CN104873973B - Purposes of the FAM135B inhibitor in treating and/or preventing esophageal squamous cell carcinoma - Google Patents
Purposes of the FAM135B inhibitor in treating and/or preventing esophageal squamous cell carcinoma Download PDFInfo
- Publication number
- CN104873973B CN104873973B CN201410068807.9A CN201410068807A CN104873973B CN 104873973 B CN104873973 B CN 104873973B CN 201410068807 A CN201410068807 A CN 201410068807A CN 104873973 B CN104873973 B CN 104873973B
- Authority
- CN
- China
- Prior art keywords
- fam135b
- cell
- squamous cell
- cell carcinoma
- purposes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to purposes of the FAM135B inhibitor in treating and/or preventing esophageal squamous cell carcinoma.On the other hand, the purposes the present invention relates to FAM135B genome mutations in for diagnosis and/or prognosis esophageal squamous cell carcinoma.
Description
Technical field
The present invention relates to FAM135B inhibitor to treat and/or prevent the technical field of esophageal squamous cell carcinoma.The opposing party
Face, the present invention relates to FAM135B gene mutations in diagnosis and/or the technical field of prognosis esophageal squamous cell carcinoma.
Background technology
The cancer of the esophagus is common tumor in digestive tract, and the whole world there are about 300,000 people and dies from the cancer of the esophagus every year, is whole world cancer
Dead the sixth-largest reason, referring to Kamangar, F., G.M.Dores, et al. (2006) " Patterns of cancer
incidence,mortality,and prevalence across five continents:defining priorities
to reduce cancer disparities in different geographic regions of the world.″J
Clin Oncol 24(14):2137-2150.The whole world about 70% oesophagus carcinogenesis in China, and China 90% the cancer of the esophagus
For esophageal squamous cell carcinoma.Up to the present, the occurrence and development mechanism to esophageal squamous cell carcinoma understands very few, therefore for food
The means and method that pipe squamous cell carcinoma is early diagnosed and treated are very limited, 5 years survival rates of patients with esophageal squamous cell
Only 10%, referring to Xu, Y., X.Yu, et al. (2012) " Neoadjuvant versus adjuvant treatment:
which one is better for resectable esophageal squamous cell carcinoma?″World
J Surg Oncol 10:173, and Zhang, S.W., M.Zhang, et al. (2012) " An Analysis of
Incidence and Mortality of Esophageal Cancer in China, 2003~2007. " China
Cancer 21(4):241-247.Molecular targeted therapy is Recent study focus, and is obtained in the treating malignant tumor of part
Breakthrough.Such as Gefitinib（Irresa）Treat non-small cell lung cancer, especially women, non-smoking, adenocarcinoma patients' prognosis more
It is good；Treatment with imatinib gastrointestinal stromal tumor, especially for there is the mutation person of Kit exons 1s 1 to obtain the effect of more preferable, join
See Nilsson B, S.K., Kindblom LG, et al. (2007) " Adjuvant imatinib treatment improves
recurrence-free survival in patients with high-risk gastrointestinal stromal
tumours(GIST).″Br J Cancer 96:1656-1658.C225 combined radiotherapies treat Locally Advanced incidence cancer, more singly
Pure radiotherapy, survival rate improves nearly 1 times, referring to Bonner JA, H.P., Giralt J, et al. (2006) " Radiotherapy
plus cetuximab for squamous-cell carcinoma of the head and neck.″N Engl J Med
354:567-578.Therefore, the molecular mechanism for illustrating esophageal squamous cell carcinoma occurrence and development is that the mankind capture esophageal squamous cell carcinoma
Premise.
FAM135B is positioned at No. 8 chromosomes, and its albumen contains 06 amino acid of Isosorbide-5-Nitrae, the FAM135B genes on NCBI
Genbank accession number is for Gene ID:51059, for its function, relevant report is had no in the literature.
The present inventor is with the sequencing of the generation of high flux two, exon trapping and Comparative genomic hybridization to China's esophageal squamous cell
The genome of shape cell cancer is studied, and is had now surprisingly been found that, missense often occurs in esophageal squamous cell carcinoma for FAM135B
Point mutation（missense）, and universal high expression, and the change of its genome and the prognosis of patients with esophageal squamous cell into
It is negatively correlated.Then our functional experiment shows, esophageal squamous cell then can be significantly lowered after suppressor FAM135B expression
Growth, Clone formation, migration and the invasive ability of cancerous cell line；Height expression wild type FAM135B can promote esophagus squameous thin
Growth, Clone formation, migration and the invasive ability of born of the same parents' cancerous cell line, and it promotes esophageal squamous cell after high expression saltant type FAM135B
The growth of shape cell carcinoma line, Clone formation, migration and invasive ability are more stronger than wild type.Therefore, gene FAM135B is expected to
The molecular marker and the target spot for the treatment of that can be diagnosed as esophageal squamous cell carcinoma.
