CN110229817A - Target siRNA and its application of KTN1 treatment breast cancer - Google Patents
Target siRNA and its application of KTN1 treatment breast cancer Download PDFInfo
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Abstract
The present invention provides a kind of siRNA and its application for targeting KTN1 and treating breast cancer.The sequence of the siRNA includes at least one of the sequence as shown in SEQ ID NO.1 and the sequence as shown in SEQ ID NO.2.The siRNA inhibits proliferation, transfer and the invasive ability of triple negative breast cancer cell, final improvement or treatment triple negative breast cancer to lower the expression of the KTN1 gene by the KTN1 gene of targeting triple negative breast cancer cell.
Description
Technical field
The present invention relates to field of gene more particularly to it is a kind of targeting KTN1 treatment breast cancer siRNA and its
Using.
Background technique
Breast cancer (Breast cancer) is one of most common female cancer, in China, the average year with breast cancer
Age is 45-55 years old, and more early than west women, the breast cancer case newly diagnosed accounts for the 12.2% of whole cancers.About 81.4% leaching
Lubricant nature patient with breast cancer will do it chemotherapy, however the recurrence of Metastasis in Breast Cancer caused by 5 years chemotherapies also increases year by year.TNBC is cream
One kind the most pernicious in gland cancer parting, due to TNBC cell surface do not express estrogen receptor (Estrogen receptor,
ER), progesterone receptor (Progesterone receptor, PR) and ErbB-2 (Human
Epidermal growth factor receptor-2, HER-2), it is still applied so far without Effective target site drug, causes TNBC
Patient's prognosis mala, the death rate increase.
Transfer and relapse is the principal element for leading to TNBC treatment failure.Firstly, TNBC is able to carry out adjacent tissue infiltration.Cream
Glandular epithelium can undergo EMT, this is a kind of transducer of hight coordinate, most early in finding in embryonic development, with cell bone
Frame remodeling, cell Basolateral polarity missing and cell are related to iuntercellular dissolution etc..Wherein, Slug, Smad, Snail,
The genes such as Twist and TGF-β take part in this process, cause TNBC cellular invasion to adjacent tissue organ；In addition, TNBC can also
It is shifted and is survived by Peripheral Circulation tumour cell (Circulating tumor cells, CTCs), cell adhesion is caused to increase
Add, causing breast cancer cell, organ is colonized at a distance.The patient of TNBC is diagnosed latter 2 years due to locally invading caused by EMT
Attack, vascular exosmosis and distal end survival field planting cause patient's cancer metastasis recur, treatment with chemotherapy drug can make patient tumors thin again
Born of the same parents generate drug resistance, so as to cause treatment failure.
Therefore, the molecular mechanism for furtheing investigate TNBC occurrence and development, studies novel target drug, carries out to TNBC patient a
Body targeted therapy is of great significance.
KTN1 is a kind of membrane receptor protein, is positioned on No. 14 chromosome q22.1, and molecular weight is 120KD and 160KD.
KTN1 majority is located on endoplasmic reticulum, is present in mitochondria on a small quantity.KTN1 can participate in endoplasmic reticulum by recruiting focal adhension
Extension；Mitochondrial metabolism function can also be increased by interacting with driving albumen (Kinesin).Hair of the KTN1 in tumour
Play a significant role in hair tonic exhibition and transfer.However, many be still in the elementary step for research of the KTN1 in tumour.Than
Such as, in triple negative breast cancer KTN1 be how to play a role it is current without research report.Meanwhile potential molecular biology
Mechanism is also unclear.
Summary of the invention
It is an object of the invention to overcome the deficiencies of existing technologies, a kind of small interference of targeting KTN1 treatment breast cancer is provided
RNA and its application.
In order to achieve the above object, first aspect present invention provides a kind of siRNA of targeting KTN1 treatment breast cancer, institute
State siRNA sequence include in the sequence as shown in SEQ ID NO.1 and the sequence as shown in SEQ ID NO.2 at least
It is a kind of.
As a further improvement of the above technical scheme, the siRNA includes at least one lock nucleic acid molecule.
Second aspect of the present invention provides a kind of siRNA as described above answering in preparation treatment breast cancer medicines
With KTN1 gene expression in breast cancer cell is lowered in the siRNA targeting.
As a further improvement of the above technical scheme, the treatment breast cancer medicines are treatment triple negative breast cancer medicine
Object.
As a further improvement of the above technical scheme, the siRNA inhibit triple negative breast cancer cell proliferation,
Transfer and invasive ability.
Third aspect present invention provides a kind of pharmaceutical composition of targeting KTN1 treatment breast cancer, including as described above small
RNA interfering and pharmaceutical acceptable carrier.
