CN105950746A - RASL12 gene serving as diagnosis and treatment target of lung squamous cell carcinoma metastasis - Google Patents
RASL12 gene serving as diagnosis and treatment target of lung squamous cell carcinoma metastasis Download PDFInfo
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Abstract
The invention discloses an RASL12 gene and an expression product thereof which can serve as molecular markers of diagnosis and treatment on lung squamous cell carcinoma metastasis. Whether a subject suffers from lung squamous cell carcinoma metastasis or not can be judged or whether the subject has the risk of suffering from lung squamous cell carcinoma or not can be diagnosed by detecting the content of the RASL12 gene and the expression product thereof in lung tissue of the subject. According to the RASL12 gene and the expression product thereof which can serve as the molecular markers of diagnosis and treatment on lung squamous cell carcinoma, it is found that RASL12 gene expression can inhibit adhesion, metastasis and invasion of lung squamous carcinoma cells by researching lung squamous carcinoma cells subjected to in vitro culture, and it is shown through research results that the RASL12 gene is a potential drug target for treating lung squamous cell carcinoma metastasis.
Description
Technical field
The present invention relates to biological technical field, the diagnosis shifted at lung squamous cancer more particularly to RASL12 gene,
Purposes in treatment.
Background technology
Tumor is one of the most topmost current cause of death, and the tumor patient of more than 90% dies from tumor
Transfer.Breast carcinoma, colorectal cancer, cervical cancer, oropharynx cancer and the esophageal carcinoma of 1/4 of about 1/3, pulmonary carcinoma,
When cancer of pancreas, wing skin cancer and gastric cancer corrective surgery, lymph node carcinoma occurs, thus affected the treatment of patient
And prognosis.And increasing the weight of along with urban air pollution, pulmonary carcinoma oneself be the important lethal tumor in developed country and city
One of.Up to the present, the treatment to extensively transfer or the cancer patient shifting late period is still in progress without obvious.
Thus, inquire into neoplasm metastasis correlation molecule mechanism, screen neoplasm metastasis Research of predicting markers, for selecting suppression transfer
Therapy target provides reliable pathology according to becoming the huge challenge that cancer pathology researcher faces.
Lung cancer pathology typing mainly includes squamous cell carcinoma, adenocarcinoma, small cell carcinoma and large cell carcinoma four kinds, and
It is major histological type at China's squamous cell carcinoma.Although in the current generation to lung squamous cancer, evolution
Hereditism changes oneself to be had disclosed, and early diagnosis, early treatment be there has also been certain research, but from clinic
From the point of view of data, neoplasm metastasis remains the major causes of death of squamous cell lung carcinoma, thus discloses lung cancer metastasis phase
Correlation gene, is predicted lung cancer metastasis and selects rational therapeutic scheme to be beneficial to improve the pre-of patients with lung cancer
Afterwards with the life span improving patient.
The transfer of tumor refers to that malignant cell departs from primary tumor, arrives secondary tissue by various branch modes
Or organ is continued propagation growth, the overall process of the secondary tumor of formation and primary tumor same nature.Tumor
Invasion and attack mainly include following basic pathology process with shifting: primary tumor growth in early days, Tumor angiogenesis, tumor
Cell detachment also invades substrate, tumor cell entrance vascular system, the formation of tumor bolt, the life of secondary tissue structures locating
Long and metastasis continues diffusion.Additionally transfer is the process of tumor cell height Immune Clone Selection, in primary tumo(u)r
There is different heterogeneous cell subpopulations, oncocyte invasive ability during tumor development of these different subgroups
Not consistent with transfer ability, and the most all of oncocyte has invasion and attack and the ability of transfer, wherein only has few
Number tumor cells have metastatic potential, oneself have experiment prove metastatic cancer cell in parental tumor cell group early
Exist, and primary tumo(u)r gene variation determines tendency or the potential of its transfer.
Neoplasm metastasis is the process that an extremely complex polygenes participates in, polygenes regulates and controls, and relates to a series of
The gene structure relevant to neoplasm metastasis and dysfunction, be in particular in the activation of some oncogenes, and some
Antioncogene may be suppressed, or the disappearance of some antioncogenes, inactivation etc., and these hereditism change
Result ultimately results in the transfer of tumor.Identify that the essence of these gene pairss announcement neoplasm metastasis is significant,
And there is clinical value widely, capture the transfer of human tumor will be in tumor research history one huge
Milestone.
