossing expression UNC5D discovery in HEKC (HEK293T), UNC5D can induce the apoptosis of HEK293T, confirms that by western b1ot the apoptosis that UNC5D induces is that caspase relies on.
According to above result, can think that UNC5D is an antioncogene, and can be used as the auxiliary characteristics of the clinical diagnosis of cancer and/or prognosis judgement, and UNC5D gene or UNC5D albumen are in treatment and/or suppress to have significant application value in tumor.
Experimental studies have found that according to of the present invention, the invention provides the application of albumen (UNC5D albumen) in the medicine of preparation treatment cancer of UNC5D gene or its coding.UNC5D sequence of the present invention can be utilized the method for any known transduction, conversion or transfection to be applied to the tumor locus of diseased individuals, recover the expression of UNC5D in the tumor cell line of UNC5D down-regulated expression or disappearance and cancerous tissue, thereby by suppressing the cell cycle progression cell growth inhibiting, the cloning efficiency of inhibition tumor cell propagation, cell migration or tumor cell can be used for the treatment of cancer.
Experimental studies have found that according to of the present invention, the present invention also provides the application of reagent in the compositions that auxiliary diagnosis and/or prognosis for the preparation of cancer judge of the albumen that detects UNC5D gene or its coding.Can be with the New Set of described UNC5D as cancer diagnosis and/or prognosis judgement, by expression or the expression of UNC5D in test sample, thereby provide new foundation for auxiliary diagnosis and/or the prognosis judgement of cancer.Can utilize any method known in the art to detect UNC5D of the present invention in the expression of nucleic acid level, also can detect at protein level the expression of UNC5D of the present invention.The reagent of the albumen of described detection UNC5D gene or its coding comprises: be used at the synthetic UNC5D gene DNA chain of PCR and/or the PCR primer of its cDNA chain or the antibody of anti-UNC5D albumen etc.
UNC5D gene of the present invention or UNC5D albumen all can obtain by conventional method.For example, the pcr amplification technology that the polynucleotide sequence of UNC5D gene can establishing criteria as template, and is chosen cDNA, mRNA or genomic DNA suitable oligonucleotide primers amplification and is obtained.The nucleotide that obtains like this can be cloned in suitable carrier, and carries out sequence description with the DNA analysis technology.Also can obtain by the standard DNA synthetic technology.UNC5D albumen of the present invention can be with various known methods by transformed host cell and after the host cell that is converted grows into suitable cell density, with suitable method evoked promoter, then continue to cultivate, after cultivation is completed, reclaim and purification UNC5D albumen of the present invention.In the present invention, as restructuring UNC5D albumen, can with or not with label.The reagent of detection UNC5D gene of the present invention or UNC5D albumen also can prepare by the whole bag of tricks known to persons of ordinary skill in the art.
Particularly, described cancer is renal carcinoma.
The present invention also provides growth and/or propagation, the clone formation of inhibition tumor cell and/or the application in tumor cell migration of the albumen of UNC5D gene or its coding at inhibition tumor cell.The present invention confirms by experiment, and the clone of the growth that the albumen of UNC5D gene or its coding can inhibition tumor cell and propagation, inhibition tumor cell forms and can the inhibition tumor cell migration.Particularly, described tumor cell is kidney cancer cell.In a specific embodiments of the present invention, described cell migration is the migration of the fibronectin kidney cancer cell of inducing.
According to specific embodiments of the present invention, UNC5D of the present invention when concrete the application, can be to be contained in carrier.Described carrier can for carrier for expression of eukaryon or virus, be preferably adenovirus.For example, in a specific embodiments of the present invention, be obviously propagation or the migration of inhibition tumor cell by adenovirus Ad-UNC5D.
On the other hand, the present invention also provides a kind of growth of cancer, inhibition tumor cell, the migration of inhibition tumor cell, the one-tenth tumor of inhibition tumor cell and/or pharmaceutical composition of cell death inducing of being used for the treatment of, and this pharmaceutical composition contains:
The albumen of UNC5D gene and/or its coding, and one or more pharmaceutical excipients or pharmaceutical carrier.
Particularly, those skilled in the art can select suitable pharmaceutical excipient or pharmaceutical carrier according to its prior art knowledge of grasping, such as can comprise normal saline, etc. ooze combination of glucose solution, buffer saline, glycerol, ethanol and mentioned solution etc.Described pharmaceutical composition can adopt suitable dosage form and use by suitable route of administration, and these need not creative work for a person skilled in the art in its ken just can determine best mode.
According to specific embodiments of the present invention, described cancer is renal carcinoma, and described tumor cell is kidney cancer cell.
On the other hand, it is a kind of for the cancer test kit of Diagnosis of Renal Cell Carcinoma and/or prognosis judgement particularly that the present invention also provides, described test kit contains the reagent for detection of the albumen of UNC5D gene or its coding, for example: be used for the PCR primer at the synthetic UNC5D gene DNA chain of PCR and/or its cDNA chain, or the antibody of anti-UNC5D albumen etc.
In sum, the present invention studies and finds that UNC5D is a kind of new potential antioncogene, particularly has important function in the generation of renal carcinoma, evolution.
Description of drawings
Figure 1A shows that PCR detects UNC5D at the expression of 16 kinds of normal structures of people, and as seen from the figure, UNC5D has all detected stronger expression in people's brain, kidney, prostate, testis, small intestinal and colon.