As a further improvement of the above technical scheme, described pharmaceutical acceptable carrier includes sugar, polyamine, amino acid, peptide, lipid
At least one of.
Fourth aspect present invention provides a kind of pharmaceutical composition as described above answering in preparation treatment breast cancer medicines
With KTN1 gene expression in breast cancer cell is lowered in described pharmaceutical composition targeting.
As a further improvement of the above technical scheme, the treatment breast cancer medicines are treatment triple negative breast cancer medicine
Object.
As a further improvement of the above technical scheme, the Pharmaceutical composition inhibits the increasing of triple negative breast cancer cell
It grows, shift and invasive ability.
Beneficial effects of the present invention:
The present invention provides a kind of siRNA and its application for targeting KTN1 and treating breast cancer.The sequence of the siRNA
Column include at least one of the sequence as shown in SEQ ID NO.1 and the sequence as shown in SEQ ID NO.2.The small interference
RNA inhibits three negative breasts to lower the expression of the KTN1 gene by the KTN1 gene of targeting triple negative breast cancer cell
Proliferation, transfer and the invasive ability of cancer cell, it is final to improve or treat triple negative breast cancer.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of the scope of the invention.
The qRT-PCR method in the embodiment of the present invention 1 that Fig. 1 shows detect respectively normal mammary epithelial MCF10A,
The cell line mcf-7 of estrogenic/progestogenic receptor positive, ZR75-1, T47D and triple negative breast cancer cell line TNBC1, MDA-
The expression of KTN1RNA in MB-231, BT549；
Fig. 2 shows Western blot analysis method in the embodiment of the present invention 1, to detect normal breast epithelial respectively thin
Born of the same parents MCF10A, the cell line mcf-7 of estrogenic/progestogenic receptor positive, ZR75-1, T47D and triple negative breast cancer cell line
The expression of KTN1 protein in TNBC1, MDA-MB-231, BT549；
Fig. 3 shows the tumor tissues of the clinical each 5 groups of TNBC patients collected, cancer beside organism in the embodiment of the present invention 1
With the expression of the KTN1RNA of lymph node tissue；
Fig. 4 show in the embodiment of the present invention 2 siNC, siKTN1_1 and siKTN1_2 transfect MDA-MB-231 cell and
After BT549 cell, the KTN1RNA expression that is detected using qRT-PCR method；
Fig. 5 shows siNC, siKTN1_1 and siKTN1_2 in the embodiment of the present invention 2 and transfects MDA-MB-231 cell
0h, for 24 hours, after 48h and 72h, the MDA-MB-231 cell Proliferation variation diagram that is detected using CCK8 method；
Fig. 6 show in the embodiment of the present invention 2 siNC, siKTN1_1 and siKTN1_2 transfect BT549 cell 0h, for 24 hours,
After 48h and 72h, the BT549 cell Proliferation variation diagram that is detected using CCK8 method；
Fig. 7 shows siNC, siKTN1_1 and siKTN1_2 in the embodiment of the present invention 3 and transfects MDA-MB-231 cell
The electron microscope of cell migration and invasion after 48h；
Fig. 8 shows siNC, siKTN1_1 and siKTN1_2 in the embodiment of the present invention 3 and transfects MDA-MB-231 cell
For the cell quantity statistical chart of the cell migration electron microscope of Fig. 7 after 48h；
Fig. 9 shows siNC, siKTN1_1 and siKTN1_2 in the embodiment of the present invention 3 and transfects MDA-MB-231 cell
For the cell quantity statistical chart of the cell invasion electron microscope of Fig. 7 after 48h；
Figure 10 is shown in the embodiment of the present invention 3 after siNC, siKTN1_1 and siKTN1_2 transfection BT549 cell 48h
Cell migration and invasion electron microscope；
Figure 11 is shown in the embodiment of the present invention 3 after siNC, siKTN1_1 and siKTN1_2 transfection BT549 cell 48h
For the cell quantity statistical chart of the cell migration electron microscope of Figure 10；
Figure 12 is shown in the embodiment of the present invention 3 after siNC, siKTN1_1 and siKTN1_2 transfection BT549 cell 48h
For the cell quantity statistical chart of the cell invasion electron microscope of Figure 10；
Figure 13 show in the embodiment of the present invention 4 transfect siNC, siKTN1_1 and siKTN1_2 after MDA-MB-231 it is thin
The protein expression situation of N-Cadherin, β-Catenin of born of the same parents, Vimentin；
Figure 14 shows the N- of BT549 cell after transfection siNC, siKTN1_1 and siKTN1_2 in the embodiment of the present invention 4
The protein expression situation of Cadherin, β-Catenin, Vimentin.