Summary of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide one and can be used for lung squamous cancer transfer early
The molecular marker of phase diagnosis.Use gene marker come Diagnosis of pulmonary scale cancer transfer have promptness, specificity and
Susceptiveness, so that patient just can know disease risks in early days in disease, for risk height, takes corresponding
Prevention and remedy measures.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the product of detection RASL12 gene expression at the instrument preparing the transfer of Diagnosis of pulmonary scale cancer
In application.
Further, the product of described detection RASL12 gene expression includes detecting RASL12 gene mRNA
The product of level and/or the product of detection RASL12 protein level.
Further, the product of described detection RASL12 gene expression includes: by RT-PCR, real-time quantitative
The expression of PCR, immune detection, in situ hybridization or chip detection RASL12 gene and expression product thereof with
The product of Diagnosis of pulmonary scale cancer transfer.
Further, the product of described RT-PCR Diagnosis of pulmonary scale cancer transfer at least includes a pair specific amplified
The primer of RASL12 gene；The product of described real-time quantitative PCR Diagnosis of pulmonary scale cancer transfer at least includes a pair
The primer of specific amplified RASL12 gene；The product of described immune detection Diagnosis of pulmonary scale cancer transfer includes: with
The antibody that RASL12 protein-specific combines；The product of described in situ hybridization Diagnosis of pulmonary scale cancer transfer includes:
Probe with the nucleic acid array hybridizing of RASL12 gene；The product of described chip Diagnosis of pulmonary scale cancer transfer includes:
Protein chip and gene chip；Wherein, protein chip includes the antibody being combined with RASL12 protein-specific,
Gene chip includes the probe of the nucleic acid array hybridizing with RASL12 gene.
A pair specific amplified that the product of described real-time quantitative PCR Diagnosis of pulmonary scale cancer transfer at least includes
The primer of RASL12 gene is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of described detection RASL12 gene expression can be the reagent, also of detection RASL12 gene expression
Can be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be the high flux using described reagent
Order-checking platform.
The instrument of described Diagnosis of pulmonary scale cancer transfer includes but not limited to that chip, test kit, reagent paper or high pass measure
Sequence platform；High-flux sequence platform is the instrument of a kind of special Diagnosis of pulmonary scale cancer transfer, along with high-flux sequence
The development of technology, will become the structure of the gene expression profile of a people and work the most easily.By contrast disease
Patient and the gene expression profile of normal population, the exception easily analyzing which gene is relevant to disease.Therefore,
In high-flux sequence, know that the exception of RASL12 gene is relevant to lung squamous cancer transfer fall within RASL12 gene
Purposes, equally within protection scope of the present invention.
Present invention also offers the instrument of a kind of Diagnosis of pulmonary scale cancer transfer, described instrument includes detecting RASL12 base
Because of the reagent expressed；Described reagent includes primer and/or probe, the detection detecting RASL12 gene mRNA
The antibody of RASL12 albumen.
Described instrument includes but not limited to chip, test kit, reagent paper or high-flux sequence platform.
Wherein, described chip includes gene chip, protein chip；Described gene chip include solid phase carrier with
And it being fixed on the oligonucleotide probe of solid phase carrier, described oligonucleotide probe includes for detecting RASL12 base
The oligonucleotide probe for RASL12 gene because of transcriptional level；Described protein chip includes solid phase carrier
And it is fixed on the specific antibody of the RASL12 albumen of solid phase carrier；Described gene chip can be used for detection bag
Include the expression at interior multiple genes (such as, relevant to lung squamous cancer transfer multiple genes) of the RASL12 gene
Level.Multiple protein that described protein chip can be used for detecting including RASL12 albumen (such as with
Multiple protein that lung squamous cancer transfer is relevant) expression.By by multiple and lung squamous cancer transfer marks
Detect simultaneously, be greatly improved the accuracy rate of lung squamous cancer transfer diagnosis.
Wherein, described test kit includes gene detecting kit and protein immunization detection kit；Described gene is examined
Test agent box includes the reagent for detecting RASL12 gene transcription level；Described protein immunization detection kit
Specific antibody including RASL12 albumen.Further, described reagent includes using RT-PCR, real-time quantitative
Needed for during PCR, immune detection, in situ hybridization or chip method detection RASL12 gene expression dose
Reagent.Preferably, described reagent includes the primer for RASL12 gene and/or probe.According to RASL12
The nucleotide sequence information of gene easily design may be used for detect RASL12 gene expression dose primer and
Probe.