Figure 1B shows that PCR detects the expression of UNC5D in the tumor cell line in Various Tissues source, and as seen from the figure, the expression of UNC5D generally is down regulation trend in the tumor cell line in Various Tissues source.UNC5D has very strong expression in nonmalignant human embryonic kidney cell line HEK293.
Fig. 1 C show the present invention by online software prediction to the UNC5D gene promoter area have a CpG island, the MSP primer that the present invention designs and the position of BGS primer mark in the drawings.
Fig. 1 D (left picture) shows the situation that methylates on UNC5D promoter region CpG island in 13 kinds of cell lines that detect by MS-PCR, the CpG island of UNC5D promoter region is methylation state in most tumors cell line, human embryonic kidney cell line HEK293 is non-methylation state; Fig. 1 D (right picture) shows by the sequencing technologies that methylates and confirms that further the CpG island of detecting UNC5D promoter region in positive several cell lines at MS-PCR is methylation state really, and the HEK293 cell is non-methylation state really.
Fig. 1 E shows that processing UNC5D with demethylation medicine 5-aza hangs down expression or express negative 786-O, Hela, SW480, SW620 and five kinds of cell lines of A549, then detect the expression of UNC5D, as seen after processing certain hour with 5-aza, the expression of UNC5D is all raised.Further confirm that by the order-checking that methylates the CpG island of UNC5D promoter region is by demethylation.
Fig. 2 A is the gel electrophoresis photo during Basic-PCR analyzes, and by for the UNC5D Auele Specific Primer, renal carcinoma and the cDNA of cancer beside organism is detected, and UNC5D mRNA down-regulated expression in the renal carcinoma tissue of pairing is described.GAPDH is system's internal reference.
Fig. 2 B shows the Real-time PCR Analysis result, by UNC5D is compared with the ratio of the detected value of GAPDH, UNC5D mRNA down-regulated expression in the renal carcinoma tissue of pairing is described.
Fig. 2 C is the tissue staining photo in immunohistochemical analysis, respectively renal carcinoma and cancer beside organism is dyeed with the antibody of UNC5D, further proves UNC5D albumen down-regulated expression in the renal carcinoma tissue of pairing.The arrow indication be exactly the UNC5D hot spot, the present invention finds that in normal and cancer beside organism, UNC5D is mainly respectively in the renal tubules kytoplasm.
Fig. 2 D shows that UNC5D is in cancer beside organism and the statistic analysis result of matching between the clinical Fuhrman classification of expression ratio (N/T ratio) in cancerous tissue and corresponding tumor tissues.Can find out, along with increasing of Fuhrman classification, N/T ratio is down regulation trend.
The methylation level on UNC5D promoter region CpG island in the renal carcinoma tissue that Fig. 2 E show to detect and pairing cancer beside organism, consistent with the situation that methylates in above-mentioned express spectra and cell line, in renal carcinoma tissue, UNC5D promoter region CpG island generally is the high methylation state, and corresponding cancer beside organism is non-and methylates or low methylation state.
Fig. 2 F shows the sequencing analysis result that methylates, and further confirms the result that shows in Fig. 2 E.
The result that the Heterozygosity disappearance (LOH) that renal carcinoma and pairing cancer beside organism are carried out of showing Fig. 2 G detects, as figure, the present invention has all detected the situation of disappearance in little some D8S1083 of three little satellites, the D8S505 in the selected UNC5D of laying respectively at upstream of coding region, middle reaches and downstream and D8S1750, Fig. 2 G is the legend that LOH occur in three sites.
Fig. 2 H is that the present invention carries out the cartogram of LOH testing result to 44 parts of pairing renal carcinomas and cancer beside organism, and in 44 pairs of specimen, the present invention has detected 14 pairs loss of heterozygosity has occured.These results suggest that, except methylating, LOH causes UNC5D that the major reason that descends occurs to express in renal carcinoma tissue.
Fig. 3 is that the enzyme action of UNC5D carrier in the embodiment of the present invention 3 is identified collection of illustrative plates.
Fig. 4 A is presented at the expression that recovers UNC5D in the renal carcinoma cell line 786-O of UNC5D down-regulated expression or disappearance and A498, then detect the cell proliferation situation of different time points with cck-8 reagent, UNC5D obviously inhibition tumor cell is the cell proliferation of 786-O and A498.
The situation of change of cell cycle before and after the renal carcinoma cell line 786-O expression UNC5D of Fig. 4 B display analysis, found no matter whether the 786-O cell line of expressing UNC5D block with Nocodazole, be in the cell quantity of G2/M phase all obviously more than matched group, this explanation UNC5D suppresses by cell tissue was realized in the G2/M phase the propagation of 786-O cell line.
Fig. 4 C display panel colony formation result illustrates that the 786-O cell after crossing expression UNC5D, exists difference in unicellular clone growth course.
Fig. 4 D shows that soft-agar cloning forms experimental result, is the swollen neoplastic experiment in vitro of research, illustrates that UNC5D has the inhibition effect to the clonality of renal carcinoma cell line 786-O.
Fig. 4 E shows the scratch test result, illustrates that UNC5D has the inhibition effect to the transfer ability of renal carcinoma cell line 786-O.
Fig. 4 F shows the further quantitative test analysis result of scratch test, cross the 786-O of expression UNC5D and the cut width of cellular control unit by measuring different time points with adenovirus, to show that UNC5D is to the inhibition effect that transfer ability was had of renal carcinoma cell line 786-O.
