CN109022584A - A kind of molecular marker of the cancer of the esophagus and application thereof - Google Patents
A kind of molecular marker of the cancer of the esophagus and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of molecular markers of cancer of the esophagus, are the expression product of FXR1 gene and/or FXR1 gene.The significantly high expression in human esophageal carcinoma of FXR1 gene can be used for esophagus cancer diagnosis as the molecular marker of the cancer of the esophagus；Highly expressed FXR1 gene and the poor prognosis of Patients With Carcinoma of Esophagus and the DISTANT METASTASES IN of the cancer of the esophagus are closely related, prompt FXR1 gene that can provide effective information as the prognostic marker of the cancer of the esophagus for the DISTANT METASTASES IN diagnosis of the cancer of the esophagus, prognosis evaluation, therapeutic effect monitoring.The invention discloses the expression inhibiting agent of FXR1 gene, including siRNA and/or shRNA, and in the expression inhibiting for FXR1 gene, interference effect is good, the application potential with clinical gene therapy.The invention discloses stablizing to strike the cell strain for subtracting FXR1 gene, it is the esophageal cancer cell of FXR1 gene stable low-expression, is advantageously implemented the further research to FXR1 gene function.
Description
Technical field
The present invention relates to oncomolecularbiology fields, and in particular to a kind of molecular marker of the cancer of the esophagus, purposes are used for
The primer sets of diagnosis of esophageal cancer, the expression inhibiting agent of FXR1 gene and stabilization strike the cell strain for subtracting FXR1 gene.
Background technique
The cancer of the esophagus (Esophageal carcinoma, EC) is one of most common malignant tumour of the mankind, and the whole world is annual
Ten thousand people die of the cancer of the esophagus about more than 40.China is one of high-incidence country and the highest country of the death rate of the cancer of the esophagus, oesophagus
Cancer death toll almost occupies the half of world's death from esophageal carcinoma total number of persons.It is shown according to the data of newest epidemiology of cancer,
China's male esophageal cancer disease incidence occupies the third position of all kinds of malignant tumours, and women occupies the 5th, and death rate men and women occupies the 4th
Position.Due to the difference of ethnic group difference between east and west and teiology, histology and the pathogenesis of Incidence of Esophageal Cancer etc., with Europe
The low hair area such as beauty is with Lower Thoracic Esophagus, the cancer of the esophagus difference that gland cancer is main pathological type, China's cancer of esophagi Neo-Confucianism class
Type is based on Middle Thoracic, squamous cell carcinoma, wherein esophageal squamous cell carcinoma (esophageal squamous cell
Carcinoma, ESCC) accounting is up to 90% or more.
Although current medical research mechanism of various countries is all in progress to the research of diagnosis, the treatment of the cancer of the esophagus, majority food
Cancer of the esophagus middle and advanced stage has been come into when pipe cancer patient assessment, the Best Times for the treatment of has often been had already passed by this time, has caused to suffer from
5 years survival rates of person are not high, and only 20%~40% or so.However according to the literature, 5 years of cancer of the esophagus Operation in early stage with
And 10 years survival rates can achieve 85.9% and 55.6%.Therefore, early discovery, early treatment are to the survival rate for improving patient with esophageal carcinoma
It is most important.
Currently, the main means of cancer of the esophagus early diagnosis include radiography, esophageal cast-off cell inspection, endoscopy, image
It learns and tumor markers inspection.In terms of radiography, x-ray canel barium meal contrast examination is clinically the most frequently used, most basic display oesophagus at present
The Imaging Method that carninomatosis becomes, can determine the upper and lower range of tumour, however the method is in early stage that cancer of the esophagus inspection is easily leaked
Examine phenomenon.Dragging in esophagus method detects cast-off cells, simple and practical, accuracy is high, is the head of China Esophageal Cancer area generaI investigation
Method is wanted, however this method patient is more painful, it is difficult to generally carry out.Endoscopy can observe directly cancerous swelling, more intuitively
It was found that Esophageal Mucosa changes, and energy biopsy tissue carries out pathologic finding, is the major diagnosis means of the nowadays cancer of the esophagus, however scope
Inspection is easy that patient is made to generate the adverse reactions such as foreign body sensation, gastric disorder causing nausea, and the review time is longer, and patient is not easily accepted by.In image
Aspect is learned, although iconography is widely used in recent years, such as CT, EUS, PET etc., these often have certain wound
Property and limitation, early-stage cancer diagnosis is also easy to exist and fails to pinpoint a disease in diagnosis phenomenon, and on the high side, this beats its application value greatly
Discount.The tumor-marker analyte detection of cancer is since its is non-invasive, the height of the rationality of prices and tumor-marker analyte detection is sensitive
Degree and high specific, the cancer of the esophagus early diagnosis and in terms of have important clinical value.
Thus, it is found that the relevant molecular marker of the new cancer of the esophagus improves food for the morning discovery of the cancer of the esophagus, early treatment
The survival rate of pipe cancer patient improves life in patients, is of great significance.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that providing a kind of diagnosis to the cancer of the esophagus and prognosis is relevant new
Molecular marker.
For this purpose, the invention provides the following technical scheme:
The present invention provides a kind of molecular marker of cancer of the esophagus, the molecular marker is FXR1 gene and/or FXR1
The expression product of gene.
Optionally, the molecular marker of the above-mentioned cancer of the esophagus, the expression product of the FXR1 gene include FXR1 mRNA
And/or FXR1 albumen.
Optionally, the molecular marker of the above-mentioned cancer of the esophagus, the cancer of the esophagus are esophageal squamous cell carcinoma.