Described reagent paper includes the reagent detecting RASL12 gene expression.
Described high-flux sequence platform includes the reagent detecting RASL12 gene expression.
Can be that DNA, RNA, DNA-RNA are embedding with the probe of the nucleic acid array hybridizing of RASL12 gene
Zoarium, PNA or other derivant.The length of described probe does not limit, if complete specific hybrid,
Specific binding with purpose nucleotide sequence, any length can.The length of described probe can be as short as 25,
20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,
300 base pairs or longer, the most whole gene.Owing to different probe length is special to hybridization efficiency, signal
The opposite sex has different impacts, the length of described probe to be typically at least 14 base pairs, the longest is usually no more than
30 base pairs, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe
Self-complementary sequences is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
Further, the specific antibody of described RASL12 albumen includes monoclonal antibody, polyclonal antibody.Institute
The specific antibody stating RASL12 albumen includes complete antibody molecule, any fragment of antibody or modifies (example
Such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described fragment can retain and RASL12
The binding ability of albumen.Well known to a person skilled in the art when the preparation of the antibody of protein level,
And the present invention can use any method to prepare described antibody.
In specific embodiments of the present invention, the primer of described detection RASL12 gene mRNA includes SEQ
Primer pair shown in ID NO.3 and SEQ ID NO.4.
Present invention also offers the accelerator of RASL12 gene and/or its expression product at preparation treatment lung squamous cancer
Application in the medicine of transfer.
Described accelerator includes the reagent promoting RASL12 gene expression and promotes RASL12 gene expression product
Reagent；The reagent of described promotion RASL12 gene expression includes promoting the reagent of genetic transcription, promoting gene
The reagent of translation, the reagent of promotion RASL12 protein content；The examination of described promotion RASL12 gene expression product
Agent includes promoting the reagent of RASL12 gene expression product stability, promoting RASL12 gene expression product activity
Reagent, promote RASL12 gene expression product function reagent.
Specifically, the reagent of described promotion RASL12 gene expression includes: reagent containing RASL12 gene,
Carry the carrier of RASL12 gene or reagent that host cell is formed, reagent containing RASL12 protein.
On the one hand the accelerator of the present invention may be used for supplementing disappearance or the deficiency of endogenic RASL12 albumen,
By improving the expression of RASL12 albumen, thus the lung squamous cancer transfer that treatment causes because of RASL12 hypoproteinosis.
On the other hand may be used for promoting activity or the function of RASL12 albumen, thus treat lung squamous cancer transfer.
The carrier carrying gene of the present invention is various carrier known in the art, such as commercially available carrier, includes
Plasmid, cosmid, phage, virus etc..
In the present invention, term " host cell " includes prokaryotic cell and eukaryotic cell.Conventional prokaryotic hosts is thin
The example of born of the same parents includes escherichia coli, bacillus subtilis etc..Conventional eukaryotic host cell includes yeast cells, insecticide
Cell and mammalian cell.It is preferred that this host cell is eukaryotic cell, as thin in Chinese hamster ovary celI, COS
Born of the same parents etc..
Present invention also offers a kind of pharmaceutical composition for treating lung squamous cancer transfer, described pharmaceutical composition bag
Include the accelerator of RASL12 gene recited above and/or its expression product.
Further, the medicine of the present invention also includes pharmaceutically acceptable carrier, carrier, this kind of carrier include (but
It is not limited to): diluent, excipient such as water etc., filler such as starch, sucrose etc.；Binding agent such as cellulose
Derivant, alginate, gelatin and polyvinylpyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as agar, carbonic acid
Calcium and sodium bicarbonate；Absorption enhancer quaternary ammonium compound；Surfactant such as hexadecanol；Absorption carrier is as high
Ridge soil and soap clay；Lubricant such as Pulvis Talci, calcium stearate and magnesium, Polyethylene Glycol etc..
The medicine of the present invention import tissue or cell mode can by be divided into external or internal in the way of.External
Mode includes importing in cell by the medicine containing RASL12 gene or the medicine containing RASL12 protein,
Again by cell transplantation or feed back to internal.Internal mode include directly by the medicine containing RASL12 gene or
In infusion of medicine in-vivo tissue containing RASL12 protein.