Specific embodiment
Term as used herein:
" by ... preparation " it is synonymous with "comprising".Term "comprising" used herein, " comprising ", " having ", " containing "
Or its any other deformation, it is intended that cover non-exclusionism includes.For example, composition, step, method comprising listed elements,
Product or device are not necessarily limited to those elements, but may include not expressly listed other elements or such composition, step
Suddenly, method, product or the intrinsic element of device.
Conjunction " by ... form " exclude any element that do not point out, step or component.If in claim,
This phrase will make claim closed, so that it is not included the material in addition to the material of those descriptions, but relative
Except customary impurities.When phrase " by ... form " be rather than immediately following theme in the clause that appears in claim main body after
When, only it is limited to element described in the clause；Other elements be not excluded the claim as a whole it
Outside.
Equivalent, concentration or other values or parameter are excellent with range, preferred scope or a series of upper limit preferred values and lower limit
When the Range Representation that choosing value limits, this should be understood as specifically disclosing by any range limit or preferred value and any range
Any pairing of lower limit or preferred value is formed by all ranges, regardless of whether the range separately discloses.For example, when open
When range " 1~5 ", described range should be interpreted as including range " 1~4 ", " 1~3 ", " 1~2 ", " 1~2 and 4~
5 ", " 1~3 and 5 " etc..When numberical range is described herein, unless otherwise stated, otherwise the range is intended to include its end
Value and all integers and score in the range.
"and/or" is used to indicate that one of illustrated situation or both may to occur, for example, A and/or B includes (A
And B) and (A or B).
As used herein, term " siRNA ", " Small interfering RNA " and " siRNA " meaning are identical.
One embodiment of the invention provides a kind of siRNA of targeting KTN1 treatment breast cancer, the small interference
The sequence of RNA includes at least one of the sequence as shown in SEQ ID NO.1 and the sequence as shown in SEQ ID NO.2.
SiRNA can be interfered by RNA, efficiently, specifically block the expression of internal homologous gene, be promoted homologous
MRNA degradation lures that cells show goes out the phenotype of specific gene missing into.
In certain embodiments of the present invention, the siRNA can target the table for lowering KTN1 gene in vivo
It reaches, to achieve the effect that targeted silent KTN1 gene.
Optionally, in the present invention, the siRNA includes at least one lock nucleic acid molecule.Lock nucleic acid (locked
Nucleic acid, LNA) herein refer to siRNA in certain nucleotide be lock nucleic acid monomer, i.e., bicyclic nucleoside or its
Analog, specifically the ribose moieties in siRNA have had more the oxygen atom and 4'- that can be connected on 2'- hydroxyl
The bridged bond of carbon atom so that the ribose moieties, which are fixed in 3'-, is configured (3'-endo conformation), thus makes small dry
It disturbs RNA to be not easy by nuclease hydrolysis, and siRNA activity is not changed, more preferably, small interference can be improved in the lock nucleic acid
Stability, activity or the therapeutic effect of RNA.
SiRNA of the invention also may include other kind of modification, such as have modification (2'-sugar at 2'- sugar
Modification), between nucleic acid molecules junction have modification (modified internucleoside linkage) or on
State the combination of different modes.
It should be understood that any largely or entirely active modification for being able to maintain the siRNA is included in this hair
In bright.
It is understood that siRNA of the invention can be synthesized by chemical mode, such as it can use nucleic acid chemistry
In polymerization reaction manufacture the siRNA.
Another embodiment of the present invention provides a kind of application of siRNA in preparation treatment breast cancer medicines, institute
It states siRNA targeting and lowers KTN1 gene expression in breast cancer cell.The sequence of the siRNA includes such as SEQ ID
At least one of sequence shown in NO.1 and the sequence as shown in SEQ ID NO.2.
Specifically, the treatment breast cancer medicines are treatment triple negative breast cancer drug.
More specifically, the siRNA inhibits proliferation, transfer and the invasive ability of triple negative breast cancer cell.
In general, the siRNA is used in the form of pharmaceutical composition in the present invention.Therefore, the present invention is another
A embodiment provides a kind of pharmaceutical composition of targeting KTN1 treatment breast cancer, including siRNA and pharmaceutical acceptable carrier,
In, the sequence of the siRNA includes in the sequence as shown in SEQ ID NO.1 and the sequence as shown in SEQ ID NO.2
At least one.
Optionally, which can prepare pharmaceutical preparations with various pharmaceutical acceptable carrier molecules.It is presently described small dry
Disturb RNA can with enhance its enter target cell ability pharmaceutical acceptable carrier molecule it is compound.This pharmaceutical acceptable carrier molecule includes but not
It is limited to sugar, polyamine, amino acid, peptide, lipid and other pairs of cells and grows indispensable molecule.For example, siRNA can be with rouge
Plastid combines to form pharmaceutical formulation.