The present invention provides the molecular markers of the above-mentioned cancer of the esophagus in following a1-a4Purposes at least one:
a1, the product of esophagus cancer diagnosis is used for as the diagnosis marker of the cancer of the esophagus, or preparation；
a2, the product of cancer of the esophagus prognosis evaluation is used for as the prognostic marker of the cancer of the esophagus, or preparation；
a3, product of the preparation for Curative Effect of Esophagus Carcinoma monitoring；
a4, the drug of the preparation treatment cancer of the esophagus.
Optionally, above-mentioned purposes, the esophagus cancer diagnosis include the diagnosis of cancer of the esophagus DISTANT METASTASES IN.
The present invention provides a kind of primer sets for esophagus cancer diagnosis, the primer sets include based on the above-mentioned cancer of the esophagus
The primer of molecular marker design.
Optionally, above-mentioned primer sets, the primer sets include following primer:
FXR1-F, nucleotide sequence is as shown in SEQ ID NO.1；
FXR1-R, nucleotide sequence is as shown in SEQ ID NO.2.
The present invention provides a kind of expression inhibiting agent of FXR1 gene, the expression inhibiting agent include siRNA and/or
shRNA。
Optionally, above-mentioned expression inhibiting agent, the nucleotide sequence of the siRNA such as SEQ ID NO.5-SEQ ID
Shown in NO.8 any sequence,
The nucleotide sequence of the shRNA is as shown in SEQ ID NO.9-SEQ ID NO.12 any sequence.
The present invention provides a kind of carrier, the carrier includes nucleotide sequence such as SEQ ID NO.9-SEQ ID NO.12
ShRNA shown in any sequence.
The present invention provides recombinant cells or pseudovirion comprising above-mentioned carrier.
The present invention provides above-mentioned expression inhibiting agent or above-mentioned carrier or above-mentioned recombinant cells or pseudovirus
Purposes of the particle in the drug of the preparation treatment cancer of the esophagus.
The cell strain for subtracting FXR1 gene is struck the present invention provides a kind of stabilization, the cell strain is using esophageal cancer cell as mesh
Cell is marked, is infected by pseudovirion, the stabilization of acquisition strikes the esophageal cancer cell for subtracting FXR1 gene.
The present invention provides it is a kind of construct above-mentioned stabilization and strike subtract the method for the cell strain of FXR1 gene, including following step
It is rapid:
(1) the shRNA segment of any shown sequence of nucleotide sequence such as SEQ ID NO.9-SEQ ID NO.12 is connected
To the viral vectors with antibiotic resistance, recombinant expression carrier is obtained；
(2) using recombinant expression carrier and viral packaging plasmid cotransfection packaging cell line, Transfected cells are cultivated, are collected
Culture medium supernatant obtains the virus liquid containing pseudovirion after concentration；
(3) esophageal cancer cell is infected using virus liquid, then carries out drug screening using antibiotic, obtained stable strike and subtract
The cell strain of FXR1 gene.
Optionally, above-mentioned construction method, the viral vectors are slow virus carrier pHBLV-U6-Scramble-
ZsGreen-Puro, the virus packaging plasmid is slow virus packaging plasmid, including pSPAX2 and pMD2G.The slow virus carries
Body has puromycin-resistant.
Optionally, above-mentioned construction method, the packaging cell line are 293T cell, and the esophageal cancer cell is
KYSE180 cell.
Unless otherwise specified, the term of claims of the present invention and specification has the meaning that
Product in claims of the present invention includes: miscellaneous by RT-PCR, real-time quantitative PCR, immune detection, original position
The product of the detection FXR1 gene expression such as friendship, chip or high-flux sequence；Wherein the product of RT-PCR and real-time quantitative PCR includes
Specific amplification FXR1 gene detects primer, the probe of FXR1mRNA level；The product of immune detection includes special with FXR1 albumen
The antibody that the opposite sex combines；The product of in situ hybridization includes the probe with the nucleic acid array hybridizing of FXR1 gene；Chip includes albumen
Chip and genetic chip, wherein protein chip includes the antibody in conjunction with FXR1 protein-specific, and genetic chip includes and FXR1
The probe of the nucleic acid array hybridizing of gene.
Drug in claims of the present invention includes: to inhibit FXR1 gene expression, inducing post-transcriptional gene silencing, inhibit
FXR1 protein active or degradation FXR1 gene, mRNA or compound, bioactive substance or the gene therapy medicament of albumen etc..
Wherein bioactive substance includes albumen, polypeptide, lipid etc..Gene therapy medicament include siRNA, miRNA, shRNA or
Carrier etc. including above-mentioned small molecule.
Technical solution of the present invention has the advantages that
1, the molecular marker of a kind of cancer of the esophagus provided by the invention, which is characterized in that the molecular marker is FXR1
The expression product of gene and/or FXR1 gene.
Fragile X chromosome related gene 1 (fragile X-related gene1, FXR1, Gene ID:8087) is brittleness
A member in X baryencephalia associated families, the FXR1 assignment of genes gene mapping are distributed mainly on nervous system and musculature in 3q26,
It plays an important role in musculature occurrence and development.Mouse and drosophila after knocking out FXR1 gene is because the defect of the muscle such as cardiac muscle exists
It is shortly dead quickly after birth.FXR1 albumen is related with most of ribosomes 60S subunit, has in nucleus and cytoplasm
Between the physiological function that shuttles.But effect of the FXR1 gene in human diseases at present also report it is less.