The medicine of the present invention also can be with the drug combination of other treatment lung squamous cancer transfer, and multi-medicament is used in combination can
Significantly to mention the success rate for the treatment of.
In the context of the present invention, " RASL12 gene " includes RASL12 gene and RASL12 base
The polynucleotide of any function equivalent of cause.RASL12 gene includes and current international public nucleic acid sequence
According to RASL12 gene (NC_000015.10) DNA sequence in the GeneBank of storehouse, there is more than 70% homology,
And coding identical function protein DNA sequence.
Preferably, the coded sequence of RASL12 gene includes any DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) the DNA sequence hybridization that limits and coding identical function protein
DNA sequence；
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% with
Source property, and coding identical function protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described RASL12 gene is SEQ ID NO.1
Shown DNA sequence.
In the context of the present invention, RASL12 gene expression product include RASL12 albumen and
The partial peptide of RASL12 albumen.The partial peptide of described RASL12 albumen contains the merit relevant to lung squamous cancer transfer
Can territory.
" RASL12 albumen " includes any function equivalent of RASL12 albumen and RASL12 albumen.
Described function equivalent includes RASL12 albumen conservative variation's protein or its active fragment, or its activity
Derivant, allelic variant, natural mutation, induced mutants, can be with under high or low stringent condition
Protein coded by the DNA of the DNA hybridization of RASL12.
Preferably, RASL12 albumen is the protein with following amino acid sequences:
(1) protein being made up of the aminoacid sequence shown in SEQ ID NO.2 in sequence table；
(2) aminoacid sequence shown in SEQ ID NO.2 is passed through the replacement of one or several amino acid residue
And/or disappearance and/or add and with the aminoacid sequence shown in SEQ ID NO.2 have identical function by
The protein that aminoacid sequence shown in SEQ ID NO.2 is derivative.Replace, lack or add is amino acid whose
Number is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.
(3) with the aminoacid sequence shown in SEQ ID NO.2, there is at least 80% homology and (be also called sequence
Homogeneity), it is highly preferred that same with the aminoacid sequence at least about 90% to 95% shown in SEQ ID NO.2
Source property, is often the polypeptide of the aminoacid sequence composition of 96%, 97%, 98%, 99% homology.
In specific embodiments of the present invention, described RASL12 albumen is to have shown in SEQ ID NO.2
The protein of aminoacid sequence.
It is known that, conventionally, in a protein, one or more amino acid whose modifications do not interfere with protein
Function.Those skilled in the art can approve change single amino acids or the aminoacid of little percentage ratio or to aminoacid sequence
Adding individually, lacking, insert, replace of row is conservative modification, and wherein the change generation of protein has similar
The protein of function.It is well known in the art for providing intimate amino acid whose Conservative substitution tables.
It is RASL12 egg by adding the example of the protein of an aminoacid or multiple Modification of amino acid residues
White fusion protein.Peptide or protein with RASL12 protein fusion is not limited, as long as gained
Fusion protein retains the biologic activity of RASL12 albumen.
It is non-conservative that the RASL12 albumen of the present invention also includes the aminoacid sequence shown in SEQ ID NO.2
Modify, as long as the protein through modifying remains able to retain the biologic activity of RASL12 albumen.?
In this type of modifying protein, the amino acid number of sudden change is typically 10 or less, such as 6 or less,
Such as 3 or less.
In the context of the present invention, " transfer of Diagnosis of pulmonary scale cancer " both include judging that experimenter has suffered from
Lung squamous cancer shifts, also includes judging whether experimenter exists the risk suffering from lung squamous cancer transfer.
In the context of the present invention, " treatment lung squamous cancer transfer " divides from the state change of disease, can wrap
Include the healing completely of the alleviation of disease, disease.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that RASL12 gene expression is relevant to lung squamous cancer transfer, by detection subject group
Knit the expression of middle RASL12, it can be determined that whether experimenter suffers from lung squamous cancer transfer or judge that experimenter is
No existence suffers from the risk of lung squamous cancer transfer, thus instructs clinicist provide prevention scheme to experimenter or control
Treatment scheme.
Present invention finds a kind of new molecular marked compound-RASL12 gene, compare traditional detection means,
Gene diagnosis more in time, more special, sensitiveer, it is possible to realize lung squamous cancer transfer early diagnosis.
