CN108467891A - Applications of the SNHG5 in breast cancer diagnosis and assessment and outcome prediction - Google Patents
Applications of the SNHG5 in breast cancer diagnosis and assessment and outcome prediction Download PDFInfo
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Abstract
The invention discloses applications of the SNHG5 in breast cancer diagnosis and assessment and outcome prediction.Present invention discover that lncRNA SNHG5 notable low expressions in breast cancer tissue and breast cancer cell line, proliferation and the migration of breast cancer cell can be enhanced by knocking out lncRNA SNHG5 genes, and apoptosis is then suppressed, it can be seen that the expression of long-chain non-coding RNA SNHG5 and the diagnosis of tumour, transfer, recurrence, patient's prognosis and Efficacy of Neoadjuvant Chemotherapy are closely related, show that lncRNA SNHG5 genes can be used as diagnosis, prognosis evaluation or the marker for predicting curative effect.The breast cancer cell model for the SNHG5 low expressions established by transfecting breast cancer cell line by siRNA can use in the preparation method of breast cancer relapse and transfer preventing preparation.
Description
Technical field
The invention belongs to oncomolecularbiology technical fields, are related to a kind of long-chain non-coding RNA SNHG5 (lncRNA
SNHG5 it) and its applies.
Background technology
Tumour is to endanger the common disease and frequently-occurring disease of human health, especially malignant tumour now to have become threat mankind's life
One of the main reason for life.And in recent years, it is No.1 " tumour killer " that breast cancer has become global women, and it is pernicious swollen to occupy women
The first place of the tumor cause of the death.It is announced according to subordinate international cancer research institution of the World Health Organization《Global cancer epidemiology in 2012
Statistical data》It has been shown that, breast cancer is also the highest tumour of China's women incidence, accounts for the 15% of whole female malignants, sternly
Women physical and mental health is endangered again.
Currently, in addition to pathology and iconography detect, the laboratory screening of breast cancer and clinical monitoring are mainly with tradition
Carbohydrate Antigen CA125, CA153 etc. and carcinomebryonic antigen (CEA), although they with relative ease and of low cost,
They lack sufficiently high sensitivity and specificity, thus limit its diagnostic value to breast cancer, in practical applications
There are certain disputes.And the cure rate of breast cancer middle and advanced stage patient is low, therefore, seeks a kind of highly sensitive and specificity swollen
Tumor markers effectively early diagnose the emphasis for having become Recent study with prognostic evaluation index as breast cancer.
Long-chain non-coding RNA (long non-coding RNA, lncRNA) refers to that Transcript Length is more than 200 nucleotide
It is right, and itself do not have a kind of RNA of coding protein ability, it can participate in chromosomal rearrangement and histone modification, choosing
The important physiology mistakes such as interference, Microrna (microRNA) structure, regulatory protein matter activity are transcribed in the modification of selecting property montage sequence
Journey possesses extensive biological function.And largely study confirmation, the expression imbalance of lncRNA and the Preventive of tumour with
And the prognosis of patient is closely related.
Small nucleolar RNA host gene 5 (small nucleolar RNA host gene 5, SHNG5) is found recently
Long-chain non-coding RNA is the host gene of small nucleolar RNA (small nucleolar RNA, snoRNA) family U50 and U50 '
The ripe spliced body of U50HG exons.SHNG5 overall length 524nt, are defined as lncRNA, are located at human chromosomal 6q14.3, by
6 exons and 5 introne compositions.Accession number of the lncRNA SHNG5 in ncbi database, Nucleotide word banks be
NR_003038, in such as SEQ ID NO of its corresponding gene sequences described in ncbi database:Shown in 1.But at present about
The research of lncRNA SNHG5 is less, and the influence to breast cancer is even more to have not been reported.
Invention content
In view of this, the purpose of the present invention is to provide long-chain non-coding RNA (lncRNA) SNHG5 as biomarker
Purposes in preparing the preparation prevented for breast cancer diagnosis, assessment, outcome prediction, recurrence and transfer.
One aspect of the present invention is that providing the reagent that can detect the SNHG5 expression of long-chain non-coding RNA is preparing use
Purposes in the preparation of breast cancer diagnosis, assessment or outcome prediction.
In some embodiments of such use of the present invention, it is preferable that the reagent includes that can quantify detection long-chain
The real-time quantitative PCR detection reagent of non-coding RNA SNHG5 expressions.