Any way that the pharmaceutical composition can be used for transporting siRNA to desired site such as mammary gland is given.Such as
It can be by way of intramuscular, intraperitoneal or intravenous injection and apply the siRNA of present embodiment.Have as one
SiRNA is injected into individual in vivo by individual limb muscle, such as arm or leg somewhere position by body embodiment.
Above-mentioned, the Pharmaceutical composition is in the application in preparation treatment breast cancer medicines, under described pharmaceutical composition targeting
Adjust breast cancer cell in KTN1 gene expression, so as to improve or treatment to breast cancer.
Specifically, the treatment breast cancer medicines are treatment triple negative breast cancer drug.
Specifically, the Pharmaceutical composition inhibits proliferation, transfer and the invasive ability of triple negative breast cancer cell.
The siRNA passes through the KTN1 gene of targeting triple negative breast cancer cell, to lower the table of the KTN1 gene
It reaches, inhibits proliferation, transfer and the invasive ability of triple negative breast cancer cell, it is final to improve or treat triple negative breast cancer.
Below with reference to experiment, technical solution of the present invention is further illustrated.
Materials and methods:
(1) cellular material: triple negative breast cancer clinical tissue sample is collected, carries out originally culture in vitro, i.e.,
TNBC1；Choose normal mammary epithelial MCF10A；Choose cell line mcf-7, the ZR75- of estrogenic/progestogenic receptor positive
1 and T47D；Triple negative breast cancer cell line MDA-MB-231 and BT549 is chosen, temperature is 37 DEG C, mass concentration is 5%
CO2Under the conditions of incubator, secondary culture is carried out in culture dish using the FBS DMEM culture medium that mass concentration is 10% respectively.
(2) KTN1 gene silencing model construction: when MDA-MB-231 and BT549 cell confluency degree it is long to 70% when, will
MDA-MB-231 and BT549 cell is respectively divided into 3 groups, i.e. siNC group, siKTN1_1 group and siKTN1_2 group.
Using 30 μ l lipofectamine2000 liposomes mix respectively 50nM 15 μ L siNC, siKTN1_1 and
SiKTN1_2, then dilute to obtain three groups of mixed liquors with serum-free antibiotic-free DMEM culture medium respectively, every group of mixing after dilution
Liquid total volume is 400 μ l.Wherein, siKTN1_1 sequence are as follows: 5'-GAGTGATCTTTCTAGCAAA-3', such as SEQ ID NO.1 institute
Show；SiKTN1_2 sequence are as follows: 5'-GAAGTCTGGTGTAATACAA-3', as shown in SEQ ID NO.2；SiNC group is negative right
According to group, siNC is provided by Guangzhou Ribo Bio Co., Ltd., and name of product is siR NC#1, and product number is
siN0000001-1-5。
Mixed liquor is stored at room temperature 15min.Then in the siNC group of MDA-MB-231 and BT549 cell, siKTN1_1 group
Be separately added into the culture dish of siKTN1_2 group volume be 4.4ml serum-free antibiotic-free DMEM culture medium, three groups are mixed
It closes liquid to be added dropwise in corresponding culture dish, keeps siNC, siKTN1_1 and siKTN1_2 transfection MDA-MB-231 and BT549 thin
Born of the same parents discard old culture medium in culture dish after cultivating 3-5h, the DMEM culture medium containing serum and antibiotic are added, and are 37 in temperature
DEG C, mass concentration be 5% CO2Continue culture under condition of culture and obtains the MDA-MB- of KTN1 gene silencing model construction completion
231 with BT549 cell.
(3) MDA- that KTN1 gene silencing model construction is completed reverse transcription and qRT-PCR reaction: is collected after 48h respectively
MB-231 and BT549 cell, are washed twice with PBS, using trypsin digestion and cell, are collected in 1.5ml centrifuge tube.Trizol
Method extracts intracellular total serum IgE, reverse transcription reaction system are as follows:
| Reagent | Usage amount |
| Total RNA | 1μg |
| Anchored Oligo(dT) | 1μl |
| 2×ES Reaction Mix | 10μl |
| EasyScript RT/TI Enzyme Mix | 1μl |
| gDNA Remover | 1μl |
| Total volume | 20μl |
After temperature is 42 DEG C of incubation 15min, then the enzymatic mixture heated in 5s inactivation reaction system at 85 DEG C, finally exist
Temperature is incubated for until cooling under the conditions of being 4 DEG C.
