CN110029107A - Target the oligonucleotides of SNHG17 treatment breast cancer - Google Patents
Target the oligonucleotides of SNHG17 treatment breast cancer Download PDFInfo
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- CN110029107A CN110029107A CN201910321663.6A CN201910321663A CN110029107A CN 110029107 A CN110029107 A CN 110029107A CN 201910321663 A CN201910321663 A CN 201910321663A CN 110029107 A CN110029107 A CN 110029107A
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Abstract
The present invention provides a kind of oligonucleotides of targeting SNHG17 treatment breast cancer, the oligonucleotides includes at least one of antisense oligonucleotides and siRNA, wherein, the sequence of antisense oligonucleotides is as shown in SEQ ID NO.1, and the sequence of siRNA is as shown in SEQ ID NO.2-4.The oligonucleotides can regulate and control proliferation, transfer and the invasive ability of triple negative breast cancer cell by the expression of SNHG17 gene in targeted silent triple negative breast cancer cell or downward SNHG17 gene, final to improve or treat triple negative breast cancer.
Description
Technical field
The present invention relates to gene therapy technology field more particularly to a kind of few nucleosides of targeting SNHG17 treatment breast cancer
Acid.
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；By the end of 2008,
Breast cancer is the 6th cause of the death that Chinese women dies of cancer, after coming lung cancer, gastric cancer, liver cancer, the cancer of the esophagus and colorectal cancer,
Age-standardized ratio (ASR) be 5.7/10 ten thousand women, the 9.6% of Zhan Quanqiu breast cancer deaths number；At the same time, due to multiple
Reason, many new drugs, which can not obtain, limits the systematic treating progress of breast cancer；New adjuvant chemotherapy is commonplace simultaneously, about
81.4% infiltrative breast carcinoma patient will do it chemotherapy, however the effect of chemotherapy is less desirable.
Due to the exploitation of molecular targeted agents in recent years, as aromatizing enzyme drug, Tamoxifen, Fulvestrant,
Trastuzumab and receptor type mammary gland can be efficiently controlled with CDK4/6, PI3K/Akt/mTOR related inhibitors use in conjunction
Carcinogenesis development.However, triple negative breast cancer (Triple negative breast cancer, TNBC) is breast cancer parting
In the most pernicious one kind, since TNBC cell surface do not express estrogen receptor (Estrogen receptor, ER), pregnant swashs
Plain receptor (Progesterone receptor, PR) and ErbB-2 (Human epidermal
Growth factor receptor-2, HER-2), cause still to be applied without Effective target site drug so far, there is TNBC patient
Very poor prognosis.TNBC patient can be since local challenge, intravascular infiltration and extravasation and distal end survival field planting be led in 2 years after being diagnosed
Its transfer and relapse is caused, treatment with chemotherapy drug can cause patient tumors cell to generate drug resistance again, so as to cause treatment failure.Cause
This, several genes access participates in the transfer of TNBC, shifts resistant characterization according to TNBC, learns to do section screening transfer drug resistance by multiple groups
Target gene develops new target treatment list medicine and/or combined chemotherapy, before providing treatment to the benefited property of clinical TNBC Patient drug
Prediction makes patient be benefited.
Small nuclear rna (small nucleolar RNAs, snoRNAs) is considered as group important in protein synthesis mechanism
At part.These non-coding RNAs play key effect in terms of regulating cell destiny and tumour generation；Meanwhile also having been reported that card
The host gene of bright snoRNAs can also promote the development of cancer, they are stored in long-chain non-coding RNA (long non-coding
RNA, lncRNA) and non-protein encoding gene include sub-district, (the small nucleolar RNA of small nuclear rna host gene 17
Host gene17, SNHG17) it is a member in these host genes.The SNHG17 assignment of genes gene mapping on No. 20 chromosome p12,
Ensembl database displaying SNHG17 gene has 9 transcripts, is primarily located in nucleus.
Summary of the invention
It is an object of the invention to overcome the deficiencies of existing technologies, a kind of few core of targeting SNHG17 treatment breast cancer is provided
Thuja acid.
To realize that the above goal of the invention, first aspect present invention provide a kind of few nucleosides of targeting SNHG17 treatment breast cancer
Acid, the oligonucleotides include at least one of antisense oligonucleotides and siRNA, wherein the antisense oligonucleotides
Sequence as shown in SEQ ID NO.1, the sequence of the siRNA is as shown in SEQ ID NO.2-4.
As a further improvement of the above technical scheme, the oligonucleotides is using nucleic acid lock modification technique or nucleic acid chains bone
Frame modification technique modified.
Second aspect of the present invention provides a kind of purposes of oligonucleotides as described above, including by few nucleosides as described above
Purposes of the acid for the expression of SNHG17 gene in SNHG17 gene in targeted silent cancer cell or downward cancer cell.
As a further improvement of the above technical scheme, the cancer cell is triple negative breast cancer cell.
As a further improvement of the above technical scheme, including proliferation, transfer and the invasive ability for inhibiting cancer cell
Purposes.
Third aspect present invention also provides a kind of pharmaceutical composition of targeting SNHG17 treatment breast cancer, including as described above
Oligonucleotides 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 also provides a kind of purposes of pharmaceutical composition, including pharmaceutical composition as described above is used
In targeted silent breast cancer cell SNHG17 gene or lower breast cancer cell in SNHG17 gene expression purposes.
As a further improvement of the above technical scheme, the breast cancer cell is triple negative breast cancer cell.
As a further improvement of the above technical scheme, including the proliferation for inhibiting triple negative breast cancer cell, transfer
With the purposes of invasive ability.
Beneficial effects of the present invention:
The present invention provides a kind of oligonucleotides of targeting SNHG17 treatment breast cancer.Oligonucleotides includes antisense oligonucleotides
At least one of with siRNA, wherein the sequence of antisense oligonucleotides as shown in SEQ ID NO.1, siRNA
Sequence is as shown in SEQ ID NO.2-4.The oligonucleotides can pass through SNHG17 base in targeted silent triple negative breast cancer cell
Cause lowers the expression of SNHG17 gene to regulate and control triple negative breast cancer cell Proliferation, transfer and invasive ability, finally changes
Kind or treatment 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.
Fig. 1 is the tumor tissues of TNBC patient in the embodiment of the present invention 1, cancer beside organism and lymph node tissue
The influence of SNHG17RNA expression；
Fig. 2 is NC ASO and SNHG17 ASO interference model in the embodiment of the present invention 2 to MDA-MB-231 cell
The influence of SNHG17RNA expression；
Fig. 3 be the embodiment of the present invention 2 in NC ASO and SNHG17 ASO handle MDA-MB-231 cell 0h, for 24 hours, 48h
After 72h, the variation diagram of CCK8 method detection MDA-MB-231 cell Proliferation is used；
Fig. 4 shows in the embodiment of the present invention 2 BT549 cell in NC ASO and SNHG17 ASO interference model
The expression of SNHG17RNA；
Fig. 5 be in the embodiment of the present invention 2 NC ASO and SNHG17 ASO handle BT549 cell 0h, for 24 hours, 48h and 72h
Afterwards, the variation diagram of BT549 cell Proliferation after interference SNHG17 is detected using CCK8 method；
Fig. 6 is the cell that NC ASO, SNHG17 ASO transfect after MDA-MB-231 cell 48h in the embodiment of the present invention 3
The electron microscope of migration and invasion；
Fig. 7 is that NC ASO, SNHG17 ASO are directed to Fig. 6 after transfecting MDA-MB-231 cell 48h in the embodiment of the present invention 3
Cell migration electron microscope cell quantity statistical chart；
Fig. 8 is that NC ASO, SNHG17 ASO are directed to Fig. 6 after transfecting MDA-MB-231 cell 48h in the embodiment of the present invention 3
Cell invasion electron microscope cell quantity statistical chart；
Fig. 9 be the embodiment of the present invention 3 in NC ASO, SNHG17 ASO transfect BT549 cell 48h after cell migration with
The electron microscope of invasion；
Figure 10 is that NC ASO, SNHG17 ASO are directed to the thin of Fig. 9 after transfecting BT549 cell 48h in the embodiment of the present invention 3
The cell quantity statistical chart of born of the same parents' migration electron microscope；
Figure 11 is that NC ASO, SNHG17 ASO are directed to the thin of Fig. 9 after transfecting BT549 cell 48h in the embodiment of the present invention 3
The cell quantity statistical chart of born of the same parents' invasion electron microscope；
Figure 12 shows in the embodiment of the present invention 4 MDA-MB-231 cell in NC ASO and SNHG17 ASO interference model
Transfer correlation factor expression；
Figure 13 shows siNC, siSNHG17_1, siSNHG17_2 and siSNHG17_3 in the embodiment of the present invention 5 and interferes
The SNHG17RNA expression of MDA-MB-231 cell in model.