Fig. 4 G shows the result of Transwell cell migration experiment, illustrates that UNC5D can suppress the migration of renal carcinoma cell line.
Fig. 4 H shows Transwell cell Matrigel result, and in order to study tumor cell Invasion and Metastasis process in vivo, presentation of results UNC5D can suppress the invasive ability of renal carcinoma cell line.
Fig. 5 A shows the cell apoptosis assay result of making of the method for Cell Image Analyzer, and the HEK293T cell line that mistake is expressed UNC5D is compared with matched group and demonstrated more karyorrhexis and apoptotic body, illustrates that UNC5D can induce the apoptosis of HEK293T cell.
Fig. 5 B shows and to confirm with the amphophilic method of Annexin V-7AAD the apoptosis experimental result that UNC5D induces the HEK293T cell, from streaming figure, be no matter early apoptosis or late period apoptosis, experimental group is all obviously more than matched group.
Fig. 5 C is the hypodiploid peak that the method with PI dyeing occurs when detecting due to the formed DNA detection by quantitative of apoptosis, and UNC5D crosses and occurred obvious hypodiploid peak in the HEK293T of expression.Find simultaneously, after adding the extensive inhibitor Z-VAD-FMK of caspase, hypodiploid peak disappearance, the apoptosis that the HEK293T cell that UNC5D induces is described is that caspase relies on.
Fig. 5 D shows the experimental result of Western blot, and UNC5D crosses and obvious PAPR can be detected in the HEK293T of expression and shear, and UNC5D is dose-dependent during this shearing.Equally, after adding the extensive inhibitor Z-VAD-FMK of caspase, PAPR shears disappearance, and the apoptosis that further illustrates the HEK293T cell that UNC5D induces is that caspase relies on.
The specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, " molecular cloning experiment guide " (third edition) (Cold Spring Harbor Laboratory (CSHL) Press, 2001) condition described in, or the condition of advising according to manufacturer.
In following examples, all commercially available acquisitions of original reagent used and material, main agents and material are:
Following cell line is used in experiment: five kinds of renal carcinoma cell lines comprise 786-O, A498, ACHN, Caki-1 and OS-RC-2; Two kinds of bladder cancer cell lines comprise RT-4 and MGH-u1; Five kinds of hepatoma cell line comprise HepG2, Bel-7402, SMMC-7721, HLE and HCC-LM3; Five kinds of lung cancer cell lines comprise NCI-H446, NCI-H460, pAa, A549 and NCI-H1299; Four kinds of digestive tract tumor's cell lines comprise SW480, SW620, HT-29 and MKN-45; Five kinds of Leukemia Cell Lines comprise U937, K562, Jurkat, Daudi and D341MED; A kind of prostate tumor cells is PC-3; A kind of breast cancer cell is MCF-7; Cervical cancer tumer line Hela; A kind of ovarian cancer cell line SK-OV6; Two kinds of melanocytoma cell line WM-451 and SK-Mel-37; And cells of tumorous bone is Saos-2.These cell lines are available from ATCC or Beijing Union Medical College cell bank.
Above cell line is used 10%FBS/RPMI-DMEM (4.5g/L glucose, the 4mM Pidolidone, the 100U/ml penicillin, 100 μ g/ml streptomycins) or 10%FBS/RPMI-1640 (4.5g/L glucose, the 4mM Pidolidone, the 100U/ml penicillin, 100 μ g/ml streptomycins) cultivate.
The other pairing tissue of 44 former of routine people renal carcinomas and cancer is provided by The Third Affiliated Hospital of Peking University's Urology Surgery.
Organization chip is available from Xinchao Biotech Co., Ltd., Shanghai;
Synthetic and the order-checking of primer (comprising that LOH detects fluorescent primer used) is entrusted Beijing to hold up Bioisystech Co., Ltd of section and is completed;
Cell Counting Kit-8 (CCK-8) is available from Dojindo company;
Decitabine (5-aza-2 '-deoxycytidine), sodium sulfite (sodium bisulfite), dyestuff 33258 (Hoechst33258), Nocodazole, propidium iodide (Propidium iodide) and ribonuclease A (RNase A) be available from Sigma-Aldrich company;
TRIZOLTM reagent, Lipofectamine 2000 are available from Invitrogen company;
Normal structure cDNA panel (Normal tissue cDNA panel) is available from Clontech company;
Real-time quantitative PCR reagent Power SYBR Green PCR master mix is available from Bio-Rad company;
Wizard Plus Miniprep DNA Purification System, Reverse Transcription System, pGEM-TEasy vector, T4DNA ligase, pan-caspase inhibitor Z-VAD-FMK, restricted enzyme BglII, EcoRI, NheI and high-fidelity DNA Go Taq polymerase are available from Promega company;
KOD DNA polymerase enzyme is available from TOYOBO company;
Anti-UNC5D antibody is available from Santa Cruz company; Two of HRP labelling goat antirabbit resists available from Promega company; Anti-GAPDH antibody is available from Proteintech company; Anti-Flag antibody is available from Sigma-Aldrich company; Anti-PARP is available from Cell Signaling company;
The Annexin V of Fibronectin, matrigel, FITC labelling and 7-AAD apoptosis staining kit are available from BDBiosciences company.
