CN108949988A - A kind of application of long-chain non-coding RNA SNHG6 in breast cancer diagnosis or treatment - Google Patents
A kind of application of long-chain non-coding RNA SNHG6 in breast cancer diagnosis or treatment Download PDFInfo
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Abstract
The invention discloses a kind of application of long-chain non-coding RNA SNHG6 in breast cancer diagnosis or treatment.The present invention is our experiments show that SNHG6 expresses significant up-regulation in breast cancer tissue and cell, it can be as competitive endogenous RNA combination miR-26a, to weaken the inhibiting effect that miR-26a translates VASP, and then promote proliferation, invasion and the migration of breast cancer cell.The present invention provides new target spot for the diagnosing and treating of breast cancer, and provides important evidence to research and develop the drug of targeted therapy breast cancer.
Description
Technical field
The present invention relates to gene therapies and area of medical diagnostics, and in particular to a kind of long-chain non-coding RNA SNHG6 is in cream
Application in gland cancer diagnosis or treatment.
Background technique
One of common female malignant is done as the whole world and Chinese women morbidity and mortality are highest pernicious swollen
One of tumor, breast cancer continue to be frequently a global challenges in women[1].Although at present for the treatment of breast cancer
Have a different treatment methods such as operation, chemotherapy, radiotherapy, ablation, hormone therapy and combination therapy, but still recurrence rate with higher and
The death rate, therapeutic effect are still undesirable[2].Therefore, carrying out research to the pathogenesis of breast cancer has important clinical meaning.
Vasodilator stimulation of phosphoprotein (vasodilator-stimulated phosphoprotein, VASP), is extensive
It is present in the relevant skelemin of one of different cells and tissue actin[3].It can promote intracellular actin monomer
It is linked into the device end of F-actin, to promote F-actin assembling, extend, is sent out in regulating cell invasion and transition process
Wave important function[4].Discovery early period VASP in breast cancer cell, which is studied, at us there is the increasing for being overexpressed and participating in cell
It grows, invade and migrates[5]。
More and more evidences show that non-coding RNA (ncRNA) can participate in various pathology and physiology course, and are based on
The length of ncRNA is divided into long-chain non-coding RNA (lncRNA) and short chain non-coding RNA[6].Have more research reports at present
LncRNAs can participate in a variety of diseases including tumour[7-9], therefore, lncRNA has become tumor research in recent years
New hot spot.SNHG6 (U87HG) is the house-keeping gene of 5 ' TOP families, it can encode two kinds of non-coding RNAs[10], SNHG6 turns
The cDNA sequence of record is as shown in SEQID NO:7.Some researches show that SNHG6 can participate in the growth and transfer of tumour, but at present still
Its effect and mechanism of action in breast cancer is not reported[11-13]。
Bibliography
[1]Siegel RL,Miller KD and Jemal A.Cancer Statistics,2017.CA Cancer J
Clin2017；67:7-30.
[2]Benedetti R,Dell'Aversana C,Giorgio C,Astorri R and Altucci
L.Breast Cancer Vaccines:New Insights.Front Endocrinol(Lausanne)2017；8:270.
[3]Zimmer M,Fink T,Fischer L,Hauser W,Scherer K,Lichter P and Walter
U.Cloning of the VASP(vasodilator-stimulated phosphoprotein)genes in human
and mouse:structure,sequence,and chromosomal localization.Genomics 1996；36:
227-233.
[4]Dertsiz L,Ozbilim G,Kayisli Y,Gokhan GA,Demircan A and Kayisli
UA.Differential expression of VASP in normal lung tissue and lung
adenocarcinomas.Thorax 2005；60:576-581.
[5]Zhang Y,Han G,Fan B,Zhou Y,Zhou X,Wei L and Zhang J.Green tea(-)-
epigallocatechin-3-gallate down-regulates VASP expression and inhibits breast
cancer cell migration and invasion by attenuating Rac1activity.Eur J
Pharmacol2009；606:172-179.
[6]Memczak S,Jens M,Elefsinioti A,Torti F,Krueger J,Rybak A,Maier L,
Mackowiak SD,Gregersen LH,Munschauer M,Loewer A,Ziebold U,Landthaler M,Kocks
C,le Noble F and Rajewsky N.Circular RNAs are a large class of animal RNAs
with regulatory potency.Nature 2013；495:333-338.