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.
" mass parts " refer to the basic measurement unit for indicating the mass ratio relationship of multiple components, and 1 part can indicate arbitrary list
Position quality, can such as be expressed as 1g, may also indicate that 2.689g etc..If we say that the mass parts of component A are a parts, the matter of B component
Measuring part is b parts, then it represents that the quality of component A and the mass ratio a:b of B component.Alternatively, indicating that the quality of component A is aK, B group
The quality divided is bK (K is arbitrary number, indicates multiplying factor).It can not misread, unlike mass fraction, all components
The sum of mass parts be not limited to 100 parts of limitation.
"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)；
In addition, indefinite article "an" before element of the present invention or component and "one" quantitative requirement to element or component
(i.e. frequency of occurrence) unrestriction.Therefore "one" or "an" should be read as including one or at least one, and odd number
The element or component of form also include plural form, unless the obvious purport of the quantity refers to singular.
As used herein, term " antisense oligonucleotides ", " antisense-oligonucleotides ", " AS-Ons ",
" ASO " meaning is identical.
Term " siRNA ", " Small interfering RNA " and " siRNA " meaning are identical.
Antisense oligonucleotides refers to that those can be combined in a manner of base pair complementarity with specific DNA, RNA, and prevents
It transcribes the short nucleotide fragments with translation.SiRNA can be interfered by RNA, efficiently, specifically be blocked homologous in vivo
The expression of gene, promotes degradation of homologous mRNA, lures that cell shows the phenotype of specific gene missing into.
In one embodiment of the invention, a kind of oligonucleotides of targeting SNHG17 treatment breast cancer is provided, it is described
Oligonucleotides includes at least one of antisense oligonucleotides and siRNA, wherein the sequence of the antisense oligonucleotides is such as
Shown in SEQ ID NO.1, the sequence of the siRNA is as shown in SEQ ID NO.2-4.
Specifically, the antisense oligonucleotides and siRNA all have targeted silent SNHG17 gene in vivo or
Lower the expression of SNHG17 gene.
Optionally, in the present invention, the oligonucleotides is using nucleic acid lock modification technique or nucleic acid chains backbone modification technology
Modified.The modification does not change the activity of oligonucleotides substantially, more preferably, described to modify the stabilization that oligonucleotides can be improved
Property, activity or therapeutic effect.
Nucleic acid lock (locked nucleic acid, LNA) typically refers to by a methylene bridge that the 2' oxygen of ribose is former
The modification technique that son and 4' carbon atom connect.LNA can extend the serum half-life of oligonucleotides, improve affine to target
Property, reduce the range and degree of effect of missing the target.
In solubility, nuclease-resistant degradation etc. has greatly to be changed the drug that modification technique based on nucleic acid chain backbone develops
It is kind, and be easy to largely synthesize.There are many nucleic acid chains backbone modification methods of oligonucleotides, including thio method, such as by deoxidation core
Thuja acid chain thio-modification is thio deoxynucleotide chain.This method is by the oxygen atom sulphur atom of the phosphate bond on DNA skeleton
Substitution can resist nuclease degradation.
It should be understood that any largely or entirely active modification for being able to maintain the oligonucleotides is included in the present invention
In.
After oligonucleotides of the present invention transfer into the human body, they can obviously lower related SNHG17 gene
Expression.
In another embodiment of the present invention, a kind of purposes of oligonucleotides is provided, the oligonucleotides can be used for target
Into silencing cancer cell SNHG17 gene or lower cancer cell in SNHG17 gene expression purposes, the oligonucleotides packet
Include antisense oligonucleotides and siRNA, wherein the sequence of the antisense oligonucleotides is described small as shown in SEQ ID NO.1
The sequence of RNA interfering is as shown in SEQ ID NO.2-4.
Specifically, the purposes of the oligonucleotides includes for inhibiting the proliferation of cancer cell, transfer and invasive ability.
More specifically, the cancer cell is triple negative breast cancer cell.
KTN1 is driving binding protein, related to cancer proliferation；N-Cadherin, β-Catenin and Vimentin are
Metastases correlation factor；Proliferation, transfer and the invasive ability of above-mentioned correlation factor control triple negative breast cancer cell.In this reality
Apply in mode, above-mentioned oligonucleotides can by targeting lower SNHG17 gene expression to inhibit KTN1, N-Cadherin,
The expression of β-Catenin and Vimentin, final proliferation, transfer and the invasive ability for inhibiting triple negative breast cancer cell.
In general, the oligonucleotides uses usually in the form of pharmaceutical composition in the present invention.Therefore, in another reality
It applies in mode, a kind of pharmaceutical composition of targeting SNHG17 treatment breast cancer, including oligonucleotides and pharmaceutical acceptable carrier is provided, it is few
Nucleotide includes antisense oligonucleotides and siRNA, wherein the sequence of antisense oligonucleotides is small as shown in SEQ ID NO.1
The sequence of RNA interfering is as shown in SEQ ID NO.2-4.
Optionally, which can prepare pharmaceutical preparations with various pharmaceutical acceptable carrier molecules.Presently described widow's core
Thuja acid can its pharmaceutical acceptable carrier molecule for entering target cell ability be compound with enhancing.This pharmaceutical acceptable carrier molecule includes but unlimited
Indispensable molecule is grown in sugar, polyamine, amino acid, peptide, lipid and other pairs of cells.For example, oligonucleotides can be with lipid
Body combination, that is, be contained in liposome and form pharmaceutical formulation.
The pharmaceutical composition can be given with any way of transport oligonucleotides to desired site such as mammary gland.
Optionally, the purposes of the pharmaceutical composition includes SNHG17 gene or downward cream in targeted silent breast cancer cell
The purposes of the expression of SNHG17 gene in adenocarcinoma cell.
Specifically, the breast cancer cell is triple negative breast cancer cell.
Specifically, the pharmaceutical composition has proliferation, transfer and the purposes of invasion for inhibiting triple negative breast cancer cell.
The pharmaceutical composition passes through targeted silent SNHG17 gene or lowers the expression of SNHG17 gene, to inhibit
The expression of KTN1, N-Cadherin, β-Catenin and Vimentin are finally reached the proliferation for inhibiting triple negative breast cancer, transfer
With invasive ability.
Below with reference to experiment, technical solution of the present invention is further illustrated.
Materials and methods:
(1) cellular material:
People's triple negative breast cancer cell line MDA-MB-231 and BT549 are chosen, temperature is 37 DEG C, mass concentration is 5%
CO2Under the conditions of incubator, use mass concentration for 10% FBS DMEM culture medium the secondary culture in 10cm culture dish.
(2) SNHG17 ASO interference model is constructed:
When MDA-MB-231 and BT549 cell confluency degree it is long to 70% when, cell is respectively divided into two groups: NC ASO at random
Groups of cells and SNHG17 ASO groups of cells.Two groups of cells are washed twice with PBS respectively；Use 15 μ l lipofectamine2000
Liposome wraps up NC ASO and the SNHG17 ASO of 50nM respectively, wherein the sequence of SNHG17 ASO are as follows: 5'-
TATTTCCGCCGGCGCGAAAC-3'；NC ASO groups of cells is negative control group, and NC ASO is had by the sharp rich biotechnology in Guangzhou
Limit company provides, and name of product is RiboTM lncRNA ASO Negative Control, and product number is
lnc3N0000001-1-5.The volume of the NC ASO and SNHG17 ASO is respectively 15 μ L, with serum-free antibiotic-free DMEM
Culture medium is diluted to obtain two groups of mixed liquors, and every group of mixed liquor total volume after dilution is 400 μ l, which is put
Set 15-20min.It is then each to cultivate the serum-free antibiotic-free for being added that volume is 4.4ml in the 10cm culture dish for having cell
DMEM culture medium the NC ASO mixed liquor after liposome is added dropwise into the culture dish of culture NC ASO groups of cells, by rouge
SNHG17 ASO mixed liquor after plastid package is added dropwise into the culture dish of culture SNHG17 ASO groups of cells, will after culture 3-5h
Liquid discards in each culture dish, the DMEM culture medium containing serum and antibiotic is added, temperature is 37 DEG C, mass concentration is
5% CO2Continue to cultivate under condition of culture.