The present invention, in the expression quantity of human esophageal carcinoma, is significantly higher than in oesophagus normal tissue by research discovery FXR1 gene
In expression quantity, the expression for illustrating FXR1 gene and the correlation of the cancer of the esophagus are significant, FXR1 gene and/or FXR1 gene
Expression product can provide effective information as the molecular marker of the cancer of the esophagus for the diagnosis and targeted therapy of the cancer of the esophagus.Meanwhile
By the analysis of COX proportional hazard model and Kaplan-Meier tracing analysis, the FXR1 gene and oesophagus of high expression level are found
The poor prognosis of carninomatosis people and the transfer of the cancer of the esophagus are closely related, and development, the transfer of FXR1 gene and the cancer of the esophagus have important pass
System is to influence the total life span of patient with esophageal carcinoma and the independent factor without transfer life span, FXR1 gene and/or FXR1 gene
Molecular marker of the expression product as the cancer of the esophagus, can be used in the DISTANT METASTASES IN of diagnosis of esophageal cancer, be patient with esophageal carcinoma
The monitoring of prognosis evaluation and therapeutic effect provides effective information, and provides foundation for the selection of its individualized treatment, has weight
The clinical value wanted.
2, the molecular marker of the cancer of the esophagus provided by the invention, the expression product of FXR1 gene include FXR1 mRNA and/or
FXR1 albumen is examined by detecting FXR1 mRNA and/or FXR1 albumen for the early diagnosis of the cancer of the esophagus, the DISTANT METASTASES IN of the cancer of the esophagus
Disconnected, curative effect monitoring and prognosis evaluation provide important clinical information.
3, the molecular marker of the cancer of the esophagus provided by the invention, the cancer of the esophagus are specifically esophageal squamous cell carcinoma, FXR1 gene and/or
The expression product of FXR1 gene can as the molecular target of oesophagus squama cancer diagnosis and treatment, be esophageal squamous cell carcinoma clinical diagnosis,
Treatment provides important information.
4, the expression product of the molecular marker of the cancer of the esophagus provided by the invention, FXR1 gene and/or FXR1 gene participates in
The expression product of generation, development and the transfer of the cancer of the esophagus, FXR1 gene and/or FXR1 gene can as cancer diagnosis, transfer,
Molecule and prognostic marker are used to prepare relevant clinical application product, to realize early discovery, the early treatment of the cancer of the esophagus, and
Prognosis prediction is carried out to patient with esophageal carcinoma in time and the therapeutic effect of the cancer of the esophagus is monitored, improves the life of patient with esophageal carcinoma
Rate is deposited, life in patients is improved.In addition, FXR1 gene and its expression product can also be used as the therapy target of the cancer of the esophagus
In the drug of the preparation treatment cancer of the esophagus, the clinical treatment of the cancer of the esophagus is assisted, improves treatment of cancer effect.
5, provided by the present invention for the primer sets of diagnosis of esophageal cancer, draw including what is designed based on cancer of the esophagus molecular marker
Object, above-mentioned primer can be used in detecting the expression of FXR1 gene, provide reliably for the diagnosis, prognosis and treatment of the cancer of the esophagus
Information.
Using primers F XR1-F and FXR1-R, by RT-PCR technology, can be realized to the FXR1 in cancerous tissue, cell
MRNA level in-site detection, and the advantage for having specificity, accuracy high.
6, the expression inhibiting agent of FXR1 gene provided by the invention, including siRNA and/or shRNA, siRNA and/or
When shRNA inhibits for gene, interference effect is good, can effectively inhibit the expression of FXR1 gene, inhibit invading for tumour cell
It attacks and shifts.Wherein, shRNA is since undershooting-effect is low, stability is high in cell, before the application with important gene therapy
Scape.
7, stabilization provided by the invention strikes the cell strain for subtracting FXR1 gene, and the cape horn fever of shRNA segment is connected with by packing
Malicious particle-lentiviral particle, and esophageal cancer cell is infected using lentiviral particle, so that shRNA sequence is inserted into esophageal cancer cell
Genome in, obtain interference RNA sequence after transcription, interference RNA sequence inhibits FXR1 base by targeting degradation FXR1 mRNA
Because of expression, obtains stabilization and strike the cell strain for subtracting FXR1 gene.Using above-mentioned cell strain, can be realized to FXR1 gene function
Further research is conducive to the generation, the development process that disclose the cancer of the esophagus, provides more clinics for the immunotherapy targeted autoantibody of the cancer of the esophagus
Application message.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
FXR1 in the human esophageal carcinoma (Tumor) and Carcinoma side normal tissue (Normal) that Fig. 1 provides for the embodiment of the present invention 1
The testing result of gene expression dose；
Fig. 2A is that the embodiment of the present invention 2 provides the correlation analysis knot of FXR1 gene expression dose Yu patients overall survival's time
Fruit；
Fig. 2 B is that the embodiment of the present invention 2 provides correlation point of the FXR1 gene expression dose with patient without transfer life span
Analyse result；
Fig. 3 is that the stabilization that the embodiment of the present invention 3 provides strikes the fluorescence detection result for subtracting the cell strain of FXR1 gene；
Fig. 4 is that the stabilization that experimental example 1 of the present invention provides strikes the cell strain (K180-sh1) for subtracting FXR1 gene and control group is thin
The testing result of the mRNA expression of FXR1 gene in born of the same parents' strain (K180-shNC)；
Fig. 5 is that the stabilization that experimental example 1 of the present invention provides strikes the cell strain (K180-sh1) for subtracting FXR1 gene and control group is thin
The testing result of the protein expression level of FXR1 gene in born of the same parents' strain (K180-shNC)；
Fig. 6 is that the stabilization that experimental example 1 of the present invention provides strikes the cell strain (K180-sh1) and control cell for subtracting FXR1 gene
The cell migration testing result of strain (K180-shNC)；
Fig. 7 is that the stabilization that experimental example 1 of the present invention provides strikes the cell strain (K180-sh1) and control cell for subtracting FXR1 gene
The cell invasion testing result of strain (K180-shNC)；
Fig. 8 is that the stabilization that experimental example 1 of the present invention provides strikes the cell strain (K180-sh1) and control cell for subtracting FXR1 gene
The cell Proliferation testing result of strain (K180-shNC).