In some embodiments of such use of the present invention, it is preferable that the reagent includes to the long-chain non-coding
RNA SNHG5 have the PCR primer of detection specificity.
In some embodiments of such use of the present invention, it is preferable that the nucleotide sequence of the PCR primer is such as
Under：
SNHG5 sense primers such as SEQ ID NO:Shown in 2；
SNHG5 downstream primers such as SEQ ID NO:Shown in 3.
Another aspect of the present invention is to provide can be by specificity interferes long-chain non-coding RNA SNHG5 genes
The siRNA for establishing the breast cancer cell model of SNHG5 low expressions is preparing the system prevented for breast cancer relapse and transfer
Purposes in agent：Interfere lncRNA SNHG5 genes, the breast cancer for establishing SNHG5 low expressions thin by siRNA specificity
Born of the same parents' model, the cell model can use in the preparation method of breast cancer relapse and transfer preventing preparation.
In some embodiments of such use of the present invention, it is preferable that the siRNA is selected from si-SNHG5-
Homo-83, si-SNHG5-Homo-208 or si-SNHG5-Homo-306, sequence are as follows：
Si-SNHG5-Homo-83 sense strand sequences are as shown in SEQ ID NO.6, antisense strand sequence such as SEQ ID NO.7 institutes
Show.
Si-SNHG5-Homo-208 sense strand sequences are as shown in SEQ ID NO.8, antisense strand sequence such as SEQ ID NO.9
It is shown.
Si-SNHG5-Homo-306 sense strand sequences are as shown in SEQ ID NO.10, antisense strand sequence such as SEQ ID
Shown in NO.11.
In the present invention, lncRNA SNHG5 expression in tissue and cell line is detected by real-time quantitative PCR, finds lncRNA
SNHG5 notable low expressions in breast cancer tissue and different breast cancer cell lines；Further, pass through specific siRNA
It transfects breast cancer cell line and knocks out lncRNA SNHG5 genes, by transfecting breast cancer cell to negative control group and siRNA
Cell Proliferation, migration, apoptosis are studied, it is found that the siRNA for knocking out lncRNA SNHG5 genes transfects breast cancer cell
(lncRNA SNHG5 low expressions) can enhance proliferation and the migration of breast cancer cell, and apoptosis is then suppressed, it is seen that the non-volume of long-chain
The expression of code RNASNHG5 and the diagnosis of tumour, transfer, recurrence, patient's prognosis and Efficacy of Neoadjuvant Chemotherapy are closely related,
Show that lncRNA SNHG5 genes can be used as diagnosis, prognosis evaluation and the marker for predicting curative effect, breast is transfected by siRNA
Gland cell system and the breast cancer cell model of SNHG5 low expressions established, can use and prevent in breast cancer relapse and transfer
In the preparation method of preparation.
Compared with prior art, the present invention has technique effect beneficial below：
The present invention proposes the new application of long-chain non-coding RNA SNHG5, finds that long-chain non-coding RNA SNHG5 exists for the first time
Differential expression in breast cancer tissue and different breast cancer cell lines, and prove the low expression meeting of long-chain non-coding RNA SNHG5
Cause breast cancer grade malignancy to increase, can be used as marker applied to diagnosis, assessment or the outcome prediction of breast cancer, transfer,
Recurrence.The breast cancer cell model for the SNHG5 low expressions established using siRNA can be used in breast cancer relapse and transfer
In the preparation method of preventing preparation.
Description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR detects normal breast tissue and lncRNA SNHG5 in cancerous tissue (tumor tissues)
The result of expression.
Fig. 2 is that real-time fluorescence quantitative PCR detects lncRNA in normal human mammary epithelial cell and different breast cancer cells
The result of SNHG5 expression.
Fig. 3 is that real-time fluorescence quantitative PCR is detected through three kinds of SNHG5 specificity sequences of small interfering RNAs transfection MCF7 breast cancer
The result of lncRNA SNHG5 expression in cell and negative control group.
Fig. 4 A~Fig. 4 D are that negative control group and sequences of small interfering RNAs (siRNA) transfect MCF7 in cell scratch experiment
The travel motion ability of breast cancer cell (si-SNHG5MCF7 cells) compares.Wherein, Fig. 4 A and Fig. 4 B are respectively negative control
For group (NC) in 0 hour, 96 hours cell growth states, amplification factor was 100 times (differences × 100), Fig. 4 C and Fig. 4 D difference
It is si-SNHG5MCF7 cells in 0 hour, 96 hours cell growth states, amplification factor is 100 times (differences × 100).