1.UNC5D the express spectra in normal structure
PCR detects the expression of UNC5D in 16 kinds of normal structures of people, and in reaction, the primer sequence is as shown in SEQID Nos.3 and 4 in table 1, and reaction condition is referring to table 2, with GAPDH as system's internal reference (the internal reference primer is seen SEQ ID Nos.5 and 6).
2. the RNA of cell or tissue extracts
According to TRIZOL
TM(Invitrogen) description of reagent is extracted respectively the total RNA of cell or tissue, and it is quantitative with ultraviolet spectrophotometer.Get the 1 total RNA of μ g, utilize Reverse Transcription System (Promega) by synthetic the first chain cDNA of Oligo dT.
3.RT-PCR reaction system
Get the 1 above-mentioned cDNA product of μ l and carry out pcr amplification as template.In reaction, the primer sequence is as shown in SEQ IDNos.7 and 8 in table 1, and reaction condition uses GAPDH as system's internal reference referring to table 2.
The present embodiment extracts RNA, preparation cDNA from 32 routine renal carcinomas and the other pairing tissue of cancer.Utilizing BIO-RAD quantitative fluorescent PCR system to carry out real-time quantitative PCR detects.Wherein increase GAPDH as internal reference.
4. the extracting genome DNA of cell or tissue
Extract the genomic DNA of cell or tissue and it is quantitative with ultraviolet spectrophotometer according to the description of DNA extraction kit (Promega).
5.UNC5D promoter region CpG island prediction
(http://www.urogene.org/methprimer/.) predicts by software, and design respectively methylation-specific primer (SEQ ID Nos.9 and 10), non-methylation-specific primer (SEQ ID Nos.11 and 12) and the sequencing primer that methylates (SEQ IDNos.13 and 14) (as shown in table 1, reaction condition is referring to table 2).
6. use bisulfite to process and detect the DNA methylation level
The genomic DNA that extracts is used the BglII digestion with restriction enzyme; Use not the restricted enzyme for the purpose fragment, the genomic DNA of the no more than 700ng of enzyme action; By boiling water decocting in water 10 minutes with cessation reaction; Add 2MNaOH to final concentration be 0.3M, place 15 minutes so that the DNA degeneration for 50 ℃; For each sample, prepare 6 EP pipes, the 300 microlitre paraffin oil of packing into are placed pre-cooling on ice.Add 2% low melting-point agarose solvent soln (using water dissolution) of 2 times of volumes in DNA solution, repeatedly blow even with " rifle "; Form the agarose pearl: draw the DNA/ agarose mixture of 10 microlitres, join in the paraffin oil of pre-cooling; (only putting a pearl in an Ep pipe) is positioned over 30 minutes on ice with pipe, to form solid pearl; Add 200-500 microlitre 2.5M Sodium Metabisulfite to each pipe; In the darkroom, placed 4-12 hour for 50 ℃; Wash pearl 3 times with 1mlTE (pH 8.0), each 15 minutes, to remove remaining Sodium Metabisulfite; Wash pearl 2 times with 500 microlitre 0.2M NaOH, each 15 minutes, to finish modification; Wash pearl 3 times with 1mlTE (pH 8.0), each 10 minutes; TE (pH8.0) with small size preserves pearl in 4 ℃; (pearl can be deposited several weeks) PCR washes pearl with the 1ml distilled water before detecting; The pearl that obtains after to process uses GoTaq DNA synzyme (Promega) as template, under the condition of touchdown, carries out pcr amplification; DNA electrophoresis detection result.
7. bisulfites-genomic DNA order-checking
The pearl that obtains after to process uses GoTaq DNA synzyme as template, under the condition of touchdown, carries out pcr amplification; DNA electrophoresis and glue reclaim, the purification amplified fragments; Reclaim with glue the fragment that obtains and be connected to pGEM-T easy carrier; Transform and carry out blue white macula and screen; The picking white colony (10) be inoculated in the LA culture medium; Send bacterium liquid to check order; Compare according to sequencing result the normal DNA sequence that NCBI provides.
8.5-aza processing cell line
The 5-aza-2 '-deoxycytidine (Sigma-Aldrich) that is 10 μ mol/L with final concentration processes 786-O, Hela, SW480, SW620 and five kinds of cell lines of A549, then detects the methylation level of UNC5D promoter region.
Result shows:
The present embodiment detects UNC5D at the expression of 16 kinds of normal structures of people with PCR, finds that UNC5D has all detected stronger expression (Figure 1A) in people's brain, kidney, prostate, testis, small intestinal and colon.
PCR detects the expression of UNC5D in the tumor cell line in Various Tissues source, finds that the expression of UNC5D generally is down regulation trend in the tumor cell line in Various Tissues source.UNC5D has very strong expression (Figure 1B) in nonmalignant human embryonic kidney cell line HEK293.
In the present embodiment, by online software prediction to the UNC5D gene promoter area have a CpG island, designed MSP primer and the position of BGS primer are referring to indicating in Fig. 1 C.MS-PCR has detected the situation that methylates on UNC5D promoter region CpG island in 13 kinds of cell lines, the CpG island of finding the UNC5D promoter region is methylation state in most tumors cell line, human embryonic kidney cell line HEK293 is non-methylation state (Fig. 1 D-is left); The sequencing technologies that methylates further confirms that the CpG island of detecting UNC5D promoter region in positive several cell lines at MS-PCR is methylation state really, and the HEK293 cell is non-methylation state (Fig. 1 D-is right) really.Process low 786-O, Hela, SW480, SW620 and the five kinds of cell lines of A549 expressing or express feminine gender of UNC5D with demethylation medicine 5-aza, then detect the expression of UNC5D, as seen after processing certain hour with 5-aza, the expression of UNC5D is all raised.Further confirm that by the order-checking that methylates the CpG island of UNC5D promoter region is by demethylation (Fig. 1 E).