KTN1 primer sequence are as follows:
HKTN1_FP:5'-ATGCAGTTGAACACCAGAGGAAGA-3'；
HKTN1_RP:5'-ATGCAACCATTCACCATAACTCAAA-3'.
(4) CCK-8 method detects cell Proliferation:
The good MDA-MB-231 of growth conditions after the completion of KTN1 gene silencing model construction is taken to give birth to BT549 logarithm respectively
Long-term cell, adjustment cell density to 6 × 104A/ml, 100 holes μ l/ are inoculated in 96 well culture plates, every group of 5 multiple holes.Point
Not in 0h, for 24 hours, after 48h and 72h, the CCK-8 solution of 10% volume is added in every hole, and avoid light place is in 37 DEG C, mass concentration
5% CO22-4h is cultivated in incubator, measures its absorbance value at 450nm with microplate reader.
(5) Transwell detects cell migration ability:
The good MDA-MB-231 of growth conditions after the completion of KTN1 gene silencing model construction is taken to give birth to BT549 logarithm respectively
Long-term cell, using trypsin digestion and cell, the PBS washing cell of 37 DEG C of pre-temperatures is twice.
Prepare 24 orifice plates and the sterile cell transwell, plasma-free DMEM medium suspension MDA-MB-231 and BT549 is thin
Born of the same parents, adjustment cell density are 1 × 105A/ml takes 200 μ l of suspension cell that cell is added.750 μ l, which are added, in 24 orifice plates serum
Complete DMEM culture medium, then cell is placed in 24 orifice plates, 37 DEG C, the CO that mass concentration is 5%2It is incubated in incubator
12-16h.3 repetitions of every group of setting.
It is cleaned 2 times after the completion of incubation with PBS, the fixed cell 2min of 3% formaldehyde；Removal formaldehyde, PBS cleaning 2 times, 100%
Methanol carries out cell permeabilization and fixes, and is incubated at room temperature 20min；Methanol is removed, PBS is cleaned 2 times；It the use of mass concentration is 0.1% knot
Crystalviolet dyeing, is incubated at room temperature 15min；After violet staining washing, cell is dried, is counted under 200 × Electronic Speculum and moves to cell bottom
The cell in face.Observe 5 high power fields, computation migration cell average.
(6) Transwell detects cell invasion ability:
The good MDA-MB-231 of growth conditions after the completion of KTN1 gene silencing model construction is taken to give birth to BT549 logarithm respectively
Long-term cell, using trypsin digestion and cell, the PBS washing cell of 37 DEG C of pre-temperatures is twice.
Prepare 24 orifice plates, while the sterile cell the transwell taking-up for completing matrigel that -20 DEG C are stored being placed in
37 DEG C of incubators are to 30min, plasma-free DMEM medium suspension MDA-MB-231 and BT549 cell, and adjustment cell density is 1 ×
105A/ml takes 200 μ l of suspension cell that cell is added.The complete DMEM culture medium that 750 μ l have serum is added in 24 orifice plates, then will
Cell is placed in 24 orifice plates, and 37 DEG C, the CO that mass concentration is 5%216-24h is incubated in incubator.3 repetitions of every group of setting.
It after the completion of incubation, is cleaned 2 times with PBS, the fixed cell 2min of 3% formaldehyde；Removal formaldehyde, PBS cleaning 2 times, 100%
Methanol carries out cell permeabilization and fixes, and is incubated at room temperature 20min；Methanol is removed, PBS is cleaned 2 times；It the use of mass concentration is 0.1% knot
Crystalviolet dyeing, is incubated at room temperature 15min；After violet staining washing, cell is dried, is counted under 200 × Electronic Speculum and moves to cell bottom
The cell in face.5 high power fields are observed, invasion cell average is calculated.
(7) Western blot analysis: 100 μ l RIPA protein lysates and 25 × PIC protease is added in each culture dish
Inhibitor freezes cracking at a temperature of -80 DEG C.Next day, sample are 12000rpm in revolving speed, are centrifuged under the conditions of 4 DEG C of temperature
Then 15min collects supernatant, i.e. protein lysate, measure concentration, carries out Western blot analysis.
(8) statistical analysis uses SPSS21.0 version software, executes independent sample one-way analysis of variance, three groups of data
Compare using variance analysis, while carrying out homogeneity of variance analysis, p < 0.001 * * p < 0.05, * * p < 0.01, * * has statistics
Meaning.