Specific embodiment
Illustrate embodiments of the present invention below by way of specific embodiment, unless otherwise stated, disclosed in this invention
Experimental method be all made of the art routine techniques, all primers synthesis and examining order are complete by Guangzhou Ying Jun company
At used reagent and raw material are available on the market in embodiment.The equal preservation of the cell related in following embodiments
In affiliated hospital of Southern Yangtze University.
Embodiment 1
The present embodiment provides FXR1 gene expressions in a kind of detection human esophageal carcinoma (Tumor) and cancer beside organism (Normal)
Horizontal method, specifically includes the following steps:
1, sample collection
The human esophageal carcinoma and oesophagus Carcinoma side normal tissue of 115 patient with esophageal carcinoma are collected, all tissue specimens come
Esophageal squamous cell carcinoma is diagnosed as derived from first outpatients, and through pathologist.It is normal by human esophageal carcinoma and the cancer of the esophagus
Tissue specimen is derived from Zhongshan Univ. Cancer Cure Center's tumour resources bank, and fresh specimens are quick-frozen in liquid nitrogen, are then stored in -80
It spends in refrigerator.
2, tissue RNA is extracted
(1) it takes tumor tissues and tumour to close on each 50mg of normal tissue, is cleaned respectively with PBS (pH 7.4), 1ml is added
Trizol is moved into 1.5ml EP pipe, is mixed, and 5min is stood；
(2) 12000rpm, 4 DEG C of centrifugation 10min；
(3) 200 μ l chloroforms are added, acutely rock 15s, stand 5min；
(4) 12000rpm, 4 DEG C of centrifugation 15min；Carefully upper strata aqueous phase is moved into new 1.5ml EP pipe, the bodies such as addition
Long-pending isopropanol is put at -20 DEG C 1 hour after mixing；
(5) 12000rpm, 4 DEG C of centrifugation 10min, carefully abandons supernatant；
(6) volumetric concentration that 1ml is added is that 75% ethyl alcohol (configuration of DEPC water) washing precipitating sinks RNA after ethyl alcohol is added
Bounce rinsing in shallow lake；
(7) 12000rpm, 4 DEG C of centrifugation 10min, pour out liquid, then remaining a small amount of of short duration centrifugation of liquid is inhaled with pipette tips
Out, it is careful not to inhale and abandons precipitating；
(8) room temperature is dried, and is dissolved with DEPC (pyrocarbonic acid diethyl ester) water of 30 μ l, is surveyed concentration.
2, cDNA is synthesized
Using the reverse transcription reagent box of TAKARA (Code No.RR047A), the RNA reverse transcription extracted in step 1 is synthesized
CDNA, specific as follows:
(1) genomic DNA is removed
Reaction mixture is prepared according to reaction system shown in table 1, RNA sample is eventually adding, then by mixed system
2min is stood at 42 DEG C, removes the genomic DNA being mixed in RNA.
The reaction mixture of the removal of table 1 DNA
| Reagent | Usage amount |
| 5*gDNA Eraser Buffer | 2μl |
| gDNA Eraser | 1μl |
| Total RNA | 1μg |
| RNase Free H2O | up to 10μl |
(2) reverse transcription reaction
According to amplification system shown in table 2, the reaction solution of reverse transcription is prepared on ice, is inverted immediately after soft mixing
Record reaction.After reacting 60min under 37 DEG C of temperature environment, the tissue RNA reverse transcription of extraction is cDNA.
2 post transcription cloning system of table
| Reagent | Usage amount |
| Reaction solution after removing genomic DNA | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer 2(for Real Time) | 4.0μl |
| RNase Free dH2O | 4.0μl |
| Total | 20μl |
(3) RT-PCR detects the relative expression quantity of FXR1 mRNA
1. passing through RT-PCR reaction detection human esophageal carcinoma (Tumor) and cancer beside organism using GAPDH as internal reference
(Normal) relative expression quantity of FXR1 mRNA in, the differential expression of measurement human esophageal carcinoma and FXR1 gene in normal tissue.
The amplimer of FXR1 mRNA is FXR1-F and FXR1-R, and the nucleotide sequence of FXR1-F is as shown in SEQ ID NO.1, FXR1-
The nucleotide sequence of R is as shown in SEQ ID NO.2；The amplimer of GAPDH mRNA is GAPDH-F and GAPDH-R, GAPDH-F
Nucleotide sequence as shown in SEQ ID NO.3, the nucleotide sequence of GAPDH-R is as shown in SEQ ID NO.4；
The reaction system of RT-PCR is as shown in table 3, reaction condition are as follows: initial denaturation: 95 DEG C of holding 3min；Denaturation: 95 DEG C of guarantors
Hold 30s；Annealing: 60 DEG C of holding 30s；Extend: 72 DEG C of holding 30s；Reaction carries out 40 circulations.60 DEG C rise to 95 DEG C, often on
It rises 0.5 DEG C of reading fluorescence data and once obtains PCR product solubility curve.
The reaction system of 3 RT-PCR of table
2. carrying out data analysis, using GAPDH as internal reference, 2 after reaction-△ctThe opposite table of method calculating FXR1 gene mRNA
Up to level.
The testing result of FXR1 gene expression dose is such as in human esophageal carcinoma (Tumor) and Carcinoma side normal tissue (Normal)
Shown in Fig. 1, the expression of the FXR1 gene in human esophageal carcinoma is significantly higher than the expression of FXR1 gene in normal tissue
(P < 0.001), the expression for illustrating FXR1 gene is significant related to the generation of the cancer of the esophagus, and FXR1 gene can be used as oesophagus
The molecular marker of cancer, diagnosis and targeted therapy for the cancer of the esophagus.