1. the RNA of cell or tissue and RT-PCR reaction system and extracting genome DNA are as described in example 1 above
The present embodiment extracts RNA, preparation cDNA from 32 routine renal carcinomas and the other pairing tissue of cancer.Utilizing BIO-RAD quantitative fluorescent PCR system to carry out real-time quantitative PCR detects.Wherein increase GAPDH as internal reference.The primer is as shown in table 1, and reaction condition is referring to table 2.
2. immunohistochemical analysis
Organization chip is through dewaxing, and gradient ethanol aquation is boiled 15min and carried out antigen retrieval in sodium citrate buffer solution, use 3%H
2O
2The room temperature lucifuge is hatched 20min and is removed endogenous peroxydase, with normal sheep serum working solution room temperature sealing 30min, then drips the anti-human UNC5D polyclonal antibody of rabbit (Santa) to final concentration 1: 100, in 37 ℃ of reaction 1h.With the normal rabbit IgG of same concentration as negative control.Fully wash the anti-room temperature reaction of the anti-goat two of goat-anti rabbit mice 30 minutes of rear dropping HRP labelling with PBS, with PBS washing three times, DAB colour developing, haematoxylin redyeing, gradient alcohol dehydration, result is observed and recorded to last medium-sized gummy mounting in microscopically.
3.UNC5D express relevant statistical analysis
Statistical analysis between the clinical Fuhrman classification of UNC5D the expression ratio (N/T ratio) in cancerous tissue and corresponding tumor tissues in cancer beside organism and pairing.The present invention is divided into three groups with the Fuhrman rank, and I/I-II, II/II-III and III/III-IV have respectively 11,37,12 parts of case correspondences.
4. detect the DNA methylation level and bisulfites-the genomic DNA sequence measurement as described in Example 1.
Detect altogether 44 parts of case specimen, every part of cancer beside organism that includes renal carcinoma and pairing.
5. loss of heterozygosity detects
The present invention has selected to lay respectively at the little point of three little satellites in UNC5D upstream of coding region, middle reaches and downstream, that is: D8S1083, D8S505 and D8S1750, and synthetic correspondingly fluorescent primer (seeing Table SEQ ID Nos.15 and 16 in 1, SEQ ID Nos.17 and 18, SEQ ID Nos.19 and 20), reaction condition determines whether to have occured LOH referring to table 2 by the size of analyzing the PCR product.Detect altogether 44 parts of case specimen, every part of cancer beside organism that includes renal carcinoma and pairing.
Result shows:
According to the bioinformatic analysis of front and the prompting of cell line result, the present invention turns to clinical samples to attention, and the present invention has selected clear cell carcinoma of kidney.Detect discovery: UNC5D by regular-PCR and express really or obviously lower (Fig. 2 A) in four pairs of renal carcinomas that detect and pairing all show as renal carcinoma tissue in cancer beside organism.Then the present invention carries out the real-time quantitative PCR detection to 32 pairs of specimen, by UNC5D is compared with the ratio of the detected value of GAPDH, UNC5DmRNA down-regulated expression (Fig. 2 B) in the renal carcinoma tissue of pairing is described.ImmunohistochemistryResults Results further confirms UNC5D albumen down-regulated expression (Fig. 2 C, in figure, the arrow indication is exactly the UNC5D hot spot, the present invention finds that in normal and cancer beside organism, UNC5D is mainly respectively in the renal tubules kytoplasm) in the renal carcinoma tissue of pairing.Carry out statistical analysis between expression ratio (N/T ratio) by UNC5D in cancer beside organism and pairing cancerous tissue and the clinical Fuhrman classification of corresponding tumor tissues, as if the present invention find significant phenomenon a: UNC5D relatively lower (Fig. 2 D of expression by inhibitation processing procedure degree in grade malignancy lower renal carcinoma tissue, along with increasing of Fuhrman classification, N/Tratio is down regulation trend).
The present invention describes the mechanism of UNC5D down-regulated expression renal carcinoma tissue from two aspects, the detection that methylates of the one, UNC5D gene promoter area (Fig. 2 E and Fig. 2 F), the 2nd, the loss of heterozygosity of UNC5D section on chromosome (Fig. 2 G and Fig. 2 H).All participate in the knowing clearly regulation and control of UNC5D down-regulated expression in renal carcinoma tissue of these two kinds of mechanism of experiment confirm.Particularly, Fig. 2 E be the renal carcinoma tissue detected with pairing cancer beside organism in the methylation level on UNC5D promoter region CpG island, consistent with the situation that methylates in above-mentioned express spectra and cell line, in renal carcinoma tissue, UNC5D promoter region CpG island generally is the high methylation state, and corresponding cancer beside organism is non-and methylates or low methylation state; Fig. 2 F further confirms the result of Fig. 2 E by the sequencing analysis that methylates.Fig. 2 G is the result that the Heterozygosity disappearance (LOH) that renal carcinoma and pairing cancer beside organism carry out is detected, as figure, the present invention has all detected the situation of disappearance in little some D8S1083 of three little satellites, the D8S505 in the selected UNC5D of laying respectively at upstream of coding region, middle reaches and downstream and D8S1750, Fig. 2 G is the legend that LOH occur in three sites.Fig. 2 H is that the present invention carries out the cartogram of LOH testing result to 44 parts of pairing renal carcinomas and cancer beside organism, and in 44 pairs of specimen, the present invention has detected 14 pairs loss of heterozygosity has occured.These results suggest that, except methylating, LOH causes UNC5D that the major reason that descends occurs to express in renal carcinoma tissue.