Embodiment of the present invention is described in detail below in conjunction with specific embodiment, but those skilled in the art
It will be understood that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the invention.It is not specified in embodiment
Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer,
It is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
The present embodiment is related to the rna expression situation of KTN1 gene in triple negative breast cancer (TNBC) specimens:
(1) collect clinical normal mammary epithelial MCF10A, estrogenic/progestogenic receptor positive cell line mcf-7,
ZR75-1, T47D and triple negative breast cancer cell line TNBC1, MDA-MB-231 and BT549 cell carry out qRT-PCR detection respectively
With Western blot analysis, as a result as shown in Figures 1 and 2 (in Fig. 1, p < 0.001 * p < 0.05, * * p < 0.01, * * *).Fig. 1
Indicate clinical MCF10A, MCF-7, ZR75-1, T47D, TNBC1, MDA-MB-231 and the BT549 collected of qRT-PCR method detection
The expression of KTN1RNA in cell；Fig. 2 indicate using Western blot analysis clinic collect MCF10A, MCF-7,
The expression of KTN1 protein and β-Actin in ZR75-1, T47D, TNBC1, MDA-MB-231 and BT549 cell.Wherein,
β-Actin is the main of one of muscle rhabdomyosarcoma fiber major protein components and muscle filament and cytoskeletal filament
Ingredient.β-Actin is good internal reference index in protein immunoblot test.Internal reference is internal referring to (Internal
Control), the albumen that expression is encoded by house-keeping gene is generally referred to for mammalian cell expression.They are in each group
Knit it is relative constant with the expression in cell, detect albumen expression variation when often make object of reference with it.
As shown in Figure 1, in TNBC1, MDA-MB-231 and BT549 cell the expression quantity of KTN1RNA be significantly larger than MCF10A,
The expression quantity of KTN1RNA in MCF-7, ZR75-1, T47D；
As shown in Figure 2, in TNBC1, MDA-MB-231 and BT549 cell KTN1 protein expression quantity be higher than MCF10A,
The expression quantity of KTN1 protein in MCF-7, ZR75-1, T47D.
(2) tumor tissues, cancer beside organism and the sentinel nodal tissue for collecting 5 groups of clinic TNBC patients, carry out respectively
QRT-PCR detection, as a result as shown in Figure 3 (in Fig. 3, p < 0.001 * p < 0.05, * * p < 0.01, * * *), in TNBC tumor tissues
The expression quantity of KTN1RNA is significantly larger than the expression quantity of KTN1RNA in cancer beside organism and sentinel nodal tissue.
Conclusion: KTN1 gene is related to the generation of TNBC.
Embodiment 2
The present embodiment be related to detect siRNA targeted silent KTN1 gene pairs TNBC cell line MDA-MB-231 and
The influence of the proliferation of BT549 cell and KTN1RNA expression.
Detected respectively using qRT-PCR method MDA-MB-231 cell after the completion of KTN1 gene silencing model construction and
The variation of the siNC group, the KTN1RNA expression of siKTN1_1 group and siKTN1_2 group of BT549 cell.Specifically such as Fig. 4 institute
Show, the KTN1RNA expression of the MDA-MB-231 and BT549 cell after transfecting siKTN1_1 and siKTN1_2 is far below control
The KTN1RNA expression of group (siNC), the success of siKTN1_1 and siKTN1_2 silencing model foundation；
Fig. 5 indicates siNC, siKTN1_1 and siKTN1_2 transfection MDA-MB-231 cell 0h, for 24 hours, after 48h and 72h, make
With the variation diagram (* p < 0.05, p < 0.001 * * p < 0.01, * * * in figure) of CCK8 method detection MDA-MB-231 cell Proliferation.It is logical
After Fig. 5 is crossed it is found that transfecting siKTN1_1 and siKTN1_2, compared with control group (siNC), with the extension of incubation time, MDA-
The proliferative capacity of MB-231 cell weakens, and illustrates that siKTN1_1 and siKTN1_2 transfection MDA-MB-231 cell can weaken MDA-
The proliferative capacity of MB-231 cell.
Fig. 6 indicates siNC, siKTN1_1 and siKTN1_2 transfection BT549 cell 0h, for 24 hours, after 48h and 72h, use CCK8
The variation diagram (* p < 0.05, p < 0.001 * * p < 0.01, * * * in figure) of method detection BT549 cell Proliferation.As shown in Figure 6, turn
After contaminating siKTN1_1 and siKTN1_2, compared with control group (siNC), with the extension of incubation time, the proliferation of BT549 cell
Ability obviously weakens, and illustrates that siKTN1_1 and siKTN1_2 transfection BT549 cell can weaken the proliferative capacity of BT549 cell.
Conclusion: KTN1 gene can regulate and control the proliferative capacity of TNBC cell line MDA-MB-231 and BT549 cell；And silencing
KTN1 gene is able to suppress the proliferative capacity of TNBC cell line MDA-MB-231 and BT549 cell.