Fig. 5 is that negative control group (being denoted as NC groups) and sequences of small interfering RNAs (siRNA) transfect MCF7 breast cancer cells (note
For si-SNHG5 groups) ability of cell proliferation compare.
Fig. 6 A are the Apoptosis figure of negative control group, and Fig. 6 B are that sequences of small interfering RNAs (siRNA) transfects MCF7 breast cancer
The Apoptosis figure of cell (si-SNHG5MCF7 cells).Wherein, in each figure horizontal axis indicate be Annexin V dyeing point
The PI dye distributions of cell are shown in cloth, the longitudinal axis.Fig. 6 C are negative control group (being denoted as NC groups) and sequences of small interfering RNAs
(siRNA) comparison of the apoptosis rate of transfection MCF7 breast cancer cells (being denoted as si-SNHG5 groups).
Specific implementation mode
With reference to specific embodiments and the drawings, the present invention is further explained.It should be understood that these embodiments are merely to illustrate
The present invention rather than limit the scope of the invention.
In the following examples, the experimental methods for specific conditions are not specified, the conventional method in this field can be used, such as join
It examines《Molecular Cloning:A Laboratory guide》(third edition, New York, CSH Press, New York：Cold Spring
Harbor Laboratory Press, 1989), or according to the condition proposed by supplier, such as be documented in product description
Condition.
Not specified various instruments, raw materials and reagents are commercial product well known in the art in the following example,
It can be obtained by commercial sources.The used specific material listed in the examples below that and its source, are merely exemplary
, it is not intended to the limitation present invention.
One, material explanation
1.1 cell line sources
Human mammary epithelial cell MCF-10A is in October, 2014 purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences basis
Medical cell center, number 3111C0001CCC000406.
Human breast cancer cell line MCF7 (being also denoted as MCF-7) is purchased from Chinese Academy of Medical Sciences's preclinical medicine in October, 2014
Research institute's preclinical medicine cell centre, number 3111C0001CCC000013.
Human breast cancer cell line MDA-MB-231 is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's base in April, 2015
Plinth medical cell center, number 3111C0001CCC000014.
Human breast cancer cell line MDA-MB-453 is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's base in September, 2016
Plinth medical cell center, number 3111C0001CCC000016.
Human breast cancer cell line BT-474 is cured in January, 2017 purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences basis
Learn cell centre, number 3111C0001CCC000129.
The address of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's preclinical medicine cell centre is：Dongcheng District, Beijing during March Dongdan
Three No. 9.
1.2 cell line cultures
DMEM/F12 (1 of the MCF-10A cell lines containing 5% (v/v) horse serum (Gibco companies of the U.S.):1) culture medium
(HyClone companies of the U.S.), in 37 DEG C, 5%CO2Culture in constant incubator, is passed on every 2~3d.
MEM culture medium (the U.S. of the MCF-7 cell lines containing 10% (v/v) fetal calf serum (Gibco companies of the U.S.)
HyClone companies), in 37 DEG C, 5%CO2Culture in constant incubator, is passed on every 2~3d.
MDA-MB-231 cell lines, which are used, contains 10% (v/v) fetal calf serum (Gibco companies of the U.S.) DMEM culture mediums (U.S.
HyClone companies), in 37 DEG C, 5%CO2Culture in constant incubator, is passed on every 2~3d.
BT-474 cell lines, which are used, contains 10% (v/v) fetal calf serum (Gibco companies of the U.S.) DMEM culture mediums (U.S.
HyClone companies), in 37 DEG C, 5%CO2Culture in constant incubator, is passed on every 2~3d.
MDA-MB-453 cell lines, which are used, contains 10% (v/v) fetal calf serum (Gibco companies of the U.S.) DMEM culture mediums (U.S.
HyClone companies), in 37 DEG C, 5%CO2Culture in constant incubator, is passed on every 2~3d.