The content of the invention
It is used to prepare in the medicine for treating and/or preventing esophageal squamous cell carcinoma the present invention relates to FAM135B inhibitor
Purposes.
Especially, it is specifically to combine and suppress FAM135B protein according to FAM135B inhibitor of the present invention
Antibody.
Term " antibody " be used for text in, refer to it is all types of specifically combine FAM135B polypeptides and suppress FAM135B work
The antibody of property.Preferably, the inhibiting antibody of the invention specifically combines the FAM135B polypeptides positioned at ligand binding domains
In epitope.Preferably, antibody of the invention be monoclonal antibody, polyclonal antibody, single-chain antibody, chimeric antibody or it is any should
The fragment or derivative of antibody.Such fragment or derivative included in term antibody are used in text, including double special anti-
Body, the antibody of synthesis, Fab, F (ab)2, in Fv or scFv fragments or these antibody any one chemical modification derivative.
In the upper and lower special combination used herein of antibody of the present invention, refer to antibody not with other polypeptide cross reactions.It can be used known
The special combination of technology for detection.
More particularly, it is Nucleic acid inhibitors according to FAM135B inhibitor of the present invention.
Preferably, described Nucleic acid inhibitors specifically combine coding FAM135B, and suppress FAM135B transcription or turn over
The polynucleotides translated.
" Nucleic acid inhibitors " are used in text, refer to such as fit nucleic acid molecules, and it similar to above-mentioned antibody by be retouched
The mode Binding peptide stated suppresses FAM135B polypeptide actives, or refer to it is complementary with the polynucleotides of coding FAM135B polypeptides and
With reference to the nucleic acid molecules of the polynucleotides, it suppresses the transcription or translation of polynucleotides.For example, inhibition nucleic acid can be by dry
The correct transcription for disturbing FAM135B genes plays a role as Triple-helix forming oligonucleotides.Moreover, inhibition nucleic acid can be core
Enzyme, it is specifically combined and FAM135B transcripts of degrading.Alternatively, its can be can combine, the transcript or at least of degrading
Suppress its antisense effectively translated（Nucleic acid）, siRNA or microRNA.The inhibition nucleic acid of latter type is characterised by it
Generally comprise the nucleotide sequence complementary with the sequence in FAM135B transcripts.The complementary series should long enough, and should include
The matching nucleotides of enough numbers, so as to allow the specific hybridization with transcript in cell.
Further, according to Nucleic acid inhibitors of the present invention be selected from ribozyme, antisense molecule, oligonucleotides inhibitor,
Fit, microRNA or siRNA.
" ribozyme " according to the present invention is the RNA molecule for including the sequence complementary with FAM135B transcripts.Ribozyme technology is
It is known in the art that those skilled in the art can design and using suitable ribozyme, without twists and turns；See, e.g.,
Khan2006,Clin.Chim.Acta367(1–2):20–27;Kalota2004,Cancer Biology&Therapy 3:1
4-12。
" antisense molecule " is used in text, refers to the therapeutic antisense RNA complementary with FAM135B transcripts or can combine
The morpholino oligonucleotide of FAM135B transcripts.Including morpholino oligonucleotide application antisense technology be it is known in the art that
See, e.g. Kalota 2004, Cancer Biology＆Therapy 3:1 4-12;Morcos 2007,Biochem
Biophys Res Commun358(2):521–7。
Inhibition oligonucleotides be used for text in, it is preferable that refer to combine the specific regions of target gene group DNA, so as to
Realize gene silencing（So-called Triple-helix forming oligonucleotides）Small double chain DNA molecule, or refer to and send out-wave work as bait
With and obstruct the oligonucleotides of transcription factor that target gene transcription specifically needs.These technologies have been successfully used to body
It is interior, and result has been obtained in the treatment to a certain extent (referring also to Kalota2004, Cancer Biology＆
Therapy 3:1 4-12.)。
Term is " fit " to be used in text, refers to the aptamer for specifically combining FAM135B polypeptides.By using, such as it is logical
Fas lignand system evolution (SELEX) technology of index concentration is crossed, fit storehouse can be generated（pool）.Selection step can be used for those
Specifically with reference to the fit of FAM135B polypeptides.Specifically combine it is fit in, blocking ligand binding, those are fit, or block
Those of interaction domain are fit, thus can be accredited as in meaning of the present invention be adapted to it is fit.It is fit for generating
Technology is well known in the art；See, e.g. Tuerk1990, Science.Aug 3;249(4968):505–510;or
Ellington 1990,Nature.Aug 30;346(6287):818-822。
" microRNA " in the sense of the present invention refers to single strand RNA molecule, itself and the nucleic acid included in FAM135B transcripts
Sequence is at least partly complementary.MicroRNA generally has about 19 to 26 length of nucleotides.MicroRNA synthesizes as precursor,
I.e. so-called pri-microRNA, the pri-microRNA there is hairpin structure and formed two of hairpin stem it is complementary from mutually
Mend region.