1.UNC5D the structure of carrier for expression of eukaryon
the present invention is take cerebral tissue cDNA as template, (amplimer sees Table SEQ ID Nos.21 and 22 in 1 to carry out pcr amplification by high-fidelity Taq enzyme K.O.D enzyme, reaction condition is referring to table 2), wherein forward primer is with the EcoRI restriction enzyme site, downstream primer is with the NheI restriction enzyme site, PCR product and pRK empty carrier are carried out the double digestion processing with EcoRI and NheI, then DNA electrophoretic separation purification, with the T4 ligase, product is connected, through after transforming-choose monoclonal-little and shaking bacterium liquid-sequence of operations such as little upgrading grain, verify (restriction enzyme mapping is seen Fig. 3) finally by order-checking and enzyme action, order-checking is correct.Correct plasmid extracts in a large number.The C end of UNC5D protein product indicates the flag label.
Adenovirus Ad-UNC5D Vector construction and virus packing
Ad-UNC5D and contrast empty carrier virus Ad-null (Mock), Ad-GFP entrust this yuan Zhenyang corporation restructuring, packing, purification to obtain, and TCID50 is also measured by the said firm.
3. the infection of renal carcinoma cell line and transfection
Cell is inoculated in 96 orifice plates, 12 orifice plates, 6 orifice plates or 60mm Tissue Culture Dish infecting the previous day with Suitable Density, carry out cell counting after 18-22 hour, with different MOI value infection cells.Suck the archeocyte culture supernatant, add the half fresh complete medium of amount, then rocked gently culture dish in every 15 minutes adding in first hour of adenovirus, supply culture medium after one hour.The cell for the treatment of transfection was inoculated in 6 orifice plates in advance in 24 hours, change culture medium into the Opti-MEM culture medium before transfection, the Opti-MEM culture medium 2504 μ l that will contain the lipofetamine2000 of 12 μ l mix with the OptiM culture medium that contains 4 μ g plasmid DNA, after room temperature 30 minutes, be added dropwise in culture medium, be changed to ordinary culture medium after 4-6 hour.Observe after 24 hours.
4.CCK-8 method is drawn the growth curve of cell
The density of cell after adenovirus infection according to 2000 cells/well is inoculated in 96 orifice plates, and every group of cell established 3 parallel multiple holes, adds the CCK-8 reagent of 10 μ l respectively at 0h, 24h, 48h, 72h, in 37 ℃ at 5%CO
2Under hatch 2 hours after, measure optical density value with ELISA Reader 450nm.To each time point not on the same group result add up the growth curve of rear drafting cell.
5. cell cycle experiment
The 786-O cell is inoculated in the 60mm culture plate with 1 * 106, and 80% converges postoperative infection.Add nocodazole (300ng/ml after 24 hours in fresh medium; Sigma-Aldrich), collect the cell that becomes circle after 16 hours, PBS washes twice, abandons supernatant, and the training basic weight is outstanding in 2x10
5/ well spreads in 6 orifice plates, is every two hours to check and accept the time to obtain cell, and PBS washes twice, adds in 1ml 70% pre-cooled ethanol, and piping and druming is evenly fixed more than 12 hours for 4 ℃.Remove ethanol with the PBS washing, 1000rpm, 5min washes twice.0.5mlPBS re-suspended cell adds PI and RNaseA to final concentration 50 μ g/ml, 37 ℃ of temperature are bathed 30min.Use the cells were tested by flow cytometry cycle.Analyze stream data with FlowJo software (Tree Star).
6. plate clone forms experiment
Respectively the cell (experimental group) of the cell (matched group) of transfection pRK and pRK-UNC5CL is inoculated in 6 orifice plates by 1000, every hole, 2000 and 10000 cells, after 24 hours, culture medium being changed into the ordinary culture medium that contains G418 cultivates, changed a not good liquor in every three days, used afterwards the methanol of pre-cooling to fix in 14 days, 0.005 violet staining.
7. soft-agar cloning forms experiment
With the cell after adenovirus infection, with 0.25% trypsinization and piping and druming gently, make it to become unicellular, make viable count, adjust cell density to 1 * 10 with the DMEM culture fluid that contains 20% hyclone
6Cell/L.Prepare respectively the LMP agar sugar liquid of 1.2% and 0.7% two concentration with distilled water, after autoclaving, maintain in 40 ℃ and make it not solidify.After in 1: 1 ratio, 1.2% agarose and 2 * DMEM culture medium (calf serum that contains 2 * antibiotic and 20%) being mixed, getting the 3ml mixed liquor, to inject diameter be that (the 10cm plate adds 7～10ml) for the plate of 6cm, cooled and solidified can be placed in CO as bottom-layer agar
2Standby in incubator.Make 0.7% agarose and 2 * DMEM culture medium mixed mutually in sterile test tube in 1: 1 ratio after, to infect the cell (matched group) of Ad-GFP and the cell (experimental group) of infection Ad-UNC5CL adds respectively in the mixture that has just prepared of 1.5ml again, abundant mixing, injection is covered with 1.2% agarose bottom plate, forms gradually two agar layer.After top-layer agar solidifies, insert 37 ℃, 5%CO
2Incubator in cultivated 10～14 days.Plate is placed under inverted microscope observation of cell clone number.Calculate formation rate.