Steps are as follows for passage：Sterile dropper absorbs culture medium in culture bottle, and appropriate PBS (phosphate buffer are added
Saline, phosphate buffered saline solution) to remove serum residence；The pancreas egg that 1ml contains 0.25% (w/v) EDTA is added after removing PBS
White enzyme (being purchased from Gibco companies, the U.S.), is placed in 37 DEG C, 5%CO2About 30 seconds in constant incubator.Observable cell under microscope
Digestible degree is added 1~2ml and cultivates completely immediately when cell rounding protrusion, gap increase, are partially disengaged culture dish bottom
Base terminates digestion；Soft piping and druming bottle wall cell, collects cell suspension in 15ml centrifuge tubes, 1000rpm, is abandoned after centrifuging 3 minutes
Supernatant；After appropriate complete medium is added, cell and mixing is resuspended in soft piping and druming.According to vitro growth rates, it is added appropriate thin
Born of the same parents' suspension is in new culture bottle, and complete medium adds to about 4~5mL in culture bottle, rocks mixing and is placed on 37 DEG C, 5%CO2It is permanent
Warm incubator culture.
Above each cell is taken when reaching exponential phase.
Two, the expression quantity of real-time fluorescence quantitative PCR step detection SNHG5
2.1 real-time fluorescence quantitative PCR step explanations
Real-time fluorescence quantitative PCR takes following steps：
(1) extraction of sample total serum IgE：According to TIANGEN companies RNAsimple Total RNA Kit (article No.s：DP419)
The Total RNA of required reagent and step extraction sample to be tested (tissue or cell)；Use NanoDrop ND-2000c nucleic acid fixed again
Measure quantitative the extracted sample total serum IgE of instrument (NanoDrop Technologies, Wilmington, Delaware) purity and
Concentration.
(2) preparation of sample cDNA：Using TaKaRa kits PrimeScriptTM RT reagent Kit(Perfect
Real Time) (article No. RR037A) by the total serum IgE reverse transcription synthesis cDNA of extraction, reaction system and condition are as follows：
By above-mentioned component 37 DEG C of incubation 15min after mixing, then 85 DEG C of inactivation 5sec are to get to cDNA.
(3) amplification of target gene：Using TAKARA SYBR Premix Ex TaqTMII (TIi RNaseH Plus) is tried
Agent box (article No. RR820L) carries out fluorescent quantitative PCR by template of the cDNA of reverse transcription (using GAPDH as internal reference).With
By inventor's designed, designed and Jikang Biotechnology Co Ltd, Shanghai is transferred to synthesize in the primer of fluorescent quantitative PCR.Draw
The nucleotide sequence of object is as follows：
SNHG5 sense primers：
5'-GCTCTGAAGATGCAAAGATACACG-3’(SEQ ID NO:2)
SNHG5 downstream primers：
5'-TTCACTGGCTACTCGTCCACACT-3’(SEQ ID NO:3)
GAPDH sense primers：
5'-GAAAAACCTGCCAAATATGATGAC-3’(SEQ ID NO:4)
GAPDH downstream primers：
5'-TGGGTGTCGCTGTTGAAGTC-3’(SEQ ID NO:5)
Dye class quantitative fluorescent PCR system and reaction condition are as follows：
Sense primer (10 μm of ol/L) 0.8 μ L；Downstream primer (10 μm of ol/L) 0.8 μ L；
2×SYBR PremexExTaq II(TIi RNaseH Plus)25μL；
The i.e. 0.6 μ L of sample cDNA (50ng/ μ L) 30ng；
Add RNase Free dH2O to 20 μ L.
Use LightCycler 480System (Roche Diagnostics) fluorescence real-time quantitative PCR program：95℃
30s pre-degenerations connect 40 cycles：95 DEG C of 5s, 60 DEG C of 30~34s.System exports Ct values automatically.By gained Ct value experiment with computing groups
Relative to control group differential expression multiple.3 multiple holes of each sample, calculate average value.Relative expression quantity calculation formula：X=
2- △ △ Ct are calculated, wherein △ △ Ct=△ E- △ C, △ E=Ctexp-CtGADPH, △ C=CtctL-CtGADPH.
2.2 real-time fluorescence quantitative PCRs detect the expression quantity of normal breast tissue and SNHG5 in tumor tissues
By normal breast tissue and breast tumor tissues (obtain patient know and the agreement of ethics), preliminary low temperature is ground respectively
It is used as sample to be tested (tissue sample) after mill processing, according to above-mentioned 2.1 real-time fluorescence quantitative PCR step, is detecting mammary gland just respectively
The often expression quantity of tissue and lncRNA SNHG5 in tumor tissues.
Testing result is as shown in Figure 1.In Fig. 1, lncRNA SNHG5 genes high expression in the normal tissue, in breast cancer group
Middle low expression is knitted, expression difference of the lncRNA SNHG5 genes in normal structure and breast cancer tissue has significant difference (P
<* * are denoted as in 0.01, Fig. 1).