Term " siRNA（siRNA）" refer to nucleic acid molecules for double-stranded RNA agent, its with FAM135B transcripts
Divide complementation, and being capable of base pairing.SiRNA is by specifically instructing the enzyme in host cell to play a role, so as to cut target
Mark RNA.By the specificity of siRNA sequence, and its homology with RNA target mark, siRNA can cause target RNA chain
Cutting, so as to inactivate target RNA molecule.Preferably, it is sufficient to which the siRNA for adjusting RNAi includes following nucleotide sequence, and it contains target
Mark gene inverted repeat and target gene coding region（Or part）.SiRNA complementary region permission siRNA is enough and target
RNA hybridization is marked, so as to adjust RNAi.In mammal, siRNA is the length of about 19-25 nucleotides.
On the other hand, the present invention relates to one or more primers for specifically identifying FAM135B genome mutations and/or spy
Pin, the purposes in preparing for the reagent of diagnosis and/or prognosis esophageal squamous cell carcinoma.Preferably, in the purposes, lead to
Cross the method identification FAM135B genome mutations of the sequencing of the generation of high flux two, exon trapping and Comparative genomic hybridization.
Preferably, the esophageal squamous cell carcinoma in mammal is referred to according to esophageal squamous cell carcinoma of the present invention.
It is highly preferred that it is people according to the wherein described mammal of the present invention.
On the other hand, the present invention relates to a kind of method treated and/or prevent mammal esophageal squamous cell carcinoma, including
To the FAM135B inhibitor of patient's drug treatment effective dose by the cancer.
Brief description of the drawings
Fig. 1 shows that FAM135B, which occurs, changes patient with changing patient's prognosis situation without FAM135B.
Fig. 2 FAM135B show the expression in esophageal squamous cell carcinoma cell line and normal esophageal cell.
Fig. 3 A and Fig. 3 B show to lower respectively FAM135B expression to the growth of KYSE150 and COLO680N esophageal cancer cells
Influence.Wherein siCtrl is control group.
Fig. 4 A and 4C show the Clone formation situation in KYSE150 cell lines；Fig. 4 B and 4D show thin in COLO680N
Clone formation situation in born of the same parents system.
Fig. 5 A-5D show to lower FAM135B expression cell migrations and the influence situation of invasion and attack.Fig. 5 A and 5C show
Cell migration and invasion and attack situation in KYSE150 cell lines；Fig. 5 B and 5D show the cell migration in COLO680N cell lines
And invasion and attack situation.
Fig. 6 A-6B show the influence situation of high expression wild type and saltant type FAM135B cell growths.
Fig. 7 A-7D show the influence situation of high expression wild type and saltant type FAM135B to cell clonal formation.Fig. 7 A and
7C shows the Clone formation situation in KYSE30 cell lines；Fig. 7 B and 7D show the Clone formation in KYSE180 cell lines
Situation.
Fig. 8 A-8D show high expression wild type and saltant type FAM135B cell migrations and the influence situation of invasion and attack.Fig. 8 A
Show the cell migration in KYSE30 cell lines and invasion and attack situation with 8C；Fig. 8 B and 8D show in KYSE180 cell lines
Cell migration and invasion and attack situation.
Embodiment
Detection method of the present invention includes DNA extractions, two generation genome sequencings, exon trapping, icp gene
Group hybridization, mass spectrum, RNA extractions, Sanger sequencings, qRT-PCR, growth curve, Clone formation and transwell etc..In view of hair
A person of good sense has found and demonstrated effects of the gene FAM135B in esophageal squamous cell carcinoma occurrence and development, in scientific research from now on
Research and clinical practice in, DNA, RNA extraction and tri- kinds of Technology applications of qRT-PCR it is more universal.These three methods are for this
The technical staff in field is routine operation.Therefore, the achievement is easily employed in scientific research and clinic.