Accompanying drawing explanation
Fig. 1 shows the expression utilizing genechip detection RASL12 gene in lung squamous cell carcinoma cancers；
Fig. 2 show utilize Western blot detect RASL12 albumen expression in lung squamous cell carcinoma cancers；
Fig. 3 shows the process LAN situation utilizing QPCR to detect RASL12 gene on transcriptional level；
Fig. 4 shows the process LAN situation utilizing Western blot detection RASL12 albumen.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are only used for
The bright present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment is logical
Often according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring HarborLaboratory Press, 1989) condition described in, or according to the bar proposed by manufacturer
Part.
The differential expression of embodiment 1RASL12 gene
1, sample acquisition: 40 example lung squamous cell carcinoma cancers (include that 20 examples have transfer sample and 20 examples without transfer sample)
Above-mentioned sample is the excision specimen of Lung Squamous Carcinoma Patients.The acquirement of above-mentioned all specimen is all entrusted by organizational ethics
The agreement of member's meeting.The clinical data of tissue samples includes: sex, age, tumor size, pathological grading
(Edmonson), whether shift, whether recur.
2, the acquisition of lung squamous cancer transfer tissue RNA
Use Trizol one-step method to extract lung squamous cancer transfer total tissue RNA, read by Nanodrop ND-1000
Take the absorbance (A) at 260nm and 280nm and measure the purity of RNA solution.Through 1% denaturing formaldehyde fine jade
Sepharose electrophoresis, observes under ultraviolet transmission light, the integrity of detection RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP labelling, the cRNAs after fluorescent labeling uses RNEASY
Mini Kit purification, enters the cRNAs that labelling is good with the RNA Fragmentation Reagents of Amhion
Row fragmentation processes.Use people's full genome chip of expression spectrum (4x44K gene) of Agilent company of the U.S.,
In chip hybridization stove, 65 DEG C of hybridization 17h, then eluting, dyeing, finally uses Agilent DNA
MicroarrayScanner scanner scanning.
4, chip data processes and analyzes
Chip after hybridization, after chip scanner reads data point, imports data to analyze software, for two groups
The natural logrithm absolute value of the ratio gene more than 2.0 or less than 0.5 is as difference expression gene.
5, statistical procedures
Using SPSS 13.0 statistical software to carry out data analysis, group difference compares employing one factor analysis of variance
Method, P < 0.05 difference has significant.
6, result
As it is shown in figure 1, compared with the lung squamous cell carcinoma cancers that transfer does not occurs, there is the lung squamous cancer group of transfer in result
The mRNA level in-site knitting middle RASL12 gene significantly reduces, and difference has statistical significance (P < 0.05).
The differential expression of embodiment 2RASL12 albumen
1, tissue total protein is extracted
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
2, Western blot detection
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch,
Two anti-hatch, develop the color.
3, statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by purpose the gray value of protein band
The gray value of informal voucher band is normalized.Result data is all to represent in the way of mean+SD,
Using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection, it is believed that
When P < has statistical significance when 0.05.
4, result
As in figure 2 it is shown, compared with the lung squamous cell carcinoma cancers that transfer does not occurs, there is the lung squamous cancer group of transfer in result
Knitting middle RASL12 protein content to reduce, difference has statistical significance (P < 0.05).
Embodiment 3RASL12 gene expression plasmid builds
1, the structure of RASL12 expression vector
Coded sequence (as shown in SEQ ID NO.1) design amplimer according to RASL12 gene.From
Become the RASL12 base of amplification total length in the cDNA library (clontech company, article No.: 638831) of Human fetal spleen
The coded sequence of cause, inserts above-mentioned cDNA sequence in eukaryotic expression vector pcDNA3.1, and connection obtains
The recombinant vector pcDNA3.1-RASL12 obtained is for subsequent experimental.
2, cell is cultivated
Lung Squamous Carcinoma Cells strain NCI-H520 is cultivated in DMEM culture medium and 10% hyclone, by people
Pulmonary epithelial cells strain BEAS-2B cultivates in BEGM culture fluid and 10% hyclone, be placed in 37 DEG C,
5%CO2In incubator.