2.3 real-time fluorescence quantitative PCRs survey each cell line (MCF-10A, MCF-7, MDA-MB-231, BT-474, MDA-MB-
453) expression quantity of SNHG5 in
According to culture MCF-10A, MCF-7, MDA-MB-231, BT-474, MDA-MB-453 in 1.2 cell line incubation steps
Cell, each cell is taken when reaching exponential phase as sample to be tested (cell sample), according to above-mentioned 2.1 real time fluorescent quantitative
PCR step detects the expression quantity of lncRNA SNHG5 in above each cell respectively.
Testing result is as shown in Figure 2.In Fig. 2, lncRNA SNHG5 genes are in normal human mammary epithelial cell MCF-10A
Height expression, is low expression in different breast cancer cells (MCF-7, MDA-MB-231, BT-474, MDA-MB-453).
Expression difference of the lncRNA SNHG5 genes in normal human mammary epithelial cell and any breast cancer cell all has statistics
Difference (P<* * * are denoted as in 0.0001, Fig. 2).
2.4 real-time fluorescence quantitative PCRs survey the expression quantity of SNHG5 in si-SNHG5MCF7 cells
(1) sequences of small interfering RNAs (siRNA) transfects MCF7 breast cancer cells
The day before transfection, by 2 × 105~4 × 105A cell inoculation makes cell density when transfection reach in 6 orifice plates
25%~40%；4ul Lipofectamine3000 and 200ul serum-free MEM culture medium mixings are taken, is stored at room temperature 5 minutes；It takes
Lipofectamine3000 and serum-free MEM culture mediums is added with after 200ul serum-free MEM culture medium mixings in 8ul siRNA
Mixed liquor in, be stored at room temperature after being sufficiently mixed 20 minutes.Then inoculating cell, the hole containing 1.6mlMEM culture mediums are transferred to
In mix well, be put into 37 DEG C of CO2In incubator, after incubator culture 8~12 hours, the culture medium in 6 orifice plates is replaced
For complete medium (the MEM culture mediums for containing 10% fetal calf serum).
Here three kinds of SNHG5 specificity sequences of small interfering RNAs (siRNA) have been respectively adopted and have carried out cell transfecting, respectively
Si-SNHG5-Homo-83, si-SNHG5-Homo-208 or si-SNHG5-Homo-306, sequence are as follows：
Si-SNHG5-Homo-83 sense strand sequences：
5'-GAUGCAAAGAUACACGAAATT-3’(SEQ ID NO:6)
Si-SNHG5-Homo-83 antisense strand sequences：
5'-UUUCGUGUAUCUUUGCAUCTT-3’(SEQ ID NO:7)
Si-SNHG5-Homo-208 sense strand sequences：
5'-GAUAAUGAAUGUCGAAUGUTT-3’(SEQ ID NO:8)
Si-SNHG5-Homo-208 antisense strand sequences：
5'-ACAUUCGACAUUCAUUAUCTT-3’(SEQ ID NO:9)
Si-SNHG5-Homo-306 sense strand sequences：
5'-GUUGCAACGAUUUCUGGCUTT-3’(SEQ ID NO:10)
Si-SNHG5-Homo-306 antisense strand sequences：
5'-AGCCAGAAAUCGUUGCAACTT-3’(SEQ ID NO:11)
In above-mentioned transfection procedure, it is also provided with negative control group (Negative Control, NC), negative control group
(NC) siRNA sequence is as follows：
NC sense strand sequences：
5'-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID NO:12)
NC antisense strand sequences
5'-ACGUGACACGUUCGGAGAATT-3’(SEQ ID NO:13)
(2) transfection efficiency of si-SNHG5MCF7 cells is measured
The expression quantity of SNHG5, comes in each si-SNHG5MCF7 cells obtained by real-time fluorescence quantitative PCR detection transfection
Transfection efficiency of three kinds of SNHG5 specificity sequences of small interfering RNAs in MCF7 breast cancer cell lines is measured, that is to say that transfection obtains
Each si-SNHG5MCF7 cells transfection efficiency.
It is glimmering in real time according to above-mentioned 2.1 using each si-SNHG5MCF7 cells for having transfected as sample to be tested (cell sample)
Fluorescent Quantitative PCR step detects the expression quantity of lncRNA SNHG5 in each si-SNHG5MCF7 cells respectively.
The results are shown in Figure 3.In Fig. 3, lncRNA SNHG5 genes high expression in negative control group (NC), different
(si-SNHG5-Homo-83, si-SNHG5-Homo-208 or si-SNHG5-Homo-306 are denoted as si- in Fig. 3 to siRNA
38, si-208, si-306) transfection each si-SNHG5MCF7 cells in be low expression.LncRNA SNHG5 genes are in feminine gender
Expression difference in control group and any si-SNHG5MCF7 cells all has significant difference (P<Remember in 0.0001, Fig. 3
For * * *).Moreover, in comparison, lncRNA SNHG5 in the si-SNHG5MCF7 cells of si-SNHG5-Homo-306 transfections
It expresses minimum, illustrates transfection efficiency highests of the si-SNHG5-Homo-306 in MCF-7 breast cancer cell lines.
It is described above：After these three SNHG5 specificity siRNAs transfect MCF7 cells, it is thin can significantly to lower MCF7
The expression of lncRNA SNHG5 in born of the same parents, after especially transfecting MCF7 cells with si-SNHG5-Homo-306, in MCF7 cells
The expression of lncRNA SNHG5 is lowered the most notable.
Three, to the research of si-SNHG5MCF7 cells
The cells in vitro locomitivity of 3.1si-SNHG5MCF7 cells is analyzed
Si-SNHG5MCF7 cells and negative control group (NC) after selecting si-SNHG5-Homo-306 to transfect are research pair
As carrying out cells in vitro scratch experiment using six orifice plates (NEST companies), (cut, passes through microscopically observation in cell monolayer
Scratch removal process is to measure of cell transfer ability).
It is grouped the si-SNHG5MCF7 cells and negative control group cell for reaching exponential phase after taking transfection, uses pancreatin
After (Gibco companies of the U.S.) digestion, by 1 × 106/ hole takes cell inoculation to enter in six orifice plates, per hole culture medium containing 2MLMEM, cell
Quantity is advisable with carrying out being paved with board bottom after necessary processing；After cell is paved with board bottom, drawn perpendicular to orifice plate using 20ul pipette tips
Trace ensures that each scratch width is consistent；Culture medium is absorbed, PBS is rinsed to not having cell fragment substantially, and serum-free MEM trainings are added
Base is supported, 37 DEG C, 5%CO are put into2It is cultivated in incubator, cell growth state was photographed to record in 0,12,24,48,72,96 hour.
As a result as shown in Fig. 4 A~4D.Fig. 4 A and Fig. 4 B are respectively negative control group (NC) in 0 hour, 96 hours cells
Growth conditions, amplification factor are 100 times (differences × 100), Fig. 4 C and Fig. 4 D be respectively si-SNHG5MCF7 cells 0 hour,
96 hours cell growth states, amplification factor are 100 times (differences × 100).By Fig. 4 A~4D as it can be seen that si-SNHG5MCF7 is thin
The cell motility of born of the same parents is significantly stronger than negative control group (NC), illustrates that transfection knocks out the lncRNA in breast cancer cell line
After SNHG5 genes, the enhancing of breast cancer cell line transfer ability.
The growth kinetics of 3.2si-SNHG5MCF7 cells are studied
Si-SNHG5MCF7 cells and negative control group (NC groups) after selecting si-SNHG5-Homo-306 to transfect are research
Object carries out cell Proliferation detection using CCK-8 methods, specific as follows：
Cell counting board and coverslip are cleaned with 75% alcohol, coverslip is placed on tally after dry；According to digestion
The si-SNHG5MCF7 cells or negative control group cell precipitation amount obtained after centrifugation (contains precipitation with appropriate complete medium
The MEM culture mediums of 10% fetal calf serum) it is resuspended, 10ul cell suspensions are drawn with pipettor delay from coverslip edge slot after mixing
Slow to be added, when sample-adding, avoids bubble from generating.The cell number being located in four block plaids of tally is counted under 10 × object lens.According to
Following formula calculates cell density：(cell suspension total cell number)/ml=(four big lattice inner cell sum/4) × extension rate
×104；Cell is spread in 96 orifice plates, ensures about 2000/hole of cell density, in 37 DEG C, 5%CO2Training in constant incubator
It supports, OD values was surveyed respectively at 24,48,72,96 hours：(it is 1 by volume ratio by CCK-8 working solutions：9 CCK-8 stostes and culture medium
Configure, CCK-8 stostes are purchased from Japanese colleague company, and culture medium is the MEM culture mediums containing 10% fetal calf serum) si- is added
In the cell orifice plate of SNHG5MCF7 cells or negative control group cell, after being incubated 1.5 hours, OD is read at wavelength 450nm
Value.