In specific embodiments of the present invention, the substantially method of FAM135B mutation and expression in testing sample is detected
It is as follows：
1）Extract the full DNA in the fresh esophageal carcinoma tissue of patient and matching blood；
2）Two generation genome sequencing technologies, exon trapping and comparative genomic hybridization hybrid chip detection tumor tissues and
With blood preparation；
3）Two generation sequencing data of whole genome, exon trapping data and chip data processing：Two generation full-length genomes are surveyed
Ordinal number evidence, exon trapping data and chip data are compared according to hg19 databases, Varscan, GATK, in-house
The methods of filter pipeline and Annovar, handles the catastrophe of gene, referring to McKenna, A., M.Hanna, et al.
(2010).″The Genome Analysis Toolkit:a MapReduce framework for analyzing next-
generation DNA sequencing data.″Genome Res 20(9):1297-1303；Wang, K., M.Li, et al.
(2010).″ANNOVAR:functional annotation of genetic variants from high-
throughput sequencing data.″Nucleic Acids Res 38(16):e164；Koboldt,D.C.,
Q.Zhang, et al. (2012) " VarScan 2:somatic mutation and copy number alteration
discovery in cancer by exome sequencing.″Genome Res 22(3):568-576.Then full genome
Group sequencing data is handled with SegSeq and DNAcopy methods and obtains gene copy number and change situation, referring to Venkatraman,
E.S.and A.B.Olshen(2007).″A faster circular binary segmentation algorithm for
the analysis of array CGH data.″Bioinformatics 23(6):657-663；Referring to Venkatraman,
E.S.and A.B.Olshen (2007).″A faster circular binary segmentation algorithm
for the analysis of array CGH data.″Bioinformatics 23(6):657-663；Chiang,D.Y.,
G.Getz, et al. (2009) " High-resolution mapping of copy-number alterations with
massively parallel sequencing.″Nat Methods 6(1):99-103；Comparative genomic hybridization hybrid chip data
Handled with SegMNT and DNAcopy methods and obtain gene copy number and change situation, referring to Olshen, A.B.,
E.S.Venkatraman, et al. (2004) " Circular binary segmentation for the analysis of
array-based DNA copy number data.″Biostatistics 5(4):557-572；Venkatraman,
E.S.and A.B.Olshen (2007).″A faster circular binary segmentation algorithm
for the analysis of array CGH data.″Bioinformatics 23(6):657-663.By tumor tissues sample
Gene mutation and copy number change gene mutation and copy number in the blood sample that situation comparison matches and change situation in this, finally
Obtain the truth that gene mutation and copy number change in tumor tissues sample.
4）Gene mutation and amplification checking：The checking of mutation is carried out using mass-spectrometric technique；The checking of amplification utilizes basis
The primer row qRT-PCR amplifications of FAM135B gene orders design.
5）Functional experiment is verified：Extract esophageal squamous cell carcinoma cell line and immortalize normal esophageal cell DNA and RNA,
The catastrophe of FAM135B in esophageal squamous cell carcinoma cell line is detected with Sanger sequencing technologies；Then according to FAM135B
The primer row qRT-PCR amplifications of gene order design, compare esophageal squamous cell carcinoma cell line and immortalize normal esophageal cell
Middle FAM135B gene magnifications situation.Appropriate cell line is selected according to qRT-PCR results and suppresses FAM135B gene expressions, height respectively
Wild type and saltant type FAM135B are expressed, FAM135B genes are studied by growth curve, Clone formation, migration and Matrigel
Effect in esophageal squamous cell carcinoma occurrence and development.
The present invention will be further described in the examples below, but the invention is not limited in these embodiments.
Embodiment 1：The method for detecting FAM135B expression conditions
1. sample Total RNA extraction-Trizol methods extraction tissue, cell total rna
（1）Sample process（Bacterium, cell do not have to grinding）；
By 1cm in aluminium foil2The tissue samples of size or so are broken into pieces, are moved in the Eppendorf pipes for having added steel ball, are made
Use beveller（30 1/s, 8min）It is ground
Note：This step operation should operate under low temperature liquid nitrogen environment as far as possible, and bacterium, cell sample do not have to grinding.