Embodiment 2
The present embodiment provides the method that the Clinical symptoms of a kind of pair of patient with esophageal carcinoma carries out statistical analysis, specifically include with
Lower content:
1, sample information acquires
Using 115 patients for being diagnosed as esophageal squamous cell carcinoma in embodiment 1 as experimental subjects, length of patient stay is
Between 2004-2007, the range of age was from 34 years old to 80 years old, average age 56 years old.The clinical data and Follow-up Data of patient comes from
Inpatient cases data, follow-up record and the Effect of follow-up visit by telephone record of patient Yu.Count gender, age, the tumour of 115 patients
Position, local Invasion degree (T is by stages), metastasis of lymph (N is by stages), DISTANT METASTASES IN (M is by stages) and pathological grading letter
Breath.
2,16.0 software of SPSS (SPSS Inc, Chicago, IL, USA) is used for data statistics and analysis.P < 0.05 is represented
Statistical result is significant.Using in the Wilcoxon method of inspection and the group of paired-samples T-test analysis patient FXR1 gene expression dose
Difference；Correlation (table 4) between FXR1 gene expression dose and clinical pathological characteristic is analyzed using Chi-square Test；
Kaplan-Meier curve and log-rank are examined to be turned for analyzing FXR1 gene expression dose and patients overall survival's time and nothing
The correlation of life span is moved, the expression of analysis FXR1 gene is used for value (Fig. 2A and the figure of cancer of the esophagus prognosis evaluation
2B)；The single factor test and multifactor survival analysis (table 5) of the cancer of the esophagus are carried out using Cox regression model.
Table 4.FXR1 gene expression dose and patient with esophageal carcinoma clinical pathological characteristic correlation analysis
*P<0.05
Fig. 2A indicates that the expression and the correlation of survival of patients time of FXR1 gene, Fig. 2 B indicate the table of FXR1 gene
Up to the horizontal correlation with patient without transfer life span, by Fig. 2A and Fig. 2 B it is found that the survival of patients of FXR1 gene high expression
Time and without transfer life span, hence it is evident that shorter than FXR1 gene low expression patient, illustrate the hair of FXR1 gene and the cancer of the esophagus
It is raw, develop and shift closely related, FXR1 gene is a kind of good cancer prognosis index.By detecting FXR1 gene expression,
It can be used in assessing patient with esophageal carcinoma prognosis, prompt survival of patients time, treatment of cancer effect monitored, in time to the clinic of cancer
Therapeutic strategy is adjusted, and is conducive to further increase clinical therapeutic efficacy, is improved life in patients.
Table 4 shows FXR1 gene expression dose and patient with esophageal carcinoma clinical pathological characteristic correlation analysis, can by table 4
Know, FXR1 gene expression dose is significant related to the DISTANT METASTASES IN that the cancer of the esophagus is suffered from, and detection FXR1 gene expression can be used in diagnosing
The DISTANT METASTASES IN of patient with esophageal carcinoma.The single factor test and multifactor Cox analysis of regression model that table 5 shows the cancer of the esophagus are as a result, can by table 5
Know, the expression of FXR1 gene can be used for Prognostic, life span prediction as the independent factor of cancer of the esophagus prognosis
And curative effect evaluation etc..
Embodiment 3
The construction method for subtracting the cell strain of FXR1 gene is struck the present embodiment provides a kind of stabilization, cell strain is specifically
KYSE180 human esophagus cancer cell strain, carrier pHBLV-U6-Scramble-ZsGreen-Puro used in building process,
PSPAX2 and pMD2G by Han Heng biotech firm obtain, construction method the following steps are included:
1, design synthesis shRNA
With four transcripts of FXR1 gene (NM_001013438.2, NM_001013439.2, NM_001363882.1,
It NM_005087.3) is molecular target, design can target the shRNA sequence of four mRNA simultaneously.Firstly, design siRNA target
Point, siRNA target spot include siRNA1, siRNA2, siRNA3 and siRNA4, the nucleotide sequence of siRNA1 such as SEQ ID NO.5
It is shown, the nucleotide sequence of siRNA2 as shown in SEQ ID NO.6, the nucleotide sequence of siRNA3 as shown in SEQ ID NO.7,
The nucleotide sequence of siRNA4 is as shown in SEQ ID NO.8.According to above-mentioned siRNA target sequence obtain shRNA1, shRNA2,
The nucleotide sequence of shRNA3 and shRNA4, shRNA1 are as shown in SEQ ID NO.9, the nucleotide sequence of shRNA2 such as SEQ ID
Shown in NO.10, the nucleotide sequence of shRNA3 is as shown in SEQ ID NO.11, the nucleotide sequence of shRNA3 such as SEQ ID
Shown in NO.12.
2, slow virus recombinant expression carrier is constructed
(1) the shRNA sequence designed according to step 1, the primer of design synthesis shRNA double-stranded segment, primer sequence are specific
It is as shown in table 6:
6 shRNA synthetic primer of table
| Primer | Sequence |
| shRNA1-F | Its nucleotide sequence is as shown in SEQ ID NO.13 |
| shRNA1-R | Its nucleotide sequence is as shown in SEQ ID NO.14 |
| shRNA2-F | Its nucleotide sequence is as shown in SEQ ID NO.15 |
| shRNA2-R | Its nucleotide sequence is as shown in SEQ ID NO.16 |
| shRNA3-F | Its nucleotide sequence is as shown in SEQ ID NO.17 |
| shRNA3-R | Its nucleotide sequence is as shown in SEQ ID NO.18 |
| shRNA4-F | Its nucleotide sequence is as shown in SEQ ID NO.19 |
| shRNA4-R | Its nucleotide sequence is as shown in SEQ ID NO.20 |
Primer annealing to form the double-stranded segment with cohesive end, the reaction system of annealing are as follows: 10XBuffer, 2 μ l；
Tris-Cl(pH7.5)100mM；NaCl 1M；EDTA 10mM；ShRNA-R/shRNA-F, 1/1 μ l；H2O, 16 μ l.Response procedures
Are as follows: 95 DEG C of holding 10min；75 DEG C of holding 10min；55 DEG C of holding 10min；35 DEG C of holding 10min；15 DEG C of holding 10min.Double-strand
The cohesive end that segment 5 ' is held is BamHI restriction enzyme site, and the cohesive end at 3 ' ends is EcoRI restriction enzyme site.