Take NCI-H520 cell by 1 × 104/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO2
In incubator, cell cultivates 24h, and transfection is according to lipofectamine 2000 (purchased from Invitrogen company)
Description transfection, experiment is divided into matched group (transfection pcDNA3.1) and experimental group (to transfect
PcDNA3.1-RASL12), the working concentration of transfected plasmids is 0.5 μ g/ml.
3, the effect of QPCR experiment detection plasmid transfection is utilized.
3.1 extract cell total rna utilizes conventional method to operate.
3.2 reverse transcription
The Reverse Transcriptase kit utilizing TAKARA company carries out the reverse transcription of RNA.
3.3QPCR
(1) design of primers
According to the coded sequence design QPCR amplification of RASL12 gene and GAPDH gene in Genbank
Primer, is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
RASL12 gene:
Forward primer is 5 '-TACAGGCAAGTCACCAAG-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-AAGTCCAGACAGGCAGAG-3 ' (SEQ ID NO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 10s, 60 DEG C of 40s) * 40 circulations.With
SYBR Green, as fluorescent marker, reacts at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument,
Determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
3.4 statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD
Representing, using SPSS13.0 statistical software to carry out statistical analysis, the difference between different groups uses t inspection
Test, it is believed that when P < has statistical significance when 0.05.
4, Western detection
Concrete steps are with embodiment 2.
5, result
As it is shown on figure 3, compared with the cell of transfection pcDNA3.1 empty carrier, transfect pcDNA3.1-RASL12
Cell in the mRNA level in-site of RASL12 significantly raise, difference has statistical significance (P < 0.05)；
As shown in Figure 4, compared with the cell of transfection pcDNA3.1 empty carrier, transfection pcDNA3.1-RASL12's
In cell, the protein level of RASL12 significantly raises, and difference has statistical significance (P < 0.05).
Embodiment 4 is studied RASL12 and is expressed the impact on Lung Squamous Carcinoma Cells adhesive capacity
1, cell is cultivated with transfection with embodiment 3.
2, cell adhesion experiments
0.25% trypsinization is used to become cell suspension, with 5 × 10 in the NCI-H520 cell of transfection 48h4Individual
/ ml is inoculated in 96 porocyte culture plates, every hole 0.1ml, and after 60min, 37 DEG C of PBS wash away nonadherent
Cell, mtt assay surveys each hole 490nm wavelength light absorption value.Represent by absorbance value size and adhere to living cells
Relative populations.
3, result
PcDNA3.1-RASL12 group relative optical density number is 0.435 ± 0.047, pcDNA3.1 empty carrier group phase
It is 1.382 ± 0.061 to optical density value.Compared with pcDNA3.1 empty carrier group, pcDNA3.1-RASL12
Group absorbance value is remarkably decreased (P < 0.05).The above-mentioned RASL12 of test result indicate that process LAN is unfavorable for lung squama
Cancer cell adhesion.
Embodiment 5 is studied RASL12 and is expressed Lung Squamous Carcinoma Cells migration, the impact of invasive ability
1, cell is cultivated with transfection with embodiment 3.
2, experiment is migrated
The NCI-H520 cell of transfection 48h uses trypsinization and counts, and takes 105Individual cell is placed in 1.5mL EP
Guan Zhong, adds 200 μ L serum-free medium re-suspended cells, adds in transwell cell, and bottom chamber adds
Enter the DMEM culture medium of 10% hyclone, put into 37 DEG C, 5%CO2Incubator cultivates 24h.Take
Transwell cell, with cotton swab erasing inside cell, and wash off gently with PBS the inside remaining cell.Gu
Take 8 random field under microscope after fixed dyeing to count.
3, Matrigel
The NCI-H520 cell of transfection 48h uses trypsinization and counts, and takes 105Individual cell is placed in 1.5mL
In EP pipe, add 200 μ L serum-free medium re-suspended cells, add the transwell through paving matrigel
In cell, bottom chamber adds the DMEM culture medium of 10%FBS, puts into 37 DEG C, 5%CO2Incubator
Cultivate 24h.Take transwell cell, with the cell inside cotton swab erasing, and in washing off gently with PBS
Face remaining cell.Take 8 random field under microscope after fixing dyeing to count.
4, result
Migrating experiment: compared with pcDNA3.1 empty carrier group, pcDNA3.1-RASL12 group is through transwell
The Leukopenia of cell basement membrane about 56%.