（2）1ml Trizol are added in Eppendorf pipes after grinding, vibration mixes；
（3）Solution after mixing is moved in a new Eppendorf pipes, adds 200 μ l chloroforms, vibration mixes；
（4）Centrifugation：4 DEG C, 12000rpm, 15min；
（5）Centrifuged supernatant is moved in a new Eppendorf pipes, 500 μ l isopropanols is added, gently mixes, room temperature
Stand 15min；
（6）Centrifugation：4 DEG C, 12000rpm, 15min；
（7）Supernatant is outwelled, adds 1ml75% ethanol, vibration mixes；
（8）Centrifugation：4 DEG C, 7500rpm, 5min；
（9）Supernatant is outwelled, and ethanol volatilizees totally in super-clean bench；
（10）Add 40~60 μ l DEPC H2O, 65 DEG C of 5min hydrotropies；
（11）- 20 DEG C of freezen protectives.
2.Total RNA quality testings
（1）NanoDrop determines Total RNA concentration（2 μ l Total RNA loadings）
（2）1.5% denaturing formaldehyde agarose gel electrophoresis detects RNA mass
Total RNA 500ng
5×Loading Buffer 2μl
The μ l of DEPC H2O to 8~9
65 DEG C of denaturation 5min, ice bath 5min
EB (500 times of dilutions) 1 μ l
Cumulative volume is about 6~8 μ l
Denaturing formaldehyde Ago-Gel：0.45g agaroses are added in 1 × TBE of 30ml Buffer, are heated in micro-wave oven molten
Change, gently shaking makes agarose fully mix（Visually observe no graininess suspension）, 600 μ l are added when being cooled to 60 DEG C or so
Formaldehyde, mix, pour into the special gel makers of RNA（7.5×5.5cm）, it is stored at room temperature 30min or so and can be used.
Deposition condition：120~130V, 15~20min.
3.RNA reverse transcriptions：
（1）Reverse transcription reaction system
（2）Reverse transcription process：16 DEG C of 30min, 37 DEG C of 30min, 70 DEG C of 10min, 4 DEG C of for ever.
4.RNA RealTime PCR react
（1）RNA RealTime PCR reaction systems
（2）RNA RealTime PCR programs
（3）RNA RealTime PCR primers 1.5% are non denatured（It is not added with formaldehyde）Agarose gel electrophoresis detects
The μ l of RNA RealTime PCR primers 2~4
2×Loading Buffer 4μl
Cumulative volume is about 6~8 μ l
Non denatured Ago-Gel：1.2g agaroses are added in 1 × TBE of 80ml Buffer, heats and dissolves in micro-wave oven,
Gently shaking makes agarose fully mix（Visually observe no graininess suspension）, 2 μ l EB are added when being cooled to 60 DEG C or so
（Stoste）Mix, pour into gel maker（15×15cm）, it is stored at room temperature 30min or so and can be used.
Deposition condition：100V, 25~30min.
5. Data Collection and processing：Using U6RNA as internal standard, it is normalized.
Embodiment 2：Influence of the FAM135B gene alterations to patient with esophageal carcinoma prognosis
Analyzed by the genome of the patient with esophageal carcinoma to Chaoshan region, using in SPSS statistical softwares
Kaplan-Meier analysis methods find there is nonsynonymous mutation and amplification, i.e. missense mutation（missense）With DNA level
Amplification changes.FAM135B change and reduce the overall survival of patient, compared with FAM135B wild type patients, there is statistics
Upper significant difference, p<0.05（Referring to Fig. 1）.
Embodiment 3：FAM135B is in esophageal squamous cell carcinoma cell line and the expression of normal esophageal cell
FAM135B genes are measured in esophageal squamous cell carcinoma cell line and normal esophageal cell using real-time PCR method
In expression（Referring to Fig. 2）.Wherein, abscissa represents mankind's esophageal carcinoma cell line.Compared with normal cell system NE3,
Expression of the FAM135B genes in esophageal carcinoma cell line adds.
Embodiment 4：Lower the influence of FAM135B expression cell growths
In the present embodiment, it is that matched siRNA sequence is transferred to by FAM135B's and the method for routine by tradition
Sequences silences and the expression for suppressing it；Wherein, siFAM135B refers to the groups of cells that FAM135B expression is lowered with siRNA sequence,
Compared with siCtrl control groups, FAM135B expression probably reduces 6-8 times, wherein, the siRNA sequence used is：5 ,-
AGAGAUAAGUAUGGAUUAGACAGGA-3,.
Detection lowers FAM135B expression to the esophageal cancer cell in KYSE150 and COLO680N esophageal cancer cells system
The influence of growth.2000 cells are counted respectively to be inoculated on 96E-Plate, and the E-Plate of inoculating cell is put into RTCA-
Cultivated in MP instruments, instrument will count a cell number, the final growth curve for obtaining cell for automatic every 15 minutes.The reality
Test and show, the downward of FAM135B expressions significantly reduces the growth of above-mentioned esophageal cancer cell system（Referring to Fig. 3 A and 3B）.