(3) reverse transcription and qRT-PCR reaction:
Cell is collected after culture 48h, is washed twice using PBS, 1mlPBS is added in each culture dish, is scraped carefully using cell scraper
After born of the same parents, intracellular total serum IgE is extracted with Trizol method, carries out reverse transcription reaction after measuring concentration.The following institute of reverse transcription reaction system
Show:
| 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 being incubated for 15min under the conditions of temperature is 42 DEG C, then the enzymatic mixture heated in 5s inactivation reaction system at 85 DEG C,
It is finally incubated under the conditions of temperature is 4 DEG C until cooling.SNHG17 primer sequence as shown in SEQ ID NO.5-6, specifically:
HSNHG17_FP_1:5'-CCCCAGGTGACGTGTCTTCA-3'；
HSNHG17_RP_1:5'-GCAACAGCCACTGAAAGCAT-3'.
(4) Western blot analysis:
The RIPA protein lysate and 25 × PIC protease inhibitors of 100 μ l-130 μ l is added in each culture dish, is placed in ice
Cracking is frozen after upper 10min 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 Western blot analysis.
(5) statistical analysis: using SPSS21.0 version software, executes independent sample one-way analysis of variance, all numbers
There is statistical significance according to double tail detections and homogeneity of variance analysis, p < 0.001 * * p < 0.05, * * p < 0.01, * * are all made of.
Embodiment 1
The present embodiment is related to the influence of SNHG17RNA expression in triple negative breast cancer (TNBC) specimens:
Collect cancerous tissue (Cancer), cancer beside organism (Peritumorial) and the sentinel of 3 groups of clinic TNBC patients
Nodal tissue (Sentinel node), extracts RNA respectively, and qRT-PCR detection is carried out after reverse transcription, as a result (Fig. 1 as shown in Figure 1
In, p < 0.001 * p < 0.05, * * p < 0.01, * * *), the expression quantity of SNHG17RNA is significantly larger than group by cancer in TNBC tumor tissues
Knit the expression quantity with SNHG17RNA in sentinel nodal tissue.
Conclusion: SNHG17 gene is related to the generation of TNBC.
Embodiment 2
The present embodiment be related to detect antisense oligonucleotides in TNBC specimens MDA-MB-231 and BT549 it is thin
The ability of regulation and control of the proliferation and SNHG17 rna expression level of born of the same parents.
NC ASO and SNHG17 ASO are established by above-mentioned experimental method in TNBC cell line MDA-MB-231 and BT549
Interference model.
Detect the MDA-MB-231 cell after transfecting NC ASO and SNHG17 ASO respectively using qRT-PCR method
The variation of SNHG17RNA expression, it is specific as shown in Fig. 2, transfecting the MDA-MB-231 cell after SNHG17 ASO
SNHG17RNA expression is far below control group (NC ASO), and SNHG17 ASO interference model is successfully established；Fig. 3 indicates NC
ASO and SNHG17 ASO handles 0h after MDA-MB-231 cell, for 24 hours, after 48h and 72h, use the detection interference of CCK8 method
The variation diagram (in Fig. 3, p < 0.001 * p < 0.05, * * p < 0.01, * * *) of MDA-MB-231 cell Proliferation, passes through Fig. 3 after SNHG17
It is found that, with the extension of incubation time, the proliferative capacity of MDA-MB-231 cell weakens after transfection SNHG17 ASO.
The SNHG17RNA of BT549 cell after detecting transfection NC ASO and SNHG17 ASO respectively using qRT-PCR method
The variation of expression, as shown in figure 4, the SNHG17RNA expression of the BT549 cell after transfection SNHG17 ASO is far below
Control group (NC ASO), SNHG17ASO interference model is successfully established；Fig. 5 indicates that NC ASO and SNHG17 ASO processing BT549 are thin
0h after born of the same parents, for 24 hours, after 48h and 72h, use variation diagram (Fig. 5 of BT549 cell Proliferation after CCK8 method detection interference SNHG17
In, p < 0.001 * p < 0.05, * * p < 0.01, * * *), as shown in Figure 5, after transfecting SNHG17 ASO, with prolonging for incubation time
Long, the proliferative capacity of BT549 cell weakens.
Conclusion: SNHG17 gene can regulate and control the proliferative capacity of TNBC cell line MDA-MB-231 and BT549 cell；And it is heavy
Silent SNHG17 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 antisense oligonucleotides to MDA-MB-231 and BT549 cell in TNBC specimens
Migration and invasive ability influence.
The present embodiment has detected MDA-MB-231 and BT549 in SNHG17 ASO interference model using Transwell technology
The migration of cell and invasive ability, specific steps are as follows:
NC ASO, SNHG17 ASO are transfected in MDA-MB-231 cell, it, will treated MDA-MB- after transfecting 48h
For 231 transferred speciess in the cell Transwell (containing matrigel in the cell of detection invasive ability), 12-16h collects cell micro-
Under the microscope, Fig. 6, Fig. 7 and Fig. 8 are please referred to, Fig. 6 is thin after NC ASO, SNHG17 ASO transfection MDA-MB-231 cell 48h
Born of the same parents migrate the electron microscope with invasion, and Fig. 7 is to be directed to the thin of Fig. 6 after NC ASO, SNHG17 ASO transfect MDA-MB-231 cell 48h
Born of the same parents migrate the cell quantity statistical chart of electron microscope, and Fig. 8 is that NC ASO, SNHG17 ASO transfect needle after MDA-MB-231 cell 48h
To the cell quantity statistical chart of the cell invasion electron microscope of Fig. 6, * p < 0.05, * * p < 0.01, * * * p in Fig. 6, Fig. 7 and Fig. 8 <
0.001.From Fig. 6, it has been observed that SNHG17 ASO transfects the transfer ability of MDA-MB-231 cell after SNHG17 under electron microscope
It reduces, while invasive ability also reduces；From Fig. 7 with Fig. 8 it can be found that SNHG17 ASO transfect MDA-MB-231 cell with compare
It is compared after group NC ASO transfection SNHG17 48h, cell number reduces about 5-6 times.
Similarly, NC ASO, SNHG17 ASO are transfected in BT549 cell, after transfecting 48h, BT549 is thin by treated
For dysuria with lower abdominal colic kind in the cell Transwell (containing matrigel in the cell of detection invasive ability), 12-16h collects cell micro-
Under the microscope, Fig. 9, Figure 10 and Figure 11 are please referred to, Fig. 9 is that NC ASO, SNHG17 ASO transfect the cell after BT549 cell 48h
The electron microscope of migration and invasion, Figure 10 are directed to the cell migration of Fig. 9 after being NC ASO, SNHG17 ASO transfection BT549 cell 48h
The cell quantity statistical chart of electron microscope, Figure 11 are directed to the cell of Fig. 9 after being NC ASO, SNHG17 ASO transfection BT549 cell 48h
Invade the cell quantity statistical chart of electron microscope；P < 0.001 * p < 0.05 in Fig. 9, Figure 10 and Figure 11, * * p < 0.01, * * *.From Fig. 9
In it can be found that the transfer ability of BT549 cell reduces after SNHG17 ASO transfection SNHG17, while invasive ability also reduces；
From Figure 10 and Figure 11 it can be found that SNHG17 ASO transfection BT549 cell and control group NC ASO transfect phase after SNHG17 48h
Than cell number reduces 9-10 times.
Conclusion: silencing SNHG17 gene can significantly inhibit the migration of TNBC cell line MDA-MB-231 and BT549 cell
And invasion.
Embodiment 4
The present embodiment is related to detecting the correlation table that antisense oligonucleotides moves TNBC specimens transfer correlation factor
The influence reached.
KTN1 is driving binding protein, related to cancer proliferation；N-Cadherin, β-Catenin and Vimentin are
Metastases correlation factor；β-Actin is the internal reference factor.Transfect NC ASO and SNHG17 respectively in MDA-MB-231 cell
ASO, then using Western blot analysis detection method detection KTN1, N-Cadherin, β-Catenin, Vimentin with
The protein expression of β-Actin.Figure 12 is please referred to, Figure 12 shows MDA-MB- in NC ASO and SNHG17 ASO interference model
The expression of the transfer correlation factor of 231 cells, it is known that SNHG17 ASO can be directly targeted KTN1, N-Cadherin, β-
Catenin's and Vimentin expresses to regulate and control proliferation, the migration, invasive ability of TNBC.
Conclusion: targeting the expression for lowering SNHG17 gene using antisense oligonucleotides, can inhibit to influence three negative breasts
Cancer cell multiplication, transfer, KTN1, N-Cadherin, β-Catenin of invasive ability and Vimentin etc. shift correlation factor
Expression, it is final to improve or treat triple negative breast cancer.
Embodiment 5
The present embodiment is related to detecting siRNA to MDA-MB-231 the and BT549 cell in TNBC specimens
The ability of regulation and control of SNHG17 expression.
It is built in TNBC cell line MDA-MB-231 by experimental method identical with building SNHG17 ASO interference model
Vertical siNC (control group) and siSNHG17_1, siSNHG17_2, siSNHG17_3 interference model.The wherein sequence of siSNHG17_1
For 5'-GGTGACGTGTCTTCAAGAA-3', as shown in SEQ ID NO.2；The sequence of siSNHG17_2 is 5'-
CGGATCCACTGTTCAATCT-3', as shown in SEQ ID NO.3；The sequence of siSNHG17_3 is 5'-
GCCTGGAATGACTTTAATA-3', as shown in SEQ ID NO.4；SiNC is mentioned by Guangzhou Ribo Bio Co., Ltd.
For name of product is siR NC#1, product number siN0000001-1-5.
After detecting transfection siNC and siSNHG17_1, siSNHG17_2, siSNHG17_3 respectively using qRT-PCR method
The variation of the SNHG17RNA expression of MDA-MB-231 cell, it is specific as shown in figure 13, transfection siSNHG17_1,
The SNHG17RNA expression of MDA-MB-231 cell after siSNHG17_2, siSNHG17_3 is far below control group (siNC),
SiSNHG17_1, siSNHG17_2, siSNHG17_3 interference model are successfully established.
Conclusion: siSNHG17_1, siSNHG17_2, siSNHG17_3 are able to suppress MDA-MB-231 cell
The expression of SNHG17RNA.
The numberical range of each technological parameter as involved in the present invention can not all embody in the above-described embodiments,
As long as but those skilled in the art's envisioned any numerical value fallen into the above-mentioned numberical range completely can be implemented this
Invention also includes any combination of occurrence in several numberical ranges certainly.Herein, due to space considerations, be omitted to
Out in certain one or more numberical range occurrence embodiment, this disclosure for being not to be construed as technical solution of the present invention do not fill
Point.
The Applicant declares that the present invention is explained by the above embodiments detailed process equipment and process flow of the invention,
But the present invention is not limited to the above detailed process equipment and process flow, that is, it is above-mentioned detailed not mean that the present invention must rely on
Process equipment and process flow could be implemented.It should be clear to those skilled in the art, any improvement in the present invention,
Addition, concrete mode selection of equivalence replacement and auxiliary element to each raw material of product of the present invention etc., fall in protection of the invention
In range.