[7]Bhan A,Soleimani M and Mandal SS.Long Noncoding RNA and Cancer:A
New Paradigm.Cancer Res 2017；77:3965-3981.
[8]Mirza AH,Kaur S and Pociot F.Long non-coding RNAs as novel players
in beta cell function and type 1diabetes.Hum Genomics 2017；11:17.
[9]Huang X,Luo YL,Mao YS and Ji JL.The link between long noncoding
RNAs and depression.Prog Neuropsychopharmacol Biol Psychiatry 2017；73:73-78.
[10]Gogolevskaya IK,Makarova JA,Gause LN,Kulichkova VA,Konstantinova
IM and Kramerov DA.U87RNA,a novel C/D box small nucleolar RNA from mammalian
cells.Gene 2002；292:199-204.
[11]Chang L,Yuan Y,Li C,Guo T,Qi H,Xiao Y,Dong X,Liu Z and Liu
Q.Upregulation of SNHG6regulates ZEB1expression by competitively binding miR-
101-3p and interacting with UPF1in hepatocellular carcinoma.Cancer Lett2016；
383:183-194.
[12]Birgani MT,Hajjari M,Shahrisa A,Khoshnevisan A,Shoja Z,Motahari P
and Farhangi B.Long Non-Coding RNA SNHG6as a Potential Biomarker for
Hepatocellular Carcinoma.Pathol Oncol Res 2018；24:329-337.
[13]Yan K,Tian J,Shi W,Xia H and Zhu Y.LncRNA SNHG6is Associated with
Poor Prognosis of Gastric Cancer and Promotes Cell Proliferation and EMT
through Epigenetically Silencing p27and Sponging miR-101-3p.Cell Physiol
Biochem2017；42:999-1012.
Summary of the invention
In order to overcome the shortcomings of the prior art, the present invention is using group by TCGA database analysis breast cancer tissue and cancer
The difference of middle SNHG6 expression is knitted, analyzing SNHG6 expression in breast cancer tissue as the result is shown has raising trend.It is based on
Data analysis result, the present invention further pass through SNHG6 expression in experiment detection breast cancer tissue and cell, the results show that with
Normal mammary glandular cell is compared with cancer beside organism, and there are significantly high expression by SNHG6 in breast cancer cell and tissue.
The present invention strikes drop SNHG6 by si-RNA, probe into SNHG6 to the expression of VASP and to Cells Proliferation of Human Breast Cancer, invade
Attack the regulation with transfer ability and mechanism of action.SNHG6 can be used as competitive endogenous RNA combination miR-26a as the result is shown,
To weaken the inhibiting effect that miR-26a translates VASP, and then promote the proliferation and migration of breast cancer cell.
Based on result above the object of the present invention is to provide the new medical usage of long-chain non-coding RNA SNHG6, that is, exist
Preparation is for the application in Computer-aided Diagnosis of Breast Cancer or curative effect predication reagent.
It is a further object of the present invention to provide long-chain non-coding RNA SNHG6 to prepare answering in breast cancer treatment drug
With.
The purpose of the present invention is achieved through the following technical solutions:
The present invention provides a kind of long-chain non-coding RNA SNHG6 in preparation for Computer-aided Diagnosis of Breast Cancer or outcome prediction
Application in reagent or kit.
The reagent, for the reagent of long-chain non-coding RNA SNHG6 expression quantity in detection biological sample.
The kit, the reagent comprising long-chain non-coding RNA SNHG6 expression quantity in detection biological sample.
The reagent of long-chain non-coding RNA SNHG6 expression quantity, is selected from: to long-chain non-coding in the detection biological sample
RNA SNHG6 has probe, genetic chip or the PCR primer of detection specificity.
The biological sample is selected from: flesh tissue or cell, formalin obtained from object are fixed or paraffin embedding group
It knits or cell, blood or body fluid.
Second aspect, the present invention provide long-chain non-coding RNA SNHG6 and are preparing the application in breast cancer treatment drug, should
Therapeutic agent refers to inhibition or reduces the reagent of long-chain non-coding RNA SNHG6 expression quantity.
The reagent of the inhibition or reduction long-chain non-coding RNA SNHG6 expression quantity, including but not limited to: siRNA,
shRNA。
Preferably, the present invention provides long-chain non-coding RNA SNHG6 and is preparing the application in breast cancer treatment drug, this is controlled
Treating drug is siRNA, and sequence is as follows:
Si-SNHG6#1 positive-sense strand: 5 '-GAGGUGCAAGAAAGCCUUUTT-3 ' (SEQ ID NO:1)
Antisense strand: 5 '-AAAGGCUUUCUUGCACCUCTT-3 ' (SEQ ID NO:2)；
Si-SNHG6#2 positive-sense strand: 5 '-GCGGCAUGUAUUGAGCAUATT-3 ' (SEQ ID NO:3)
Antisense strand: 5 '-UAUGCUCAAUACAUGCCGCTT-3 ' (SEQ ID NO:4)；
Si-SNHG6#3 positive-sense strand: 5 '-GCUUCGUUACCUCAAGUGUTT-3 ' (SEQ ID NO:5)
Antisense strand: 5 '-ACACUUGAGGUAACGAAGCTT-3 ' (SEQ ID NO:6)；
In above-mentioned sequence, 3 ' ends addTTIt is the stability in order to reinforce siRNA.
The therapeutic agent is for inhibiting the proliferation of breast cancer cell, invasion and migration.
The present invention illustrates mechanism of action of the SNHG6 in breast cancer, provides new target for the diagnosing and treating of breast cancer
Point.And si-RNA interference is carried out to inhibit proliferation, invasion and the migration of breast cancer cell to SNHG6, this means is for opening
The therapeutic effect of the breast cancer treatment drug and raising breast cancer of sending out new is of great significance, and has great application prospect and passes through
Ji value.
Detailed description of the invention
Fig. 1 is the expression of SNHG6, miR-26a and VASP in breast cancer tissue and cancer beside organism in TCGA database
Figure；
Fig. 2 is the expression figure of the prognosis SNHG6, VASP and miR-26a of patient with breast cancer in TCGA database；
Fig. 3 is the expression of SNHG6, miR-26a and VASP in the breast cancer tissue and cancer beside organism of the invention detected
Figure；
Fig. 4 is the table of SNHG6, miR-26a and VASP in the breast cancer cell and normal mammary glandular cell of the invention detected
Up to level view；
Fig. 5 is the expression figure that 3 si-SNHG6 of synthesis can significantly strike drop SNHG6；
Fig. 6 is that CCK-8 experiment strikes drop SNHG6 to MCF-7 and MDA-MB-231 ability of cell proliferation influence diagram；
Fig. 7 is that plate clone experiment strikes drop SNHG6 to MCF-7 and MDA-MB-231 ability of cell proliferation influence diagram；
Fig. 8 is that Edu Coloration experiment strikes drop SNHG6 to MCF-7 and MDA-MB-231 ability of cell proliferation influence diagram；
Fig. 9 is that cell cycle detection strikes drop SNHG6 to MCF-7 and MDA-MB-231 ability of cell proliferation influence diagram；
Figure 10 is that scratch experiment detection strikes drop SNHG6 to the transfer ability influence diagram of MCF-7 and MDA-MB-231 cell；
Figure 11 is that Transwell experiment detection strikes drop SNHG6 to the influence of the invasive ability of MCF-7 and MDA-MB-231 cell
Figure；
Figure 12 is that Transwell experiment detection strikes drop SNHG6 to the influence of the transfer ability of MCF-7 and MDA-MB-231 cell
Figure；
Figure 13 is to strike drop SNHG6 to the expression shadow of VASP and miR-26a in MCF-7 and MDA-MB-231 cell
Ring figure；
Figure 14 is expression influence diagram of the miR-26a to VASP and SNHG6 in MCF-7 and MDA-MB-231 cell；
Figure 15 is that Western blotting experiment detection strikes drop SNHG6 to the VASP of MCF-7 and MDA-MB-231 cell
Protein expression level influence diagram；
Figure 16 is VASP of the Western blotting experiment detection miR-26a to MCF-7 and MDA-MB-231 cell
Protein expression level influence diagram；
Figure 17 is that immunofluorescence dyeing detection strikes drop SNHG6 to the albumen table of the VASP of MCF-7 and MDA-MB-231 cell
Up to horizontal influence diagram；
Figure 18 is the binding sequence of bioinformatic analysis miR-26a and VASP and miR-26a and SNHG6, and mutation
Binding sequence；
Figure 19 is shadow of the miR-26a to 3 ' UTRmut reporter gene activity of 3 ' UTR reporter gene activity of VASP and VASP
As figure；
Figure 20 is striograph of the miR-26a to SNHG6 reporter gene activity；
Figure 21 is to strike drop SNHG6 to the striograph of 3 ' UTR reporter gene activity of VASP；
Figure 22 is to interfere SNHG6 or miR-26a inhibitor to the albumen of the VASP of MCF-7 and MDA-MB-231 cell
Expression influence diagram；
Figure 23 is to strike drop VASP or SNHG6 or VASP or combinations thereof to the influence diagram of the proliferation of MDA-MB-231 cell；
Figure 24 is to strike drop VASP or SNHG6 or VASP or combinations thereof to the influence diagram of the migration of MDA-MB-231 cell；
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.These realities
Example is applied to be only illustrative of the invention and is not intended to limit the scope of the invention.The test of actual conditions is not specified in the following example
Method, usually according to normal condition.
[embodiment 1] SNHG6 differential expression in breast cancer tissue and cell
1, breast cancer high-flux sequence data in TCGA database are downloaded, are analyzed in breast cancer tissue and cancer beside organism
The expression difference of SNHG6, miR-26a and VASP.Pass through the pre- of Kaplan-Meier survival analysis patient with breast cancer simultaneously
Relationship between the expression of SNHG6, VASP and miR-26a afterwards.
Such as Figure 1A -1C, compared with cancer beside organism, the expression of VASP is obviously raised in breast cancer tissue, miR-26a's
Expression is obviously lowered, and the expression of SNHG6 also presents although there was no significant difference and increases trend.Such as Fig. 2A -2C, mammary gland
The prognosis of cancer patient and the expression of VASP are negatively correlated, are positively correlated with miR-26a, and the expression with SNHG6 is although without statistics
Difference, but there are still negatively correlated trend.
2, RNA is extracted
The breast cancer of frost is extracted with Trizol reagent (Invitrogen, Carlsbad, CA) according to operation manual step
And its RNA of normal adjacent tissue, and the RNA for extracting breast cancer cell and normal breast cell is analyzed.
3, reverse transcription and real-time fluorescence quantitative PCR
It is pressed with RevertAid First Strand Cdna Synthesis Kit (Thermo SCIENTIFIC, USA)
Operation manual to mRNA carry out reverse transcription, with real-time fluorescence quantitative PCR (Applied Biosystem Inc.) come to mRNA into
Row quantitative analysis.The upstream primer of the real-time fluorescence quantitative PCR of SNHG6 is 5 '-CCTACTGACAACATCGACGTTGAAG-3 '
Downstream primer is 5 '-GGAGAAAACGCTTAGCCATACAG-3 '；The upstream primer of the real-time fluorescence quantitative PCR of internal reference GAPDH
For 5 '-AATGGACAACTGGTCGTGGAC-3 ', downstream primer 5 '-CCCTCCAGGGGATCTGTTTG-3 '.
As shown in Fig. 3 A-3C, 4A-4C, relative to normal mammary glandular cell and tissue, in breast cancer cell and breast cancer group
The mRNA expression for knitting middle VASP and SNHG6 increases, and the expression of miR-26a significantly reduces.
4, it designs si-RNA and transfects
The gene sequence of SNHG6 is searched in gene order query web (http://www.ncbi.nlm.nih.gov/gene)
Column, then design is directed to the si-RNA of SNHG6, while design and Negative Control (si- of the target gene without homology
NC)。
It selects 3 to design obtained si-RNA and si-NC sequence to be synthesized, sequence is as follows:
Si-SNHG6#1 positive-sense strand: 5 '-GAGGUGCAAGAAAGCCUUUTT-3 '
Antisense strand: 5 '-AAAGGCUUUCUUGCACCUCTT-3 '；
Si-SNHG6#2 positive-sense strand: 5 '-GCGGCAUGUAUUGAGCAUATT-3 '
Antisense strand: 5 '-UAUGCUCAAUACAUGCCGCTT-3 '；
Si-SNHG6#3 positive-sense strand: 5 '-GCUUCGUUACCUCAAGUGUTT-3 '
Antisense strand: 5 '-ACACUUGAGGUAACGAAGCTT-3 '；
Si-NC positive-sense strand: 5 '-UUCUCCGAACGUGUCACGUTT-3 '
Antisense strand: 5 '-ACGUGACACGUUCGGAGAATT-3 '.
Above-mentioned RNA is purchased from Suzhou GenePharma Co., Ltd..3 si-SNHG6 are turned respectively by transfection reagent
Dye detects its effect for striking drop SNHG6 into MDA-MB-231 and MCF-7 cell.As shown in figure 5,3 si-SNHG6 of synthesis
It can significantly strike the expression of drop SNHG6.In follow-up test, by si-SNHG6#1, si-SNHG6#2 and si-SNHG6#3
It is used in combination and (is indicated with si-SNHG6), to lower the expression of SNHG6.
The proliferation and transfer ability of [embodiment 2] SNHG6 promotion breast cancer cell
1, cell culture
Human breast cancer cell line MDA-MB-231 is cultivated with complete RPMI-1640 culture medium culture, MCF-7 with complete DMEM
Base culture.All culture mediums include 10% fetal calf serum and antibiotic (10000U/ml penicillin, 10ug/ml streptomysin).Carefully
Born of the same parents' incubator environment is 37 DEG C, is moistened, 5%CO2。
2, CCK-8 is tested
MDA-MB-231 and MCF-7 cell culture is in 35mm culture dish, with SNHG6 siRNA and negative control RNA
Transiently transfect MDA-MB-231 cell and MCF-7 cell.It is dispelled, is inoculated into 96 porocyte culture plates, cell with pancreatin digestion
Suspension concentration is about 3 × 103/ mL, every 100 μ L of hole.It is placed in cell incubator and continues to cultivate for 37 DEG C, respectively in 0,24,48h
The CCK-8 solution of 10 μ L is added in every hole, continues to cultivate 2h after mixing well, and measures every hole absorbance at 450nm with microplate reader.
As a result such as Fig. 6 is compared with control group, and the increasing of MCF-7 and MDA-MB-231 cell can be significantly inhibited by striking drop SNHG6
Grow ability.
3, plate clone is tested
MDA-MB-231 and MCF-7 cell culture is in 35mm culture dish, with SNHG6 siRNA and negative control RNA
Transiently transfect MCF-7 and MDA-MB-231 cell.Continue culture about one week, calculates the clone formation number of cell.
As shown in fig. 7, comparing with control group, the increasing of MCF-7 and MDA-MB-231 cell can be significantly inhibited by striking drop SNHG6
Grow ability.
4, Edu Coloration experiment
Edu Coloration experiment is frequently used to the proliferation proliferative capacity of detection cell.By cell (4 × 103~1 × 105Cell/
Hole) it is inoculated in 24 orifice plates and cultivates 24 hours, then containing debita spissitudo Edu (Cell-LightTM EdU ApoIIo567
In Vitro Kit, Ribobio, China) cell culture medium in continue culture 2 hours.Based on EdU and Apollo594 fluorescence
The specific reaction of dyestuff can be used to quickly detect ability of cell proliferation.
As shown in figure 8, comparing with control group, the increasing of MCF-7 and MDA-MB-231 cell can be significantly inhibited by striking drop SNHG6
Grow ability.
5, the cell cycle is detected
MDA-MB-231 and MCF-7 cell culture is in 35mm culture dish, with SNHG6 siRNA and negative control RNA
Transiently transfect MCF-7 and MDA-MB-231 cell.It is dispelled after culture 48h with pancreatin digestion, according to cell cycle detection kit
The step of standardization, operates, and then uses the flow cytomery cell cycle.
As a result such as Fig. 9, striking drop SNHG6 can inhibit significantly by MCF-7 and MDA-MB-231 cell block in the G0/G1 phase
The proliferative capacity of cell.
6, scratch experiment
MDA-MB-231 and MCF-7 cell culture is in 100mm culture dish, with SNHG6 siRNA and negative control
RNA transiently transfects MCF-7 and MDA-MB-231 cell.Culture is to cell density about 90%, in culture dish bottom 200ul pipette tips
A scratch is done, continues to observe scratch healing when culture 0,24,48h respectively, the faster migration for representing cell of scratch healing
Ability is stronger.
It is compared as shown in Figure 10 with control group, MCF-7 and MDA-MB-231 cell can be significantly inhibited by striking drop SNHG6
Transfer ability.
7, Transwell is tested
MDA-MB-231 and MCF-7 cell culture is in 35mm culture dish, with SNHG6 siRNA and negative control RNA
Transiently transfect MCF-7 and MDA-MB-231 cell.It is dispelled with pancreatin digestion, it is about 1 × 10 that concentration, which is made,5The cell suspension of/mL,
100 μ L cell suspensions are added in the upper chamber of each cell, and the complete medium that 500 μ L contain 20%FBS is added in lower room.It is placed in cell
Continue to cultivate 12h for 37 DEG C in incubator.Cell is taken out, is contaminated again with 0.5% crystal violet solution after 4% paraformaldehyde solution is fixed
Color.The cell that upper chamber internal surface is not passed through film is wiped with cotton swab, then microscopically observation is taken pictures.Transwell is tested both
The invasive ability that can detecte cell also can detecte the transfer ability of cell, the difference is that it is for detecting invading for cell
When attacking ability, matrigel is coated in the cell that aperture is 8 μm.
It is compared as shown in figure 11 with control group, MCF-7 and MDA-MB-231 cell can be significantly inhibited by striking drop SNHG6
Invasive ability；It is compared as shown in figure 12 with control group, MCF-7 and MDA-MB-231 cell can be significantly inhibited by striking drop SNHG6
Transfer ability.
Study on mechanism of [embodiment 3] SNHG6 in breast cancer
1, real-time fluorescence quantitative PCR
Laboratory operating procedures are as described in Example 1, and wherein VASP upstream primer sequence is 5 '-
ATGGCAACAAGCGATGGCT-3 ', downstream primer sequence 5 '-CGATGGCACAGTTGATGACCA-3 '.The upstream miR-26a
Primer sequence is 5 '-TTCAAGTAATCCAGGATAGGCT-3 ', downstream primer sequence be universal primer (Qiagen,
Germany).In addition, being respectively synthesized miR-26a mimics and miR-26a inhibitor, it is transfected into cell and was used to table
Reach and strike drop miR-26a.
MiR-26a mimic positive-sense strand: 5'-UUCAAGUAAUCCAGGAUAGGCU-3'
Antisense strand: 5'-AGCCUAUCCUGGAUUACUUGAA-3'
MiR-26a inhibitor:5'-AGCCUAUCCUGGAUUACUUGAA-3'
As shown in Figure 13 A, 13B, in MCF-7 and MDA-MB-231 cell, strike drop SNHG6 can significantly inhibit VASP and
The expression of miR-26a；As shown in Figure 14 A, 14B, in MCF-7 and MDA-MB-231 cell, being overexpressed miR-26a can
The expression for significantly inhibiting VASP and SNHG6 strikes the expression that drop miR-26a then remarkably promotes VASP and SNHG6.
2, Western blotting is tested
SDS- polyacrylamide gel is prepared, the protein sample prepared is added in loading hole.By electrophoresis, transferring film by egg
White matter is integrated on pvdf membrane, after the non-specific sites in 5% milk close membrane, with 5% bovine serum albumin(BSA) press than
Example dissolution primary antibody (GAPDH 1:1000, VASP 1:1000) shaking table is incubated overnight.Next day recycles antibody, is washed away with 1xTBST more
Remaining antibody, room temperature are incubated for secondary antibody, film are washed after two hours three times.Develop after ECL luminescent solution is added on film, and analyzes result.
As shown in figure 15, in MCF-7 and MDA-MB-231 cell, the albumen of VASP can be significantly inhibited by striking drop SNHG6
Expression；As shown in figure 16, in MCF-7 and MDA-MB-231 cell, the egg of VASP can be significantly inhibited by being overexpressed miR-26a
White expression strikes the protein expression level that drop miR-26a then remarkably promotes VASP.
3, immunofluorescence dyeing
On a glass by logarithmic growth phase MCF-7 and MDA-MB-231 cell inoculation, and by sterile round slide it is placed in
In 24 hole plates, 37 DEG C of cultures.When cell is successfully inoculated with, cell 48h is transfected, after fixed, penetrating, Seal treatment, is added 1:
500 VASP antibody diluent, in 4 DEG C of one nights of incubation.Second day, glass plate is cleaned, secondary antibody (this step of fluorescent marker is added
Start to be protected from light), 2h is handled, is then rinsed.With mountant mounting, and in fluorescence microscopy microscopic observation coloration result.
As a result such as Figure 17, in MCF-7 and MDA-MB-231 cell, the albumen of VASP can be significantly inhibited by striking drop SNHG6
Expression.
4, bioinformatic analysis and reporter plasmid building
As shown in figures 18a and 18b, pass through bioinformatic analysis miR-26a's and VASP and miR-26a and SNHG6
Then the binding sequence of binding sequence and mutation is cloned into Reporter gene vector pMIR-report by binding sequence respectively,
Building obtains 3 ' UTR of pMIR-report-VASP, pMIR-report-VASP3 ' UTRmut, pMIR-report-SNHG6,
PMIR-report-SNHG6mut and negative control vector pMIR-report-con.
5, Luciferase Assay detects promoter activity
By miR-26a mimics and miR-26a inhibitor respectively with 3 ' UTR of pMIR-report-VASP, pMIR-
Report-VASP 3 ' UTRmut, pMIR-report-SNHG6, pMIR-report-SNHG6mut and negative control vector
PMIR-report-con is transfected into HEK293T cell, continues to cultivate 48h.According to luciferase reporter gene detection reagent
Box specification (Promega, Madison, WI, USA) detects each group reporter gene activity.
As shown in figure 19,3 ' UTR reporter gene activity of VASP can be significantly inhibited by being overexpressed miR-26a, and strike drop miR-
26a then significantly increases 3 ' UTR reporter gene activity of VASP, but on 3 ' UTRmut reporter gene activity of VASP without influence.Such as
Shown in Figure 20, SNHG6 reporter gene activity can be significantly inhibited by being overexpressed miR-26a, and is struck drop miR-26a and then significantly increased
SNHG6 reporter gene activity, but on SNHG6mut reporter gene activity without influence.In addition, as shown in figure 21, striking drop SNHG6
It can significantly inhibit and inhibit 3 ' UTR reporter gene activity of VASP.The above result shows that miR-26a can target VASP 3 ' simultaneously
UTR and SNHG6 inhibits 3 ' UTR and SNHG6 reporter gene activity of VASP.
In addition, as shown in figure 22, interference SNHG6 can significantly inhibit the protein expression level of VASP, and miR-26a
Inhibitor can significantly reverse interference SNHG6 to the inhibiting effect of VASP protein expression.As shown in figs. 23-24, drop is struck respectively
VASP or SNHG6 can dramatically the proliferation and migration for inhibiting MDA-MB-231 cell, and miR-26a inhibitor can significantly promote
Into the proliferation and migration of MDA-MB-231 cell.MiR-26a inhibitor can significantly reverse SNHG6 or VASP to strike drop to MDA-
The inhibiting effect of MB-231 cell Proliferation and migration.The above result shows that SNHG6 can be used as ceRNA combination miR-26a, thus
Weaken the inhibiting effect that miR-26a translates VASP, and then promotes the proliferation and migration of breast cancer cell.
The present invention demonstrates regulation of the non-coding long-chain RNA SNHG6 to Cells Proliferation of Human Breast Cancer transfer ability, and probes into
SNHG6 to the regulation of breast cancer progression be by raising the expression of VASP to promote breast cancer proliferation, invasion and migration, this
New target spot is provided for the Clinics and Practices of breast cancer, the development of target medicines for treatment of breast cancer new to exploitation and promotion breast cancer
Therapeutic effect is of great significance.
Sequence table
<110>Wuhan University
<120>application of a kind of long-chain non-coding RNA SNHG6 in breast cancer diagnosis or treatment
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<213>artificial sequence (Artificial Sequence)
<400> 2
aaaggcuuuc uugcaccuc 19
<210> 3
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gcggcaugua uugagcaua 19
<210> 4
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 4
uaugcucaau acaugccgc 19
<210> 5
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gcuucguuac cucaagugu 19
<210> 6
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
acacuugagg uaacgaagc 19
<210> 7
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<212> DNA
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tgaggtgaag gtgtatgaaa gtcatcataa cagatgtttt ccaaaaactt gtagaaggtt 180
gtgaaaaaac tactaggatc acgcggcatg tattgagcat ataggttgct gtagatgaat 240
gttcttagct gtcatgttta aaaatacttc tgcttcgtta cctcaagtgt ggcatgcagc 300
attttggaag gaaaattgaa gacgtgttca agaaaacatg aacagaagca aatgatgaaa 360
atgagcattt tacttgatgt tgataacatc acaataaatt atggagaaaa atacatattt 420
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cattctcata gtcactagtc agacctgttt tgaacatatt cgaaagatta taatcttgtc 540
aataattagc ttatttatgg gtggtgattc tcattgaggc tgacagctgg ggagacattg 600
cttgtacctc taggttccct gtctggcttc cccttcagag cctgctgttg taccaggtgg 660
ttgaatctta aaactcttta ataccaaata gcaatcaaat tcccccttac aaaaaaaaaa 720
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<400> 10
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Claims (9)
1. a kind of long-chain non-coding RNA SNHG6 is in preparation for Computer-aided Diagnosis of Breast Cancer or curative effect predication reagent or kit
In application.
2. application according to claim 1, which is characterized in that the reagent is long-chain non-coding in detection biological sample
The reagent of RNA SNHG6 expression quantity.
3. application according to claim 1, which is characterized in that the kit is non-comprising long-chain in detection biological sample
The reagent of coding RNA SNHG6 expression quantity.
4. application according to claim 2 or 3, which is characterized in that long-chain non-coding RNA in the detection biological sample
The reagent of SNHG6 expression quantity, is selected from: there is probe, the genetic chip of detection specificity to long-chain non-coding RNA SNHG6, or
PCR primer.
5. application according to claim 2 or 3, which is characterized in that the biological sample is selected from: obtained from the fresh of object
Tissue or cell, formalin are fixed or paraffin-embedded tissue or cell, blood or body fluid.
6. long-chain non-coding RNA SNHG6 is preparing the application in breast cancer treatment drug, which is characterized in that the therapeutic agent is
Refer to the reagent for inhibiting or reducing long-chain non-coding RNA SNHG6 expression quantity.
7. application according to claim 6, which is characterized in that the inhibition reduces long-chain non-coding RNA SNHG6
The reagent of expression quantity, including siRNA, shRNA.
8. application according to claim 7, which is characterized in that the therapeutic agent is siRNA, and sequence is as follows:
Si-SNHG6#1 positive-sense strand: 5 '-GAGGUGCAAGAAAGCCUUUTT-3 ' (SEQ ID NO:1)
Antisense strand: 5 '-AAAGGCUUUCUUGCACCUCTT-3 ' (SEQ ID NO:2)；
Si-SNHG6#2 positive-sense strand: 5 '-GCGGCAUGUAUUGAGCAUATT-3 ' (SEQ ID NO:3)
Antisense strand: 5 '-UAUGCUCAAUACAUGCCGCTT-3 ' (SEQ ID NO:4)；
Si-SNHG6#3 positive-sense strand: 5 '-GCUUCGUUACCUCAAGUGUTT-3 ' (SEQ ID NO:5)
Antisense strand: 5 '-ACACUUGAGGUAACGAAGCTT-3 ' (SEQ ID NO:6).
9. application according to claim 8, which is characterized in that the therapeutic agent is for inhibiting breast cancer cell
Proliferation, invasion and migration.
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| CN113957152A (en) * | 2021-12-14 | 2022-01-21 | 浙江大学 | Detection kit for SNHG6 gene and application thereof |
| RU2807480C1 (en) * | 2022-12-29 | 2023-11-15 | Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт общей патологии и патофизиологии" (ФГБНУ "НИИОПП") | Method for predicting breast cancer metastasis based on set of long non-coding rna genes |
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| CN112575081A (en) * | 2019-09-28 | 2021-03-30 | 中国医学科学院肿瘤医院 | Application of long-chain non-coding RNA molecule in diagnosis and/or treatment of triple negative breast cancer |
| CN112575081B (en) * | 2019-09-28 | 2022-08-19 | 中国医学科学院肿瘤医院 | Application of long-chain non-coding RNA molecule in diagnosis and/or treatment of triple negative breast cancer |
| CN111973744A (en) * | 2020-07-15 | 2020-11-24 | 北京大学深圳医院 | Application of PLCE1-AS2 in breast cancer |
| CN111973744B (en) * | 2020-07-15 | 2022-07-01 | 北京大学深圳医院 | Application of PLCE1-AS2 in breast cancer |
| CN113957152A (en) * | 2021-12-14 | 2022-01-21 | 浙江大学 | Detection kit for SNHG6 gene and application thereof |
| RU2807480C1 (en) * | 2022-12-29 | 2023-11-15 | Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт общей патологии и патофизиологии" (ФГБНУ "НИИОПП") | Method for predicting breast cancer metastasis based on set of long non-coding rna genes |
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