(2) using restriction enzyme BamHI and EcoRI to slow virus carrier pHBLV-U6-Scramble-ZsGreen-
Puro carries out digestion, then connects the double-strand shRNA segment that cohesive end is had in slow virus carrier and step (1), respectively
To the recombinant expression carrier pHBLV-U6-shRNA1-ZsGreen- for being connected with shRNA1, shRNA2, shRNA3 and shRNA4
Puro, pHBLV-U6-shRNA2-ZsGreen-Puro, pHBLV-U6-shRNA3-ZsGreen-Puro and pHBLV-U6-
shRNA4-ZsGreen-Puro.Coupled reaction system are as follows: shRNA double-stranded segment, 1 μ l；Carrier after digestion, 100-200ng；
Ligase buffer, 2 μ l；T4ligase, 1 μ l；H2O, polishing to 20 μ l.Condition of contact are as follows: 16 DEG C overnight.
(3) carrier after connection shRNA segment is converted into DH5 α competent bacteria, the LB of the ammonia benzyl resistance of 37 DEG C of coating is flat
Plate culture medium, overnight incubation.Next day picking single colonie is inoculated in containing 5ml, the LB culture solution of 100 μ g/ml Ampicillin resistances
In, 250rpm, 37 DEG C constant-temperature table culture 14 hours, with mini-scale plasmid extraction agent box extract plasmid, and with BamHI with
Digestion is identified that correct recombinant clone send sequence verification by EcoRI digestion identification, and the correct plasmid of sequencing result is to construct successfully
Slow virus recombinant expression carrier.
3, slow virus is packed
(1) using DMEM culture medium (being purchased from Invitrogen) culture containing 10% fetal calf serum (being purchased from Invitrogen)
293T cell, cell density grow to the cotransfection that virus is carried out when about 50%-60% converges rate.The plasmid of cotransfection includes disease
Malicious packaging plasmid pSPAX2, pMD2G and recombinant expression carrier (pHBLV-U6-shRNA1-ZsGreen-Puro, pHBLV-
U6-shRNA2-ZsGreen-Puro, pHBLV-U6-shRNA3-ZsGreen-Puro and pHBLV-U6-shRNA4-ZsGreen-
At least one of Puro), transfection uses LipofiterTMTransfection reagent (is purchased from Chinese Hang Seng object).
(2) replacement contains the fresh complete medium of 10% fetal calf serum FBS after transfecting, if turned in that morning
Dye, 6h carries out changing liquid after transfection, if that afternoon is transfected, after transfection the next morning about transfect after 16h carry out changing liquid.
(3) 48h and 72h collects vial supernatant twice respectively after transfecting (48h collects rear substitution fresh complete medium).
When 48h receives poison, the culture medium in 100mm culture dish is poured into 50ml centrifuge tube, notices that culture dish wall not contact centrifugation
Nozzle then fills into the fresh complete medium that 10ml contains 10% fetal calf serum FBS, is steadily placed in prevent there is germ contamination
37 DEG C, 5%CO2Constant incubator in continue to cultivate.When 72h receives poison, directly the culture medium in 100mm culture dish is poured into
In 50ml centrifuge tube, note also that culture dish wall not contact centrifugation nozzle, to prevent there is germ contamination.
By the viral supernatants in 50ml centrifuge tube, 4 DEG C, 3000rpm, 10min, cell fragment is removed；Then virus is collected
Stoste supernatant is placed in ultracentrifugation pipe, 4 DEG C, 25000rpm, is centrifuged 120 minutes, the virus liquid after being concentrated.
3, target cell is infected, stabilization is obtained and strikes the cell strain for subtracting FXR1 gene
(1) thin using RPMI-1640 culture medium (being purchased from Invitrogen) culture KYSE180 containing 10% fetal calf serum
Born of the same parents, when cell density grow to about 60% converge rate when carry out cell infection.The virus liquid infection obtained using step 2
KYSE180 cell is observed under fluorescence microscope after cultivating 72h, can observe intracellular green fluorescent protein GFP
(ZsGreen) expression (Fig. 3).
Not carry out KYSE180 cellular control unit (K180-shNC) that clpp gene subtracts as control, wherein do not carry out base
Because strike the KYSE180 cell subtracted be under the same experimental conditions of embodiment 3, be not connected with shRNA segment viral vectors
The control cell strain that pHBLV-U6-Scramble-ZsGreen-Puro is packed, infected.Fig. 3 slow virus as the result is shown
Particle successfully infects KYSE180 cell, has obtained stabilization and has struck the KYSE180 cell strain (K180-sh1) for subtracting FXR1 gene；And it is thin
Born of the same parents' form is normal, and the death rate is lower, infection rate 90%.
(2) screening is passed on the culture medium containing final concentration of 1.5 μ g/ml puromycin, obtains to stablize to strike subtracting
The cell strain of FXR1 gene, -80 DEG C of refrigerators freeze spare.
Experimental example 1
This experimental example, which strikes the stabilization constructed in embodiment 3, to be subtracted the clpp gene reduction fruit of the cell strain of FXR1 gene and tests
Card, and the influence of FXR1 gene pairs esophagus carcinoma proliferation, invasion and transfer is detected, particular content is as follows:
1, clpp gene subtracts effect detection
(1) the PT-PCR amplification method detection stabilization provided using embodiment 1 strikes the KYSE180 cell strain for subtracting FXR1 gene
(K180-sh1), the mRNA expression of FXR1 gene in the KYSE180 cellular control unit (K180-shNC) that clpp gene subtracts and is not carried out
Horizontal (Fig. 4).
(2) detected by Western blot detection stablize strike the KYSE180 cell strain (K180-sh1) for subtracting FXR1 gene and not into
The protein expression level (Fig. 5) of FXR1 gene in the KYSE180 cellular control unit (K180-shNC) that row clpp gene subtracts:
A, sample dissociation: before RIPA cracking, cell is cleaned 3 times to remove and remain serum, by 10 μ l/cm2 with the PBS of pre-cooling
Cell grows area and lysate is added, and cell scraper scraping takes supernatant after 4 DEG C of centrifugations, and cell lysate is surveyed using Bradford method
Determine concentration.
B, albuminous degeneration: supernatant object is mixed with 5 × PAGE electrophoresis sample-loading buffer, DTT after taking 30 μ l to be centrifuged, in 99 DEG C
5min is heated, sample adds in 12% polyacrylamide gel loading hole.
C, electrophoresis: setting constant pressure 100V, electrophoresis 120min.
D, transferring film: the albumen in PAGE glue is transferred to 0.45 μm of pvdf membrane by setting constant current 250mA, transferring film 3h.
E, close: 5% skimmed milk power-PBST solution, room temperature shaker close 1h.
F, primary antibody is incubated for: primary antibody is incubated overnight in 4 DEG C.
G, it washes film: washing film 3 times with PBST, each 5min.
H, secondary antibody is incubated for: the secondary antibody (1/5000 dilution) of horseradish peroxidase-labeled is incubated for film 45min in room temperature.
I, it washes film: washing film 4 times with PBST, each 5min.
J, 50 μ l/cm chemiluminescence: are pressed2Chemical luminescence for liquid is added in membrane area, rinses after x-ray film autoradiography and obtains albumen one
Band.
2, cell migration and Matrigel
A, before experiment 48h with 2 × 106/ ware is by tumor cell inoculation in 10cm culture dish.
B, trypsin digestion cell, adjustment cell concentration are 2 × 105/ml。
C、4×104With 8 × 104It is small that a cell (respectively at 200 μ l and 400 μ l serum-free RPMI-1640) is inoculated in 24 holes
Room carries out migration and Matrigel respectively, which is the film in the aperture 8ul, to imitate the hole on basilar memebrane, invades
There is preparatory coated matrigel glue to contain to imitate the collagen of extracellular matrix below cell for 0.7ml when experiment on film
The RPMI-1640 of 10%FBS.
D, cell is placed in regular growth condition of culture after culture 48h, fixes 5min, 0.5% violet staining with methanol
1h, after being cleaned with tap water, with cotton swab removal without migration and the cell invaded.After room temperature is dried, under optical microscopy
(200X) randomly selects 5 visual field computation migrations, invasion cell number.
3, cell proliferation experiment
A, logarithmic phase cell is collected, concentration of cell suspension is adjusted, 100ul is added in every hole, and bed board makes cell tune density to be measured
To the hole 1000-10000, (edge hole is filled with sterile PBS).
B, 5%CO2, 37 DEG C are incubated for, until cell monolayer is paved with bottom hole (96 hole flat underside), the drug of concentration gradient is added,
In principle, after cell is adherent can dosing or two hours or time half a day, but in this experimental example using in noon before that day
Bed board, the dosing of morning next day.General 5-7 gradient, every hole 100ul, if 3-5 multiple holes.It is recommended that setting 5, otherwise it is difficult to react
Truth
C, 5%CO2, 37 DEG C incubation 16-48 hours, observe under inverted microscope.
D, 20ul MTT solution (5mg/ml, i.e. 0.5%MTT) is added in every hole, continues to cultivate 4h.If drug and MTT can
Reaction, can first be centrifuged and discard culture solution afterwards, carefully rush 2-3 after with PBS, add the culture solution containing MTT.
E, culture is terminated, culture solution in hole is carefully sucked.
F, 150ul dimethyl sulfoxide is added in every hole, sets low-speed oscillation 10min on shaking table, dissolves crystal sufficiently.In enzyme
Join the light absorption value in each hole of measurement at immune detector OD490nm.
G, it is arranged zeroing hole (culture medium, MTT, dimethyl sulfoxide) simultaneously, (cell, the drug of same concentrations are molten for control wells
Solve medium, culture solution, MTT, dimethyl sulfoxide)
Experimental result:
The mRNA expression for striking FXR1 gene in the cell strain for subtracting FXR1 gene and control cell strain is stablized in Fig. 4 display,
As shown in Figure 4, compared with cellular control unit (mRNA relative expression quantity > 80%), in the stable cell line constructed in embodiment 3
FXR1 gene expression significantly reduces (mRNA relative expression quantity < 20%), illustrates FXR1 clpp gene in the cell strain of the offer of embodiment 3
Subtract success, the construction method provided using embodiment 3 can screen to obtain the esophageal cancer cell of FXR1 gene stable low-expression.
The protein expression situation for striking FXR1 gene in the cell strain for subtracting FXR1 gene and control cell strain is stablized in Fig. 5 display,
As shown in Figure 5, it is able to detect that the high FXR1 protein band of expression quantity in control cell strain group, and subtracts FXR1 base stablizing to strike
In the cell strain of cause, apparent FXR1 protein band is not detected, further demonstrates the KYSE180 cell strain in embodiment 3
Middle successfully strike subtracts FXR1 gene.
The cell migration testing result for striking the cell strain for subtracting FXR1 gene and control cell strain is stablized in Fig. 6 display.It can by Fig. 6
Know, after striking and subtracting FXR1 gene, the transfer ability of cell significantly reduces esophageal cancer cell, illustrate the migration of esophageal cancer cell with
The significant correlation of FXR1 gene expression.The cell invasion inspection for striking the cell strain for subtracting FXR1 gene and control cell strain is stablized in Fig. 7 display
Survey result.As shown in Figure 7, after striking and subtracting FXR1 gene, the invasive ability of cell significantly reduces esophageal cancer cell, illustrates the cancer of the esophagus
The invasion of cell are significant related to FXR1 gene expression.Fig. 8 display, which is stablized, strikes the cell strain for subtracting FXR1 gene and control cell strain
Cell Proliferation testing result.As shown in Figure 8, FXR1 gene expression does not generate esophageal cancer cell increase and significantly affects.By scheming
6- Fig. 8 is potential as dlinial prediction esophagus cancer invasion metastatic potential it is found that FXR1 gene participates in the occurrence and development of the cancer of the esophagus
Index realizes that the prognosis evaluation to the cancer of the esophagus, therapeutic effect monitor, is the diagnosis of the cancer of the esophagus by detecting FXR1 gene expression
Effective information, which is provided, with targeted therapy improves the survival rate and life quality of patient to realize the individualized treatment of cancer.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Sequence table
<110>affiliated hospital of Southern Yangtze University
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Claims (15)
1. a kind of molecular marker of the cancer of the esophagus, which is characterized in that the molecular marker is FXR1 gene and/or FXR1 gene
Expression product.
2. the molecular marker of the cancer of the esophagus according to claim 1, which is characterized in that the expression product of the FXR1 gene
Including FXR1mRNA and/or FXR1 albumen.
3. the molecular marker of the cancer of the esophagus according to claim 1 or 2, which is characterized in that the cancer of the esophagus is esophageal squamous cell
Cancer.
4. the molecular marker of the described in any item cancer of the esophagus of claim 1-3 is in following a1-a4Purposes at least one:
a1, the product of esophagus cancer diagnosis is used for as the diagnosis marker of the cancer of the esophagus, or preparation；
a2, the product of cancer of the esophagus prognosis evaluation is used for as the prognostic marker of the cancer of the esophagus, or preparation；
a3, product of the preparation for Curative Effect of Esophagus Carcinoma monitoring；
a4, the drug of the preparation treatment cancer of the esophagus.
5. purposes according to claim 4, which is characterized in that the esophagus cancer diagnosis includes that cancer of the esophagus DISTANT METASTASES IN is examined
It is disconnected.
6. a kind of primer sets for esophagus cancer diagnosis, which is characterized in that the primer sets include any based on claim 1-3
The primer of cancer of the esophagus molecular marker design described in.
7. primer sets according to claim 6, which is characterized in that the primer sets include following primer:
FXR1-F, nucleotide sequence is as shown in SEQ ID NO.1；
FXR1-R, nucleotide sequence is as shown in SEQ ID NO.2.
8. a kind of expression inhibiting agent of FXR1 gene, which is characterized in that the expression inhibiting agent includes siRNA and/or shRNA.
9. expression inhibiting agent according to claim 8, which is characterized in that the nucleotide sequence of the siRNA such as SEQ ID
Shown in NO.5-SEQ ID NO.8 any sequence,
The nucleotide sequence of the shRNA is as shown in SEQ ID NO.9-SEQ ID NO.12 any sequence.
10. a kind of carrier, which is characterized in that the carrier includes that nucleotide sequence such as SEQ ID NO.9-SEQ ID NO.12 appoints
ShRNA shown in one sequence.
11. including the recombinant cell or pseudovirion of carrier described in any one of claim 10.
12. described in expression inhibiting agent, carrier described in any one of claim 10 described in claim 8 or 9 or claim 11
The purposes of recombinant cell or pseudovirion in the drug of the preparation treatment cancer of the esophagus.
13. a kind of stabilization strikes the cell strain for subtracting FXR1 gene, which is characterized in that the cell strain is using esophageal cancer cell as target
Cell, is infected by the pseudovirion described in claim 11, and the stabilization of acquisition strikes the esophageal cancer cell for subtracting FXR1 gene.
14. stabilization described in a kind of building claim 13 strikes the method for subtracting the cell strain of FXR1 gene, which is characterized in that including
Following steps:
(1) the shRNA segment of any shown sequence of nucleotide sequence such as SEQ ID NO.9-SEQ ID NO.12 is connected to tool
There is the viral vectors of antibiotic resistance, obtains recombinant expression carrier；
(2) using recombinant expression carrier and viral packaging plasmid cotransfection packaging cell line, Transfected cells are cultivated, collect culture
Base supernatant obtains the virus liquid containing pseudovirion after concentration；
(3) esophageal cancer cell is infected using virus liquid, then carries out drug screening using antibiotic, obtained stable strike and subtract FXR1 base
The cell strain of cause.
15. construction method according to claim 14, which is characterized in that the viral vectors is pHBLV-U6-
Scramble-ZsGreen-Puro, the virus packaging plasmid includes pSPAX2 and pMD2G.
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| CN110512004A (en) * | 2019-10-11 | 2019-11-29 | 新乡医学院 | It is a kind of to be migrated and the molecular marker of invasive ability and its application for detecting esophageal cancer cell |
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| CN112397146A (en) * | 2020-12-02 | 2021-02-23 | 广东美格基因科技有限公司 | Microbial omics data interaction analysis system based on cloud platform |
| CN112397146B (en) * | 2020-12-02 | 2021-08-24 | 广东美格基因科技有限公司 | Microbial omics data interaction analysis system based on cloud platform |
| CN113789377A (en) * | 2021-05-12 | 2021-12-14 | 山西医科大学 | Molecular marker of esophageal cancer and application thereof |
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