Embodiment 3
The present embodiment is related to detecting siRNA to the migration of TNBC cell line MDA-MB-231 and BT549 cell and invade
Attack the influence of ability.
The MDA-MB-231 cell after the completion of KTN1 gene silencing model construction is had detected using Transwell technology
SiNC group, the migration of siKTN1_1 group and siKTN1_2 group and invasive ability, please refer to Fig. 7, Fig. 8 and Fig. 9, Fig. 7 siNC,
The electron microscope of cell migration and invasion after siKTN1_1 and siKTN1_2 transfection MDA-MB-231 cell 48h, Fig. 8 siNC,
For the cell quantity system of the cell migration electron microscope of Fig. 7 after siKTN1_1 and siKTN1_2 transfection MDA-MB-231 cell 48h
The cell invasion Electronic Speculum of Fig. 7 is directed to after meter figure, Fig. 9 siNC, siKTN1_1 and siKTN1_2 transfection MDA-MB-231 cell 48h
The cell quantity statistical chart of figure, p < 0.001 * p < 0.05, * * p < 0.01, * * * in Fig. 7, Fig. 8 and Fig. 9.As shown in Figure 7, it transfects
After siKTN1_1 and siKTN1_2, compared with control group (siNC), the transfer ability of MDA-MB-231 cell is reduced, and is invaded simultaneously
Ability also reduces；From Fig. 8 and Fig. 9 it is found that after transfection siKTN1_1 and siKTN1_2, compared with control group (siNC), MDA-MB-
231 cell numbers reduce about 2-5 times.
Had detected using Transwell technology the BT549 cell after the completion of KTN1 gene silencing model construction siNC group,
The migration of siKTN1_1 group and siKTN1_2 group and invasive ability, please refer to Figure 10, Figure 11 and Figure 12, Figure 10 siNC,
The electron microscope of cell migration and invasion after siKTN1_1 and siKTN1_2 transfection BT549 cell 48h, Figure 11 siNC,
The cell quantity statistical chart of the cell migration electron microscope of Figure 10 is directed to after siKTN1_1 and siKTN1_2 transfection BT549 cell 48h,
Figure 12 is directed to the cell of the cell invasion electron microscope of Figure 10 after being siNC, siKTN1_1 and siKTN1_2 transfection BT549 cell 48h
Quantity statistics figure；P < 0.001 * p < 0.05 in Figure 10, Figure 11 and Figure 12, * * p < 0.01, * * *.As shown in Figure 10, it transfects
After siKTN1_1 and siKTN1_2, compared with control group (siNC), the transfer ability of BT549 cell is reduced, while invasive ability
Also it reduces；By Figure 11 and Figure 12 it is found that after transfection siKTN1_1 and siKTN1_2, compared with control group (siNC), BT549 cell
Number reduces 9-10 times.
Conclusion: siKTN1_1 and siKTN1_2 being capable of targeted silent KTN1 gene；Silencing KTN1 gene can significantly inhibit
The migration of TNBC cell line MDA-MB-231 and BT549 cell and invasive ability.
Embodiment 4
The present embodiment is related to the correlation point that KTN1 and EMT is expressed in TNBC cell line MDA-MB-231 and BT549 cell
Analysis.
N-Cadherin (N- cadherin) is also referred to as cadherin -2 (CDH2) or neural cadherin
(NCAD), it is protein that human body is encoded by CDH2 gene；N- cadherin is to express and play mediation in Various Tissues carefully
The transmembrane protein of born of the same parents-cell adherence function.β-Catenin (beta-catenin) is predominantly located at cell membrane, and in endochylema middle reaches
Less from measuring, beta-catenin is the subunit of cadherin complex, and plays intracellular signal transduction in Wnt signal pathway
Effect.Vimentin (vimentin) is responsible for control and transmits the gallbladder as derived from low-density lipoprotein (LDL) from lysosome
Sterol is to esteratic site.N-Cadherin, β-Catenin, Vimentin are used as metastases correlation factor in epithelial cell-
It plays an important role during mesenchyma conversion (EMT).