Embodiment 5：Lower influence of the FAM135B expression to cell clonal formation
In cell clonal formation experiment, count 2000 cells and be inoculated on big ware, be placed in 5%CO2 constant incubators
In, 37 DEG C of cultures, cultivate 5 days, form macroscopic cell colony.Stopping culture, PBS is rinsed 2 times, and methanol fixes 10min,
With 0.5% violet staining 20min, flowing water is rinsed, is air-dried.Take a picture and count clone's number.Application of results t-test is united
Meter.P<0.05 is to have significant difference.
Should be test result indicates that lowering FAM135B expression significantly reduces KYSE150 and COLO680N esophageal cancer cells system
Colony counts（Fig. 4 A to 4D）.
Embodiment 6：Lower the influence of FAM135B expression cell migrations and invasion and attack
In cell invasion experiment, the matrigel of 2% concentration is prepared with serum free medium（Matrigel is melted in 4 DEG C
Solution, at the same by the culture medium of serum-free, EP pipe, pipette tips precooling treatment）；The 2% matrigel culture medium prepared is added
Transwell upper chamber（Each 100ul）, it is put into incubator and is incubated more than 1 hour afterwards；By the cell of pretreatment（Nature enemy 6
After hour）After being digested with pancreatin, cell is resuspended with serum free medium, piping and druming is uniform, cell count；Under Transwell
The culture medium 600-800ul containing 20% serum is added in room, the cell counted is added in upper chamber, and the volume of upper chamber is used
Serum free medium constant volume is in 100-200ul；Transwell plates are put into after being incubated 6-10 hours in incubator, taken out
Transwell plates, and the cell that upper chamber is not passed through above scrapes off；Upper chamber is put into 70% methanol and fixes about 20 minutes,
1xPBS is washed 5 minutes；0.25% violet staining 40 minutes, deionized water wash cell.Micro- sem observation is just being put to be perforated through
Cell, and taken pictures, count cell.
In Cell migration assay, after cell count, the culture medium containing 20% serum is added in Transwell lower room
600-800ul, by the cell counted add upper chamber in, and by the volume of upper chamber with serum free medium constant volume in 100-
200ul；Transwell plates are put into after being incubated 6-10 hours in incubator, take out Transwell plates, and will not have above upper chamber
The cell passed through scrapes off；Upper chamber is put into in 70% methanol fixed about 20 minutes, 1xPBS washings 5 minutes；0.25% crystal violet
Dyeing 40 minutes, deionized water wash cell.The cell that micro- sem observation is perforated through just is being put, and is being taken pictures, is counting cell.Knot
Fruit is counted with t-test.P<0.05 is to have significant difference.
Should be test result indicates that lowering FAM135B expression significantly reduces KYSE150 and COLO680N esophageal cancer cells system
Migration and invasion and attack（Fig. 5 A to 5D）.
Embodiment 7：The influence of height expression wild type and saltant type FAM135B cell growths
In the present embodiment, the plasmid realization of FAM135B wild types, the zero load of relative transfection are transfected with liposome method
Plasmid, it is more about 60-200 times that it ultimately results in the expression of the FAM135B in cell；Same FAM135B-mut is mutated
FAM135B a certain site（Point mutation）Plasmid afterwards, is transfected into cell with liposome method and realizes and allow FAM135B-mut height
Expression, it does not have differential expression relative to wild type.
The influence of high expression FAM135B cell growths is have detected in KYSE30 and KYSE180 esophageal cancer cells system.Its
In, FAM135B-wt-representative is transferred to the FAM135B plasmids of wild type；FAM135B-mut-- is represented and is transferred to wild type
FAM135B plasmids.Count 2000 cells to be inoculated on 96E-Plate, and the E-Plate of inoculating cell is put into RTCA-MP
Cultivated in instrument, instrument will count a cell number, the final growth curve for obtaining cell for automatic every 15 minutes.
The result shows that FAM135B high expression significantly promotes the growth of KYSE30 and KYSE180 esophageal cancer cells（Ginseng
See Fig. 6 A-6B）.
Embodiment 8：The influence of height expression wild type and saltant type FAM135B to cell clonal formation
In cell clonal formation experiment, count 2000 cells and be inoculated on big ware, be placed in 5%CO2 constant incubators
In, 37 DEG C of cultures, cultivate 5 days, form macroscopic cell colony.Stopping culture, PBS is rinsed 2 times, and methanol fixes 10min,
With 0.5% violet staining 20min, flowing water is rinsed, is air-dried.Take a picture and count clone's number.Application of results t-test is united
Meter.P<0.05 is to have significant difference.