8. migration, invasion and attack and cut and cut quantitative experiment
The employing aperture is that the 24-well Transwell (Corning company) of the polycarbonate membrane (polycarbonate membrane) of 8 μ m carries out chemotaxis assay (chemotaxis assay).Be placed in lower chamber with 10%FBS, fibronectin (fibronectin) (BD-Pharmingen company) be applied to the lower floor of polycarbonate membrane.The cell of results exponential phase is washed cell 2 times with 0.1%BSA/DMEM, adjusts cell density to 1 * 10
6/ ml, 50 μ l/ holes, every kind of cell is established 3 parallel multiple holes.In 37 ℃ at 5%CO
2Under hatch 7 hours after, winder is used the methanol fixed cell, Giemsa dyeing 20 minutes scrapes off with cotton swab the cell that does not move on the upper strata gently, washes 3 times the optical microphotograph Microscopic observation with PBS.Every hole is chosen at random 3 high power lens visuals field and is counted, and the count value in the high power lens visual field, 9, every kind of cell 3 parallel multiple holes is got arithmetical average add up.Coat the Matrigel (BD Biosciences) of 30 μ g on the basis of the tangible Transwell migration of Matrigel experiment on the cell film.. scratch experiment carries out in 6 orifice plates, metainfective cell is laid in the hole until cover with one deck, uses aseptic rifle head with the cell cut, washes 3 times with PBS, hatches to corresponding time point and observe photograph under mirror.In the cut quantitative test, every three hours is a time point, carries out Taking Pictures recording with phase contrast microscope, then measures width and the curve plotting figure of cut.
Result shows:
Recover the expression of UNC5D in the renal carcinoma cell line 786-O of UNC5D down-regulated expression or disappearance and A498, then detect the cell proliferation situation of different time points with cck-8 reagent, UNC5D obviously inhibition tumor cell is the cell proliferation (Fig. 4 A) of 786-O and A498.The variation of cell cycle before and after the renal carcinoma cell line 786-O expression UNC5D that analyzes, found no matter whether the 786-O cell line of expressing UNC5D block with Nocodazole, be in the cell quantity of G2/M phase all obviously more than matched group, this explanation UNC5D suppresses by cell tissue is realized (Fig. 4 B) in the G2/M phase the propagation of 786-O cell line.Form in experiment in plate clone, the 786-O cell exists difference (Fig. 4 C) in unicellular clone growth course after crossing expression UNC5D.Equally, form in experiment at soft-agar cloning, UNC5D has inhibition effect (Fig. 4 D) to the clonality of renal carcinoma cell line 786-O.Scratch test shows that UNC5D has inhibition effect (Fig. 4 E) to the transfer ability of renal carcinoma cell line 786-O.To the further quantitative test of scratch test, cross the 786-O of expression UNC5D and the cut width of cellular control unit by measuring different time points with adenovirus, to show that UNC5D is to the inhibition effect that transfer ability was had (Fig. 4 F) of renal carcinoma cell line 786-O.The presentation of results UNC5D of Transwell cell migration experiment can suppress the migration (Fig. 4 G) of renal carcinoma cell line, Transwell cell Matrigel, in order to study tumor cell Invasion and Metastasis process in vivo, presentation of results UNC5D can suppress the invasive ability (Fig. 4 H) of renal carcinoma cell line.
Embodiment 4, UNC5D apoptosis induction and the correlation molecule mechanism thereof to HEKC HEK293T
1. Cell Image Analyzer detects apoptosis
Results UNC5D crosses 293T cell and the cellular control unit of expression, the PBS washed twice, with the PBS fixed cell that contains 4% formalin 30 minutes, the PBS washed twice, with Hoechst33258 (Sigma) dyeing 5 minutes, the PBS washed twice, observed and recorded under the UV of fluorescence microscope passage, the cell that karyorrhexis and apoptotic body occur is considered to apoptotic cell.
2. fluidic cell is learned and is detected apoptosis
The amphophilic method of Annexin V-7AAD is crossed 293T cell and the cellular control unit of expression for results UNC5D, and the PBS washed twice detects according to the description of Apoptosis Detection Kit (BD Biosciences).In the test at the hypodiploid peak that occurs when the method with PI dyeing detects due to apoptosis formed DNA detection by quantitative, enter UNC5D and MOCK plasmid at the 293T transit cell, add or do not add the extensive inhibitor Z-VAD-FMK of caspase, after 48 hours, results UNC5D crosses 293T cell and the cellular control unit of expression, the PBS washed twice, then add in 1ml 70% pre-cooled ethanol, piping and druming is evenly fixed more than 12 hours for 4 ℃.Remove ethanol with the PBS washing, 1000rpm, 5min washes twice.0.5mlPBS re-suspended cell adds PI and RNaseA to final concentration 50 μ g/ml, 37 ℃ of temperature are bathed 30min.Use cells were tested by flow cytometry.