Detect the MDA-MB- after the completion of KTN1 gene silencing model construction respectively using Western blot analysis method
The siNC group of 231 cells and BT549 cell, siKTN1_1 group and siKTN1_2 group N-Cadherin, β-Catenin,
The protein expression of Vimentin and β-Actin, wherein β-Actin is internal reference.Specifically as shown in Figure 13 and 14, wherein scheming
13 show transfection siNC, siKTN1_1 and siKTN1_2 after MDA-MB-231 cell N-Cadherin, β-Catenin,
The protein expression situation of Vimentin and β-Actin, after Figure 14 shows transfection siNC, siKTN1_1 and siKTN1_2
The protein expression situation of N-Cadherin, β-Catenin of BT549 cell, Vimentin and β-Actin.It knows under targeting
Adjust KTN1 expression, can inhibit influence triple negative breast cancer cell Proliferation, transfer, invasive ability N-Cadherin, β-
The expression of Catenin and Vimentin, it is final to improve or treat triple negative breast cancer.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
In addition, it will be appreciated by those of skill in the art that although some embodiments in this include institute in other embodiments
Including certain features rather than other feature, but the combination of the feature of different embodiment means in the scope of the present invention
Within and form different embodiments.For example, in claims above, embodiment claimed it is any it
One can in any combination mode come using.The information disclosed in the background technology section is intended only to deepen to the present invention
General background technology understanding, and be not construed as recognizing or imply that information composition has been this field skill in any form
The prior art well known to art personnel.
SEQUENCE LISTING
<110>Shenzhen people's hospital
<120>siRNA of targeting KTN1 treatment breast cancer and its application
<130> 2019
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> RNA
<213>artificial synthesized
<400> 1
gagtgatctt tctagcaaa 19
<210> 2
<211> 19
<212> RNA
<213>artificial synthesized
<400> 2
gaagtctggt gtaatacaa 19
Claims (10)
1. a kind of siRNA of targeting KTN1 treatment breast cancer, which is characterized in that the sequence of the siRNA includes such as
At least one of sequence shown in SEQ ID NO.1 and the sequence as shown in SEQ ID NO.2.
2. siRNA as described in claim 1, which is characterized in that the siRNA includes at least one lock nucleic acid point
Son.
3. application of the siRNA as described in claim 1 in preparation treatment breast cancer medicines, which is characterized in that described
KTN1 gene expression in breast cancer cell is lowered in siRNA targeting.
4. application of the siRNA as claimed in claim 3 in preparation treatment breast cancer medicines, which is characterized in that described
Treating breast cancer medicines is treatment triple negative breast cancer drug.
5. application of the siRNA as claimed in claim 4 in preparation treatment breast cancer medicines, which is characterized in that described
Proliferation, transfer and the invasive ability of siRNA inhibition triple negative breast cancer cell.
6. a kind of pharmaceutical composition of targeting KTN1 treatment breast cancer, which is characterized in that including as described in claim 1 small dry
Disturb RNA and pharmaceutical acceptable carrier.
7. pharmaceutical composition as claimed in claim 6, which is characterized in that described pharmaceutical acceptable carrier includes sugar, polyamine, amino
At least one of acid, peptide, lipid.
8. application of the pharmaceutical composition as claimed in claim 6 in preparation treatment breast cancer medicines, which is characterized in that described
KTN1 gene expression in breast cancer cell is lowered in pharmaceutical composition targeting.
9. application of the pharmaceutical composition as claimed in claim 8 in preparation treatment breast cancer medicines, which is characterized in that described
Treating breast cancer medicines is treatment triple negative breast cancer drug.
10. application of the pharmaceutical composition as claimed in claim 9 in preparation treatment breast cancer medicines, which is characterized in that institute
State proliferation, transfer and invasive ability that Pharmaceutical composition inhibits triple negative breast cancer cell.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013154422A1 (en) * | 2012-04-13 | 2013-10-17 | Erasmus University Medical Center Rotterdam | Biomarkers for triple negative breast cancer |
| CN106668863A (en) * | 2017-02-21 | 2017-05-17 | 南方医科大学 | Medicine targeting KTN1 to treat skin squamous cell carcinoma |
| CN109715802A (en) * | 2016-03-18 | 2019-05-03 | 卡里斯科学公司 | Oligonucleotide probe and application thereof |
-
2019
- 2019-06-20 CN CN201910537696.4A patent/CN110229817B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013154422A1 (en) * | 2012-04-13 | 2013-10-17 | Erasmus University Medical Center Rotterdam | Biomarkers for triple negative breast cancer |
| CN104471402A (en) * | 2012-04-13 | 2015-03-25 | 鹿特丹伊拉斯谟大学医疗中心 | Biomarkers for triple negative breast cancer |
| CN109715802A (en) * | 2016-03-18 | 2019-05-03 | 卡里斯科学公司 | Oligonucleotide probe and application thereof |
| CN106668863A (en) * | 2017-02-21 | 2017-05-17 | 南方医科大学 | Medicine targeting KTN1 to treat skin squamous cell carcinoma |
Non-Patent Citations (1)
| Title |
|---|
| MOAMER A ET AL.: "A role for kinesin-1 subunits KIF5B/KLC1 in regulating epithelial mesenchymal plasticity in breast tumorigenesis", 《EBIOMEDICINE》 * |
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