The result shows that FAM135B high expression significantly increases clone's number of KYSE30 and KYSE180 esophageal cancer cells
（Referring to Fig. 7 A-7D）.
Embodiment 9：Height expression wild type and the influence of saltant type FAM135B cell migrations and invasion and attack
In cell invasion experiment, the matrigel of 2% concentration is prepared with serum free medium（Matrigel is melted in 4 DEG C
Solution, at the same by the culture medium of serum-free, EP pipe, pipette tips precooling treatment）；The 2% matrigel culture medium prepared is added
Transwell upper chamber（Each 100ul）, it is put into incubator and is incubated more than 1 hour afterwards；By the cell of pretreatment（Nature enemy 6
After hour）After being digested with pancreatin, cell is resuspended with serum free medium, piping and druming is uniform, cell count；Under Transwell
The culture medium 600-800ul containing 20% serum is added in room, the cell counted is added in upper chamber, and the volume of upper chamber is used
Serum free medium constant volume is in 100-200ul；Transwell plates are put into after being incubated 6-10 hours in incubator, taken out
Transwell plates, and the cell that upper chamber is not passed through above scrapes off；Upper chamber is put into 70% methanol and fixes about 20 minutes,
1xPBS is washed 5 minutes；0.25% violet staining 40 minutes, deionized water wash cell.Micro- sem observation is just being put to be perforated through
Cell, and taken pictures, count cell.
In Cell migration assay, after cell count, the culture medium containing 20% serum is added in Transwell lower room
600-800ul, by the cell counted add upper chamber in, and by the volume of upper chamber with serum free medium constant volume in 100-
200ul；Transwell plates are put into after being incubated 6-10 hours in incubator, take out Transwell plates, and will not have above upper chamber
The cell passed through scrapes off；Upper chamber is put into in 70% methanol fixed about 20 minutes, 1xPBS washings 5 minutes；0.25% crystal violet
Dyeing 40 minutes, deionized water wash cell.The cell that micro- sem observation is perforated through just is being put, and is being taken pictures, is counting cell.Knot
Fruit is counted with t-test.P<0.05 is to have significant difference.
The result show FAM135B high expression significantly increase KYSE30 and KYSE180 esophageal cancer cells migration and
Invasion and attack（Referring to Fig. 7 A-7D）.
Claims (9)
1.FAM135B inhibitor is used to prepare the purposes in the medicine for the treatment of and/or prevention esophageal squamous cell carcinoma.
2. purposes according to claim 1, wherein the FAM135B inhibitor is specifically to combine and suppress FAM135B
The antibody of protein.
3. purposes according to claim 1, wherein described FAM135B inhibitor is Nucleic acid inhibitors.
4. purposes according to claim 3, wherein described Nucleic acid inhibitors specifically combine coding FAM135B, and press down
The polynucleotides of FAM135B processed transcription or translation.
5. purposes according to claim 3, wherein the Nucleic acid inhibitors are selected from ribozyme, antisense molecule, oligonucleotides suppression
Preparation, fit, microRNA or siRNA.
6. one or more primers for specifically identifying FAM135B genome mutations and/or probe preparing for diagnosing and/or
Purposes in the reagent of prognosis esophageal squamous cell carcinoma.
7. purposes according to claim 6, wherein miscellaneous by the sequencing of the generation of high flux two, exon trapping and Comparative genomic strategy
The method identification FAM135B genome mutations of friendship technology.
8. according to the purposes described in claim any one of 1-7, wherein the esophageal squamous cell carcinoma refers to the food in mammal
Pipe squamous cell carcinoma.