Matrigel: compared with pcDNA3.1 empty carrier group, pcDNA3.1-RASL12 group is through having spread
Cross the Leukopenia 52% of the transwell cell basement membrane of matrigel.
Above-mentioned test result indicate that, RASL12 gene overexpression can significantly inhibit NCI-H520 cell migration,
Invasive ability.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It is right to it should be pointed out that,
For those of ordinary skill in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention
Carrying out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the product of detection RASL12 gene expression application in the instrument preparing the transfer of Diagnosis of pulmonary scale cancer.
Application the most according to claim 1, it is characterised in that described product includes: by RT-PCR,
Real-time quantitative PCR, immune detection, in situ hybridization chip or high-flux sequence detection of platform RASL12 gene
Express with the product of Diagnosis of pulmonary scale cancer transfer.
Application the most according to claim 2, it is characterised in that described RT-PCR Diagnosis of pulmonary scale cancer
The product of transfer at least includes the primer of a pair specific amplified RASL12 gene；Described real-time quantitative PCR
The product of Diagnosis of pulmonary scale cancer transfer at least includes the primer of a pair specific amplified RASL12 gene；Described immunity
The product of checkout and diagnosis lung squamous cancer transfer includes: the antibody being combined with RASL12 protein-specific；Described with former
The product of position hybridization Diagnosis of pulmonary scale cancer transfer includes: with the probe of the nucleic acid array hybridizing of RASL12 gene；Institute
State and include with the product of chip Diagnosis of pulmonary scale cancer transfer: protein chip and gene chip；Wherein, protein chip bag
Including the antibody being combined with RASL12 protein-specific, gene chip includes and the nucleotide sequence of RASL12 gene
The probe of hybridization.
Application the most according to claim 3, it is characterised in that described real-time quantitative PCR Diagnosis of pulmonary squama
The primer such as SEQ ID NO.3 of a pair specific amplified RASL12 gene that the product of cancerometastasis at least includes and
Shown in SEQ ID NO.4.
5. the instrument of a Diagnosis of pulmonary scale cancer transfer, it is characterised in that described instrument includes detecting RASL12
The reagent of gene expression；Described reagent includes primer and/or probe, the detection detecting RASL12 gene mRNA
The antibody of RASL12 albumen.
Instrument the most according to claim 5, it is characterised in that described detection RASL12 gene mRNA
Primer include the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4.
The accelerator of 7.RASL12 gene and/or its expression product is in the medicine of preparation treatment lung squamous cancer transfer
Application.
Application the most according to claim 7, it is characterised in that described promotion RASL12 gene expression
Reagent, the reagent of promotion RASL12 gene expression product stability, promotion RASL12 gene expression product activity
Reagent, promote RASL12 gene expression product function reagent.
Application the most according to claim 8, it is characterised in that promote the reagent of RASL12 gene expression
Reagent that the carrier including the reagent containing RASL12 gene, carrying RASL12 gene or host cell are formed,
Reagent containing RASL12 protein.
10. the pharmaceutical composition being used for treating lung squamous cancer transfer, it is characterised in that described pharmaceutical composition
Including the accelerator according to any one of 7-9.
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| CN103874706A (en) * | 2011-04-28 | 2014-06-18 | 贝勒医学院 | Recurrent chimeric RNAs enriched in human prostate cancer as biomarkers |
| CN104053788A (en) * | 2011-11-28 | 2014-09-17 | 加泰罗尼亚调查和高级研究机构 | Methods and kits for prognosis of colorectal cancer |
| CN105603117A (en) * | 2016-03-31 | 2016-05-25 | 北京泱深生物信息技术有限公司 | Application of miR-3613 as miRNA marker in distinguishing metastasis and non-metastasis of lung squamous carcinoma |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103874706A (en) * | 2011-04-28 | 2014-06-18 | 贝勒医学院 | Recurrent chimeric RNAs enriched in human prostate cancer as biomarkers |
| CN104053788A (en) * | 2011-11-28 | 2014-09-17 | 加泰罗尼亚调查和高级研究机构 | Methods and kits for prognosis of colorectal cancer |
| CN105603117A (en) * | 2016-03-31 | 2016-05-25 | 北京泱深生物信息技术有限公司 | Application of miR-3613 as miRNA marker in distinguishing metastasis and non-metastasis of lung squamous carcinoma |
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