3.Wertern blot detects apoptosis
Enter UNC5D and MOCK plasmid at the 293T transit cell, add or do not add the extensive inhibitor Z-VAD-FMK of caspase, after 48 hours, results UNC5D crosses 293T cell and the cellular control unit of expression, the UNC5D of results equal number crosses the 293T cell of expression and the total cell protein of cellular control unit, Wertern blot detects, PAPR and shear protein thereof, GAPDH and flag.
Result shows:
In the cell apoptosis assay of making of the method for Cell Image Analyzer, cross the HEK293T cell line express UNC5D and compare with matched group and demonstrate more karyorrhexis and apoptotic body, illustrate that UNC5D can induce the apoptosis (Fig. 5 A) of HEK293T cell.Confirm with the amphophilic method of Annexin V-7AAD the apoptosis that UNC5D induces the HEK293T cell, be no matter early apoptosis or late period apoptosis, experimental group is all obviously more than matched group (Fig. 5 B).The hypodiploid peak that occurs when detecting due to the formed DNA detection by quantitative of apoptosis with the method for PI dyeing, UNC5D crosses and has occurred obvious hypodiploid peak in the HEK293T of expression.Find simultaneously, after adding the extensive inhibitor Z-VAD-FMK of caspase, hypodiploid peak disappearance, the apoptosis that the HEK293T cell that UNC5D induces is described is that caspase relies on (Fig. 5 C).In the experimental result of Western blot, UNC5D crosses and obvious PAPR can be detected in the HEK293T of expression and shear, and UNC5D is dose-dependent during this shearing.Equally, after adding the extensive inhibitor Z-VAD-FMK of caspase, PAPR shears disappearance, and the apoptosis that further illustrates the HEK293T cell that UNC5D induces is that caspase relies on (Fig. 5 D).
The list of table 1-primer
Table 2-PCR reaction condition
Annotate: the generic condition of PCR reaction is: 94 ℃, unwind in 4 minutes; 94 ℃ 30 seconds, lower 30 seconds of annealing temperature (Tm), setting in 1000bp/ minute is pressed in 72 ℃ of extensions, period is generally 28-32; 72 ℃ 10 minutes.The Taq enzyme that the PCR reaction is used and Tm value and the extension time of reaction see Table 2.
Claims (10)
1.UNC5D the application of the albumen of gene and/or its coding in the medicine of preparation treatment cancer.
2. detect the application of reagent in the compositions that auxiliary diagnosis and/or prognosis for the preparation of cancer judge of the albumen of UNC5D gene or its coding;
Preferably, the reagent of the albumen of described detection UNC5D gene or its coding comprises: be used at the synthetic UNC5D gene DNA chain of PCR and/or the PCR primer of its cDNA chain or the antibody of anti-UNC5D albumen.
3. application according to claim 1 and 2, wherein, described cancer is renal carcinoma.
4.UNC5D the albumen of gene and/or its coding has application in the preparation of effect of the one-tenth tumor of migration, inhibition tumor cell of growth, the inhibition tumor cell of inhibition tumor cell and/or cell death inducing in preparation.
5. application according to claim 4, wherein, described tumor cell is kidney cancer cell.
6. application according to claim 4, wherein, described migration is the migration of the extracellular matrix fibronectin kidney cancer cell of inducing.
7. according to claim 1 and 2 or 4 or 5 or 6 described application, wherein, described UNC5D gene is contained in carrier;
Preferably, described carrier is carrier for expression of eukaryon or virus, more preferably adenovirus.
8. one kind is used for the treatment of the growth of cancer, inhibition tumor cell, the migration of inhibition tumor cell, the one-tenth tumor of inhibition tumor cell and/or the pharmaceutical composition of cell death inducing, and this pharmaceutical composition contains:
The albumen of UNC5D gene and/or its coding, and one or more pharmaceutical excipients or pharmaceutical carrier.
9. pharmaceutical composition according to claim 8, wherein, described cancer is renal carcinoma, described tumor cell is kidney cancer cell.
10. test kit that is used for cancer diagnosis and/or prognosis judgement, described test kit contains: be used for the PCR primer at the synthetic UNC5D gene DNA chain of PCR and/or its cDNA chain, or the antibody of anti-UNC5D albumen;
Preferably, described cancer is renal carcinoma.
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| Title |
|---|
| HONG WANG ET AL: "A newly identified dependence receptor UNCSH4 is induced during DNA damage-mediated apoptosis and transcriptional target of tumor suppressor p53", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》, vol. 370, 8 April 2008 (2008-04-08), pages 594 - 598, XP022654740, DOI: doi:10.1016/j.bbrc.2008.03.152 * |
| WANG,H. ET AL: "Homo sapiens unc-5 homolog D (C. elegans) (UNC5D), mRNA", 《NCBI REFERENCE SEQUENCE: NM_080872.2》, 30 June 2012 (2012-06-30) * |
| 朱育焱 等: "Identification of UNC5D as a tumor suppressor for unfavorable neuroblastomas", 《生命的分子机器及其调控网络—2012年全国生物化学与分子生物学学术大会摘要集》, 24 August 2012 (2012-08-24) * |
| 王弘 等: "Unc5D基因在高_低危神经母细胞瘤中的表达及其意义", 《中国医科大学学报》, vol. 38, no. 3, 31 March 2009 (2009-03-31), pages 225 - 233 * |
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