SEQUENCE LISTING
<110>Shenzhen people's hospital
<120>oligonucleotides of targeting SNHG17 treatment breast cancer
<130> 2019
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
tatttccgcc ggcgcgaaac 20
<210> 2
<211> 19
<212> RNA
<213>artificial sequence
<400> 2
ggtgacgtgt cttcaagaa 19
<210> 3
<211> 19
<212> RNA
<213>artificial sequence
<400> 3
cggatccact gttcaatct 19
<210> 4
<211> 19
<212> RNA
<213>artificial sequence
<400> 4
gcctggaatg actttaata 19
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
ccccaggtga cgtgtcttca 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<400> 6
gcaacagcca ctgaaagcat 20
Claims (10)
1. a kind of oligonucleotides of targeting SNHG17 treatment breast cancer, which is characterized in that the oligonucleotides includes antisense widow's core
At least one of thuja acid and siRNA, wherein the sequence of the antisense oligonucleotides is described as shown in SEQ ID NO.1
The sequence of siRNA is as shown in SEQ ID NO.2-4.
2. oligonucleotides as described in claim 1, which is characterized in that the oligonucleotides is using nucleic acid lock modification technique or core
Sour chain backbone modification technique modified.
3. a kind of purposes of oligonucleotides as described in claim 1, which is characterized in that including the oligonucleotides is used for target
Into silencing cancer cell SNHG17 gene or lower cancer cell in SNHG17 gene ability to express purposes.
4. the purposes of oligonucleotides as claimed in claim 3, which is characterized in that the cancer cell is that triple negative breast cancer is thin
Born of the same parents.
5. the purposes of oligonucleotides as claimed in claim 3, which is characterized in that including for inhibiting cancer cell proliferation, turn
Move the purposes with invasive ability.
6. a kind of pharmaceutical composition of targeting SNHG17 treatment breast cancer, which is characterized in that including as described in claim 1 few
Nucleotide 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. a kind of purposes of pharmaceutical composition as claimed in claim 6, which is characterized in that including using described pharmaceutical composition
In targeted silent breast cancer cell SNHG17 gene or lower breast cancer cell in SNHG17 gene expression purposes.
9. the purposes of pharmaceutical composition as claimed in claim 8, which is characterized in that the breast cancer cell is three negative breasts
Cancer cell.
10. the purposes of pharmaceutical composition as claimed in claim 8, which is characterized in that including for inhibiting triple negative breast cancer
Proliferation, the purposes of transfer and invasive ability of cell.
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Cited By (5)
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| CN111057766A (en) * | 2019-12-26 | 2020-04-24 | 中国人民解放军军事科学院军事医学研究院 | Application of SNHG17 in screening of drugs for regulating and controlling radiation-induced pulmonary epithelial interstitial transformation and/or pulmonary fibrosis |
| CN111454944A (en) * | 2020-04-03 | 2020-07-28 | 深圳市人民医院 | Method for synthesizing separated RNA and DNA template thereof |
| CN112553199A (en) * | 2020-11-30 | 2021-03-26 | 深圳市人民医院 | Construction method and application of snhg17-KO gene knockout mouse model |
| CN114990117A (en) * | 2022-06-20 | 2022-09-02 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | Antisense oligonucleotide of targeted long-chain non-coding RNA and application thereof |
| WO2023221324A1 (en) * | 2022-05-18 | 2023-11-23 | 浙江省肿瘤医院 | Use of no-coding rna snhg17 as marker and treatment target |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111057766A (en) * | 2019-12-26 | 2020-04-24 | 中国人民解放军军事科学院军事医学研究院 | Application of SNHG17 in screening of drugs for regulating and controlling radiation-induced pulmonary epithelial interstitial transformation and/or pulmonary fibrosis |
| CN111454944A (en) * | 2020-04-03 | 2020-07-28 | 深圳市人民医院 | Method for synthesizing separated RNA and DNA template thereof |
| CN112553199A (en) * | 2020-11-30 | 2021-03-26 | 深圳市人民医院 | Construction method and application of snhg17-KO gene knockout mouse model |
| WO2023221324A1 (en) * | 2022-05-18 | 2023-11-23 | 浙江省肿瘤医院 | Use of no-coding rna snhg17 as marker and treatment target |
| CN114990117A (en) * | 2022-06-20 | 2022-09-02 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | Antisense oligonucleotide of targeted long-chain non-coding RNA and application thereof |
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