9. purposes according to claim 8, wherein the mammal is people.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410068807.9A CN104873973B (en) | 2014-02-27 | 2014-02-27 | Purposes of the FAM135B inhibitor in treating and/or preventing esophageal squamous cell carcinoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410068807.9A CN104873973B (en) | 2014-02-27 | 2014-02-27 | Purposes of the FAM135B inhibitor in treating and/or preventing esophageal squamous cell carcinoma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104873973A CN104873973A (en) | 2015-09-02 |
| CN104873973B true CN104873973B (en) | 2017-11-10 |
Family
ID=53941823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410068807.9A Active CN104873973B (en) | 2014-02-27 | 2014-02-27 | Purposes of the FAM135B inhibitor in treating and/or preventing esophageal squamous cell carcinoma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104873973B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055360B (en) * | 2018-09-14 | 2022-10-11 | 皖南医学院 | Method for extracting anopheles total RNA |
| CN114075562A (en) * | 2021-03-10 | 2022-02-22 | 北京肿瘤医院(北京大学肿瘤医院) | MAGE-C3 inhibitor and application thereof in preparation of drugs for treating and/or preventing esophageal squamous cell carcinoma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1868536A (en) * | 2006-07-04 | 2006-11-29 | 中国医学科学院肿瘤研究所 | Use of cell cycle regulation protein Cyclin B1 for preparing medicine for anti-human esophagus cancer invasion and transference |
| CN101074254A (en) * | 2007-03-05 | 2007-11-21 | 中国医学科学院肿瘤研究所 | Sequence against human esophagus cancer invasion and transfer |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012031008A2 (en) * | 2010-08-31 | 2012-03-08 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
-
2014
- 2014-02-27 CN CN201410068807.9A patent/CN104873973B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1868536A (en) * | 2006-07-04 | 2006-11-29 | 中国医学科学院肿瘤研究所 | Use of cell cycle regulation protein Cyclin B1 for preparing medicine for anti-human esophagus cancer invasion and transference |
| CN101074254A (en) * | 2007-03-05 | 2007-11-21 | 中国医学科学院肿瘤研究所 | Sequence against human esophagus cancer invasion and transfer |
Non-Patent Citations (1)
| Title |
|---|
| Novel human genetic variants associated with extrapulmonary tuberculosis: a pilot genome wide association study;Noffisat O Oki et al;《BMC Research Notes》;20110131;第4卷(第28期);http://www.biomedcentral.com/1756-0500/4/28 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104873973A (en) | 2015-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6032616B2 (en) | KIF5B gene and RET gene fusion gene and method for determining the effectiveness of cancer treatment targeting the fusion gene | |
| Wu et al. | MiR-19a is correlated with prognosis and apoptosis of laryngeal squamous cell carcinoma by regulating TIMP-2 expression | |
| CN111778338B (en) | Application of circular RNA biomarker | |
| Okada et al. | Oncogenic role of NALP7 in testicular seminomas | |
| KR102138131B1 (en) | Animal model of brain tumor and manufacturing method of animal model | |
| Gong et al. | LncRNA HAND2‐AS1 represses cervical cancer progression by interaction with transcription factor E2F4 at the promoter of C16orf74 | |
| CN109797151B (en) | Application of Circ-CDH1 inhibitor | |
| CN105980576A (en) | Method For The Prognosis And Treatment Of Cancer Metastasis | |
| Zhang et al. | CircRNA_0000502 promotes hepatocellular carcinoma metastasis and inhibits apoptosis through targeting microRNA-124 | |
| JP2023123748A (en) | Methods for diagnosing and treating metastatic cancer | |
| Ju et al. | A novel intronic circular RNA, circGNG7, inhibits head and neck squamous cell carcinoma progression by blocking the phosphorylation of heat shock protein 27 at Ser78 and Ser82 | |
| CN109266743B (en) | A kind of cancer markers and application thereof | |
| CN108841955B (en) | Application of C22orf41 as pancreatic cancer tumor marker | |
| CN104873973B (en) | Purposes of the FAM135B inhibitor in treating and/or preventing esophageal squamous cell carcinoma | |
| Lee et al. | Mutations in K-ras and epidermal growth factor receptor expression in Korean patients with stages III and IV colorectal cancer | |
| CN106676183A (en) | ZFHX4 as biomarker for prognosis of esophagus cancer | |
| Zhang et al. | Oncogenic Neuregulin 1 gene (NRG1) fusions in cancer: A potential new therapeutic opportunities | |
| CN110358834B (en) | Application of lncRNA, kit and medicine | |
| CN104873984B (en) | Purposes of the microRNA 548k inhibitor in treating and/or preventing esophageal squamous cell carcinoma | |
| CN116103403B (en) | Biomarker for diagnosis and prognosis of ovarian cancer and application thereof | |
| CN106729756B (en) | Application of biomarker as target in diagnosis and treatment of lung adenocarcinoma | |
| TW201204393A (en) | Diagnostic agent and therapeutic agent of cancer | |
| CN106755373B (en) | Application of LMOD3 in preparation of oral squamous cell carcinoma diagnosis and treatment preparation | |
| CN110577952B (en) | Application of siRNA interfering long non-coding RNA in preparation of medicine for treating breast cancer | |
| CN111172161B (en) | Long non-coding RNA and application thereof in diagnosis/treatment of preeclampsia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |