CN108841965A - Application of the TLCD1 in the diagnosis of Metastasis in Breast Cancer, prognosis and treatment - Google Patents
Application of the TLCD1 in the diagnosis of Metastasis in Breast Cancer, prognosis and treatment Download PDFInfo
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Abstract
The invention discloses application of the TLCD1 in the diagnosis of Metastasis in Breast Cancer, prognosis and treatment.Present invention discover that TLCD1 high expression in the tissue and cell that Bone of Breast Cancer shifts, and imply shorter no Bone tumour life span, internal experiment in vitro confirms that the expression of TLCD1 gene deregulation is able to suppress breast cancer migration and invasion, so that Bone of Breast Cancer be inhibited to shift；Based on above-mentioned discovery, the present invention proposes that the reagent that will test TLCD1 gene expression dose is applied in the kit whether preparation detection breast cancer shifts and/or the prognosis kit of prediction Metastasis in Breast Cancer tendency；The inhibition reagent of TLCD1 is applied in the drug of preparation prevention and/or treatment Metastasis in Breast Cancer, provides new thinking for the diagnosis, prognosis and treatment of Metastasis in Breast Cancer.
Description
Technical field
The invention belongs to fields of biomedicine, relate more specifically to TLCD1 in the diagnosis, prognosis and treatment of Metastasis in Breast Cancer
In application.
Background technique
Transfer is the underlying cause of death of tumor patient, and the tumour associated death more than 90% is related with distant metastasis of human.Bone
It is one of most common DISTANT METASTASES IN organ of entity tumor, after Bone tumour occurs for tumour, incident is bone related complication,
Such as pain, pathologic fracture, hypercalcinemia, spinal compression, loss of motor function, eventually lead to death.It is directed at present
The treatment of Bone tumour belongs to Palliative, either operation, radiotherapy and drug therapy, only alleviates and improve corresponding symptom, and
The existence that patient cannot be significantly improved is expected.Thus, once Bone tumour occurs, not only seriously affected the quality of life of patient with
Service life also brings great difficulty to clinical treatment.
Breast cancer (breast cancer：It BC) is the tumour that Bone tumour most easily occurs.In China, as trophic structure changes
Become and aging trend, the incidence and The dead quantity of Bone of Breast Cancer transfer have the tendency that significantly increasing.In most cases, mammary gland
Cancer is not before causing apparent clinical symptoms, after the generation or primary tumo(u)r treatment that have just caused Bone tumour stove
Do not recur, but it has been found that Bone tumour stove formation, bring great difficulty to clinical treatment.Breast cancer turns once bone occurs
It moves, shows that patient has entered advanced tumor, seriously affected quality of life and the service life of patient, increased the economy of patient home
Burden, also consumes a large amount of medical resource.Therefore, targetedly exploitation detection breast cancer whether shift product,
It predicts the prognosis product of Metastasis in Breast Cancer tendency, and inhibits the drug of Metastasis in Breast Cancer, for diagnosis, prognosis and treatment mammary gland
Metastasis of cancer improves survival and has great importance.
Summary of the invention
The purpose of the present invention is to provide application of the TLCD1 in the diagnosis of Metastasis in Breast Cancer, prognosis and treatment.
The technical solution adopted by the present invention is that：
For detecting the reagent of TLCD1 gene expression dose in the kit whether preparation detection breast cancer shifts
Application.
Wherein, the reagent of the detection TLCD1 gene expression dose is selected from：Primer, probe, chip, antibody.
Wherein, the transfer includes Bone tumour, hepatic metastases, Lung metastases, pleura metastasis, brain metastes and Lymph Node Metastasis.
For detecting the reagent of TLCD1 gene expression dose in the prognosis kit of preparation prediction Metastasis in Breast Cancer tendency
Application.
Wherein, the reagent of the detection TLCD1 gene expression dose is selected from：Primer, probe, chip, antibody.
Wherein, the transfer includes Bone tumour, hepatic metastases, Lung metastases, pleura metastasis, brain metastes and Lymph Node Metastasis.
Application of the inhibition reagent of TLCD1 in the drug of preparation prevention and/or treatment Metastasis in Breast Cancer.
Wherein, the inhibition reagent of the TLCD1 is selected from：The substance of TLCD1 gene expression can completely or partially be inhibited,
The substance of TLCD1 protein exhibits effect can completely or partially be inhibited.
Wherein, the inhibition reagent of the TLCD1 is selected from：Protein, oligonucleotides, oligonucleotides expression vector, small point
Sub- compound.
Wherein, the transfer includes Bone tumour, hepatic metastases, Lung metastases, pleura metastasis, brain metastes and Lymph Node Metastasis.
The beneficial effects of the invention are as follows：
Present invention discover that TLCD1 high expression in the tissue and cell that Bone of Breast Cancer shifts, and imply shorter nothing
Bone tumour life span.Internal experiment in vitro confirms that the expression of TLCD1 gene deregulation is able to suppress breast cancer migration and invasion, from
And Bone of Breast Cancer is inhibited to shift；Based on above-mentioned discovery, the present invention proposes that the reagent that will test TLCD1 gene expression dose is applied to
In the prognosis kit of kit and/or prediction Metastasis in Breast Cancer tendency that whether preparation detection breast cancer shifts；It will
The inhibition reagent of TLCD1 is applied in the drug of preparation prevention and/or treatment Metastasis in Breast Cancer, for examining for Metastasis in Breast Cancer
Disconnected, prognosis and treatment provide new thinking.
Detailed description of the invention
Fig. 1：The mRNA and expressing quantity of TLCD1 in breast cancer tissue and cell；Wherein, figure A is Bone of Breast Cancer transfer
The mRNA content of TLCD1, figure B are that Bone of Breast Cancer shifts tissue (4) and bone-free transfer tissue in tissue and bone-free transfer tissue
The protein content of TLCD1 in (4)；Scheme the mRNA content that C is TLCD1 in various cell lines, figure D is in various cell lines
The protein content of TLCD1；BC/nBM indicates that bone-free metastatic breast cancer tissue, BC/BM indicate Bone tumour breast cancer tissue in figure；
Fig. 2：TLCD1 high expression group and TLCD1 low expression group patient without Bone tumour life span；TLCD1 H table in figure
Show TLCD1 high expression group, TLCD1 L indicates TLCD1 low expression group；
Fig. 3：Influence of the RNA interference plasmid to the mRNA and expressing quantity of TLCD1 in MDA-MB-231 and MCF7 cell,
Vector is the cell line that transfection has control plasmid in figure, and TLCD1 RNAi#1 is TLCD1 stable low-expression breast cancer cell line；
Fig. 4：Influence of the RNA interference plasmid to MDA-MB-231 and MCF7 cell migration and invasion, vector is to turn in figure
It is infected with the cell line of control plasmid, TLCD1 RNAi#1 is TLCD1 stable low-expression breast cancer cell line；
Fig. 5：There is the TLCD1 stable low-expression MDA-MB-231 cell construction mammary gland of mouse of RNA interference plasmid using transfection
Cancer Bone tumour model；Wherein, figure A is BLI imaging schematic diagram, and figure B is bone X ray picture, and figure C is bone HE colored graph, schemes D and turns for bone
Appraisal result is moved, figure E is BLI signal results figure, and figure F is total survivorship curve, and figure G is no Bone tumour survivorship curve；In figure
Vector is the cell line that transfection has control plasmid, and TLCD1 RNAi#1 is TLCD1 stable low-expression breast cancer cell line；
Fig. 6：There is the TLCD1 stable low-expression MCF7 cell construction mouse breast cancer bone of RNA interference plasmid to turn using transfection
Shifting formwork type；Wherein, figure A is BLI imaging schematic diagram, and figure B is bone X ray picture, and figure C is bone HE colored graph, schemes D for Bone tumour scoring
As a result, figure E is BLI signal results figure, figure F is total survivorship curve, and figure G is no Bone tumour survivorship curve；Vector is to turn in figure
It is infected with the cell line of control plasmid, TLCD1 RNAi#1 is TLCD1 stable low-expression breast cancer cell line.
Specific embodiment
Inventor using the sequencing of two generations, tumour Relational database and analysis of biological information means, by long-time research and
Screening, discovery TLCD1 can diagnosis as Metastasis in Breast Cancer and prognostic marker, and be applied to the prevention of Metastasis in Breast Cancer with
In treatment.
For this purpose, the present invention proposes：
For detecting the reagent of TLCD1 gene expression dose in the kit whether preparation detection breast cancer shifts
Application.
Wherein, the reagent of the detection TLCD1 gene expression dose is selected from：Primer, probe, chip, antibody, but be not limited to
This.Those skilled in the art can design specifically primer, probe, core according to TLCD1 gene or its sequence for encoding albumen
Piece, antibody, and the modification of the reagent should also be included in protection scope.
Wherein, the transfer includes Bone tumour, hepatic metastases, Lung metastases, pleura metastasis, brain metastes and Lymph Node Metastasis.
For detecting the reagent of TLCD1 gene expression dose in the prognosis kit of preparation prediction Metastasis in Breast Cancer tendency
Application.
Wherein, the reagent of the detection TLCD1 gene expression dose is selected from：Primer, probe, chip, antibody, but be not limited to
This.Those skilled in the art can design specifically primer, probe, core according to TLCD1 gene or its sequence for encoding albumen
Piece, antibody, and the modification of the reagent should also be included in protection scope.
Wherein, the transfer includes Bone tumour, hepatic metastases, Lung metastases, pleura metastasis, brain metastes and Lymph Node Metastasis.
Application of the inhibition reagent of TLCD1 in the drug of preparation prevention and/or treatment Metastasis in Breast Cancer.
Wherein, the inhibition reagent of the TLCD1 is selected from：The substance of TLCD1 gene expression can completely or partially be inhibited,
The substance of TLCD1 protein exhibits effect can completely or partially be inhibited.
It further illustrates, the substance of TLCD1 gene expression can completely or partially be inhibited to can be by interrupting TLCD1 base
The transcription of cause and/or the mRNA translation realization for blocking TLCD1 gene；TLCD1 protein exhibits effect can completely or partially be inhibited
Substance be at least understood as inhibiting the substance of the activity of TLCD1 albumen, useful effect duration, stability.
Wherein, the inhibition reagent of the TLCD1 is selected from：Protein, oligonucleotides, oligonucleotides expression vector, small point
Sub- compound, but not limited to this.It further illustrates, viral vectors or eukaryotic vector can be selected in expression vector.
Wherein, the transfer includes Bone tumour, hepatic metastases, Lung metastases, pleura metastasis, brain metastes and Lymph Node Metastasis.
Below with reference to specific experiment, thinking that the present invention is further explained, but protection scope of the present invention but be not limited to
This.
Experimental method used in following experimental examples or experiment condition, unless otherwise specified, according to a conventional method or factory
Family's specification carries out, and material, reagent used in following experimental examples etc. is commercially available unless otherwise specified.
Materials and methods
1, experimental material
(1) cell line：People's normal mammary epithelial (NMEC1), non-metastatic human breast cancer cell line (MDA-MB-
415, T47D, BT549), metastatic breast cancer cell line (MDA-MB-453, MCF-7,4T1, MDA-MB-231)；Wherein,
Kerationcyte culture medium (serum-free) culture of the NMEC1 cell added with epidermal growth factor and bovine brain pituitary extract, and
120 μ g/mL streptomysins and 120 μ g/mL penicillin are added in the medium；MDA-MB-415 cell, which is used, is added with 15% tire ox blood
Clear L15 culture medium culture；The RPMI-1640 culture medium culture of BT549,4T1 added with 10% fetal calf serum；T47D,
MCF-7, MDA-MB-453, MDA-MB-231 cell use added with 10% fetal calf serum, 200mg/ml penicillin, 250mg/
The DMEM culture medium culture of ml chloramphenicol, 500mg/ml glutamine, the above cell culture condition are：Gas concentration lwevel is
5%, temperature is 37 DEG C；Routine observation cell state and cell density regularly replace culture medium, suitable when cell density is to 80%
Preferably test use.
(2) tissue samples：Breast cancer tissue's sample that 263 operations or aspiration biopsy obtain, wherein 231 non-bone turn
Breast cancer tissue, 32 Bone tumour breast cancer tissues are moved, the above tissue samples derive from Jiangmen city central hospital, have corresponding
Clinical pathology information.
(3) experimental animal：5~6 week old, the BALB/c-nu nude mice of 18~20g.
(4) primer：For detecting the mRNA of people's TLCD1 gene and its primer of reference gene by the sharp rich biological section in Guangzhou
Skill Co., Ltd design and synthesis.
(5) antibody：Primary antibody, secondary antibody for detecting people TLCD1 gene coded protein and its internal reference albumen is purchased from Santa
Cruz company.
(6) RNA interference plasmid：People TLCD1 shRNA plasmid (abbreviation RNA interference for lower mediator TLCD1 gene expression
Plasmid) and its control plasmid by Guangzhou Ribo Bio Co., Ltd.'s design and synthesis.
2, experimental method
(1) fluorescence quantitative PCR detection TLCD1 mRNA
Test serum/the cell for taking about 100mg, is pulverized in liquid nitrogen with mortar, and TRizol lysis at room temperature 10min is added,
0.2ml chloroform is added in 1ml TRizol lysate, acutely shakes 30s, stands 5min；4 DEG C, 12,000g centrifugation 15min are drawn
Isometric isopropanol is added into new centrifuge tube in supernatant, and piping and druming uniformly, stands 10min, 4 DEG C, 12,000g centrifugation 5min are used
The water-soluble washing RNA precipitate of DEPC containing 75% ethyl alcohol 3 times after pausing drying, dissolves RNA with DEPC water.Measure after concentration by
The operation of PrimeScript RT reagent Kit specification, carries out reverse transcription：It is said by SYBR Premix Ex Taq II Kit
Bright book selects 2ddCt relative quantification method to be tested and analyzed on 7500 real time quantitative PCR systems, reference gene GAPDH,
Seek the opposite mrna expression amount for calculating target gene.
(2) Western blotting detects TLCD1 protein expression level
Test serum/the cell for taking about 100mg, is pulverized in liquid nitrogen with mortar, and RIPA lysate is added and cracks on ice
30min collects lysate, and 4 DEG C, 12,000g centrifugation 15min collect supernatant, carry out by BCA protein quantification kit specification
5 × sample-loading buffer of volume 1/4, after boiling water bath 10min, ice bath 5min are added to supernatant for protein quantification.Use SDS
PAGE protein electrophoresis system, applied sample amount are 30 μ g total proteins, and electrophoretic parameters are 10% separation gel, 5% concentration glue, 100V electrophoresis
90min expects that blue indicator is migrated to bottom to bromine, carries out electrotransfer, and using 0.45 μm of pvdf membrane, 300mAl ice bath electricity turns
90min.After electricity turns, film is closed using 5% skimmed milk power, and TBST rinses 3 times 5min, and anti-TLCD1 primary antibody (1 is added:
1000), 4 DEG C of overnight incubations, TBST rinse 3 times 5min, and the diluted secondary antibody of confining liquid is added, and are incubated at room temperature lh, and TBST is rinsed 3 times
ECL luminescent solution is added in 5min, is placed in the photosensitive lmin to 20min of exograph in piece pressing clip, developed fixing processing：Immunoblotting
Detection is control with α-tubulin.
(3) plasmid transfection
By breast cancer cell with 10 before transfection5A/hole is inoculated in culture plate, RNA interference plasmid, control after addition dilution
Plasmid and liposome, are screened after transfection using antibiotic, final to obtain the cell line for stablizing expression, specific transfection experiment ginseng
It is carried out according to Lipofectamine3000 product description.
(4) invade and migrate experiment
Invaded and migrated experiment using the cell transwell method, in brief, the cell transwell coating or not
Coating substrate glue, will be cells trypsinised, and is suspended in serum free medium, upper chamber addition 1.5 × 105It is a thin
Born of the same parents, lower room are that the culture medium containing 10% fetal calf serum is fixed, and use hematoxylin after being incubated for 24~48h with 4% paraformaldehyde
Dyeing counts (100 ×) under the microscope.
(5) Bone of Breast Cancer shifts animal model
5-6 week old, the BALB/c-nu nude mice of 18~20g are used to test, be anaesthetized before experiment, and left ventricle inoculation 1 ×
105A (100 μ L PBS) transfects MDA-MB-231 the or MCF-7 cell by control plasmid and RNA interference plasmid respectively.With biology
Luminescence imaging system (BLI) monitors Bone tumour progress, and X-ray monitors the transmittance damage of osteolytic lesion, schemed with Metamorph
As the area of analysis system and software measurement osteolytic lesion, the osteoclasia degree of every animal is expressed as square millimeter, according to
Following standard scores to Bone tumour：0：Without transfer；1：Bone injury covers bone width less than 1/4；2：It is wide that bone injury covers bone
Degree 1/4~1/2；3：Bone injury covers bone width 1/2~3/4；4：Bone injury covers bone width and is greater than 3/4；The bone of every animal
Transfer scoring monitors the sign of animal, when there is weight loss 10%, paralysis, head skew etc. from four limbs scoring synthesis daily
Euthanasia is selected when painful symptom.
Embodiment 1, TLCD1 shift high expression in tissue and cell in Bone of Breast Cancer
In the tissue samples and cell line of collection, detected respectively by quantitative fluorescent PCR and Western blotting
The mRNA and protein content of TLCD1, as a result as shown in Figure 1, compared with bone-free metastatic breast cancer tissue, Bone tumour breast cancer tissue
The mRNA and protein content of middle TLCD1 increases (Figure 1A and Figure 1B), compared with people's normal breast epithelial, breast cancer cell
The mRNA of TLCD1 and protein content increase in system, and the mRNA of TLCD1 and protein content increase in metastatic breast cancer cell line
It is mostly more significant, especially MCF-7 and MDA-MB-231 cell line (Fig. 1 C and Fig. 1 D).
The above result shows that：TLCD1 shifts high expression in tissue and cell in Bone of Breast Cancer, therefore, can pass through detection
TLCD1 expression, whether diagnosis occurs Bone of Breast Cancer transfer, for example, with TLCD1 in subject breast cancer tissue sample
MRNA or expressing quantity are TLCD1 mRNA or expressing quantity and control group in index or subject breast cancer tissue sample
Knitting fiducial value is index, if index is more than given threshold, is then determined as Bone tumour.
Embodiment 2, TLCD1 high expression imply that no Bone tumour life span is short
By 231 bone-free metastatic breast cancer tissue samples according to TLCD1 expression quantity median be divided into TLCD1 high expression group and
TLCD1 low expression group further analyzes no Bone tumour life span in conjunction with follow-up of patients's data of breast cancer tissue's sample, as a result
As shown in Fig. 2, TLCD1 high expression group is more without Bone tumour life span.
The above result shows that：TLCD1 high expression implies that no Bone tumour life span is short, therefore, can pass through detection
TLCD1 expression, using no Bone tumour life span as prognostic indicator, to have predicted whether Bone of Breast Cancer metastasis tendency；Example
Such as, using TLCD1 mRNA in subject breast cancer tissue sample or expressing quantity as index or subject breast cancer tissue sample
TLCD1 mRNA or expressing quantity and control tissue fiducial value are index in this, if index is more than given threshold, are then determined as
Bone tumour risk is larger, and life span is shorter.
Embodiment 3, downward TLCD1 gene expression can inhibit breast cancer cell to migrate and invade
MDA-MB- is transfected respectively using the RNA interference plasmid and control plasmid with downward TLCD1 gene expression function
231 or MCF-7 cell, as a result as shown in figure 3, having transfected TLCD1 in MDA-MB-231 the and MCF-7 cell of RNA interference plasmid
The mRNA and expressing quantity of gene are significantly lowered.
Experiment is invaded and migrated respectively to MDA-MB-231 and MCF-7 cell after transfection, shown in result figure 4, is turned
The migration and invasive ability for having contaminated MDA-MB-231 the and MCF-7 cell of RNA interference plasmid are significantly inhibited.
Breast cancer cell can be inhibited to migrate and invade the above result shows that lowering TLCD1 gene expression.
Embodiment 4, downward TLCD1 gene expression can inhibit mouse breast cancer Bone tumour
MDA-MB-231 and MCF-7 cell after transfection is respectively used to Bone of Breast Cancer transfer animal model, is as a result distinguished
As shown in Figure 5 and Figure 6, BLI imaging and signal result illustrate that RNA interference plasmid is able to suppress Bone of Breast Cancer transfer, X-ray knot
Fruit illustrates that RNA interference plasmid can reduce osteolytic lesion, and tibial neoplasms slice coloration result illustrates that RNA interference plasmid can drop
Tumor load in low bone, in addition, transfected RNA interference plasmid mouse Bone tumour scoring it is lower, without Bone tumour life span
Longer, total life span is also longer.
Embodiment 3 and 4 the result shows that, it is no matter external or in vivo, lower the RNA interference of TLCD1 gene expression function
Plasmid is able to suppress breast cancer migration and invasion, so that Bone of Breast Cancer is inhibited to shift, it therefore, can be by inhibiting TLCD1 gene
The substance of expression inhibits the substance of TLCD1 protein exhibits effect to be able to suppress Bone of Breast Cancer transfer, for preventing and treating
Bone of Breast Cancer transfer.
Claims (10)
1. the reagent for detecting TLCD1 gene expression dose is in the kit whether preparation detection breast cancer shifts
Using.
2. application according to claim 1, it is characterised in that：The reagent of the detection TLCD1 gene expression dose is selected from：
Primer, probe, chip, antibody.
3. application according to claim 1, it is characterised in that：The transfer includes Bone tumour, hepatic metastases, Lung metastases, chest
Film transfer, brain metastes and Lymph Node Metastasis.
4. the reagent for detecting TLCD1 gene expression dose is in the prognosis kit of preparation prediction Metastasis in Breast Cancer tendency
Using.
5. application according to claim 1, it is characterised in that：The reagent of the detection TLCD1 gene expression dose is selected from：
Primer, probe, chip, antibody.
6. application according to claim 1, it is characterised in that：The transfer includes Bone tumour, hepatic metastases, Lung metastases, chest
Film transfer, brain metastes and Lymph Node Metastasis.
Application of the inhibition reagent of 7.TLCD1 in the drug of preparation prevention and/or treatment Metastasis in Breast Cancer.
8. application according to claim 7, it is characterised in that：The inhibition reagent of the TLCD1 is selected from：Can all or
Part inhibits the substance of TLCD1 gene expression, can completely or partially inhibit the substance of TLCD1 protein exhibits effect.
9. application according to claim 7, it is characterised in that：The inhibition reagent of the TLCD1 is selected from：Protein, widow
Nucleotide, oligonucleotides expression vector, small molecule compound.
10. application according to claim 7, it is characterised in that：The transfer includes Bone tumour, hepatic metastases, Lung metastases, chest
Film transfer, brain metastes and Lymph Node Metastasis.
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Cited By (5)
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| CN110218796A (en) * | 2019-06-27 | 2019-09-10 | 江门市中心医院 | New target drone PCDHB2 for Bone of Breast Cancer transfer diagnosis and treatment |
| CN111269987A (en) * | 2020-03-27 | 2020-06-12 | 吉林大学中日联谊医院 | Diagnostic prognostic marker MAPK8IP1P2 for thyroid cancer and application thereof |
| CN114574583A (en) * | 2022-03-28 | 2022-06-03 | 中国医科大学附属第一医院 | Application of TMC5 in diagnosis and treatment of breast cancer specific bone metastasis |
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| CN109507161A (en) * | 2018-11-28 | 2019-03-22 | 中山大学孙逸仙纪念医院 | Application of the TIMP-1 in the kit and suppression cancer drug of preparation Prognosis in Breast Cancer diagnosis |
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| CN111269987A (en) * | 2020-03-27 | 2020-06-12 | 吉林大学中日联谊医院 | Diagnostic prognostic marker MAPK8IP1P2 for thyroid cancer and application thereof |
| CN111269987B (en) * | 2020-03-27 | 2023-05-05 | 吉林大学中日联谊医院 | Diagnostic prognosis marker MAPK8IP1P2 for thyroid cancer and application thereof |
| CN114574583A (en) * | 2022-03-28 | 2022-06-03 | 中国医科大学附属第一医院 | Application of TMC5 in diagnosis and treatment of breast cancer specific bone metastasis |
| CN114854747A (en) * | 2022-04-25 | 2022-08-05 | 南通大学附属医院 | Application of KIAA1467 gene |
| CN114854747B (en) * | 2022-04-25 | 2024-04-26 | 南通大学附属医院 | Application of KIAA1467 gene |
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