The results are shown in Figure 5.Fig. 5 is that negative control group (being denoted as NC groups) and siRNA transfect MCF7 breast cancer cells and (be denoted as
Si-SNHG5 groups) ability of cell proliferation compare.From figure 5 it can be seen that the proliferation of si-SNHG5 groups is higher than NC groups.Culture first day,
The proliferation level ratio NC groups of si-SNHG5 groups improve 26.5% (P<0.01)；The proliferation of culture second day, si-SNHG5 groups is horizontal
65.6% (P is improved than NC groups<0.01)；Third day is cultivated, the proliferation level ratio NC groups of si-SNHG5 groups improve 61.4% (P<
0.05)；It cultivates the 4th day, the proliferation level ratio NC groups of si-SNHG5 groups improve 55.9% (P<0.001).
Explanation：The expression of interference (reduction) SNHG5 promotes the proliferation of breast cancer cell MCF-7, that is, it is thin to knock out breast cancer
After lncRNA SNHG5 genes in born of the same parents system, breast cancer cell line proliferative capacity enhancing, breast cancer cell line growth becomes faster.
The apoptosis rate of 3.3si-SNHG5MCF7 cells detects
Si-SNHG5MCF7 cells and negative control group (NC) after selecting si-SNHG5-Homo-306 to transfect are research pair
As using flow cytomery each group apoptosis rate.It is specific as follows：
Si-SNHG5MCF7 groups cell and cellular control unit are collected in grouping, are placed in centrifuge tube, and 800 × rpm centrifuges 5min,
Supernatant is removed, is operated according to Annexin V-FITC/PI apoptosis kit specifications, often 500ul buffer solutions are added in pipe, according to
Secondary addition 5ul Annexin V-FITC, 5ul PI, room temperature, which is protected from light, is incubated 10min, using flow cytomery each group cell
Apoptosis rate.
As a result as figs. 6 a to 6 c.Fig. 6 A are the Apoptosis figure of negative control group, and Fig. 6 B are that siRNA transfects MCF7
The Apoptosis figure of breast cancer cell.Wherein, in each figure horizontal axis indicate be Annexin V dye distribution, what the longitudinal axis was shown
It is the PI dye distributions of cell.Fig. 6 C are that negative control group (being denoted as NC groups) and siRNA transfect MCF7 breast cancer cells and (be denoted as
Si-SNHG5 groups) apoptosis rate comparison.It can be seen that the apoptosis rate ratio NC groups drop of si-SNHG5 groups from Fig. 6 A~Fig. 6 C
Low about 20% (P<0.05), explanation：The expression inhibiting of the SNHG5 apoptosis of breast cancer cell MCF-7 is interfered, that is, knocks out mammary gland
After lncRNA SNHG5 genes in cancerous cell line, breast cancer cell line apoptosis weakens.
In summary Germicidal efficacy and verification, lncRNA SNHG5 in tissue and cell line are detected by real-time quantitative PCR
Expression finds lncRNA SNHG5 notable low expressions in breast cancer tissue and different breast cancer cell lines；Further, pass through
Specific siRNA transfection breast cancer cell line knocks out lncRNA SNHG5 genes, by turning to negative control group and siRNA
The cell Proliferation of dye breast cancer cell, migration, apoptosis are studied, and find the siRNA transfections for knocking out lncRNA SNHG5 genes
Breast cancer cell (lncRNA SNHG5 low expressions) can enhance proliferation and the migration of breast cancer cell, and apoptosis is then suppressed, can
It is good at the expression of chain non-coding RNA SNHG5 and the diagnosis of tumour, transfer, recurrence, patient's prognosis and new adjuvant chemotherapy is treated
Imitate it is closely related, show lncRNA SNHG5 genes can be used as diagnosis, prognosis evaluation and predict curative effect marker.By small dry
The breast cancer cell model of SNHG5 low expressions disturbed RNA transfection breast cancer cell line and established, can use in breast cancer relapse
In the preparation method of transfer preventing preparation.
It can be seen that the purpose of the present invention is achieved completely and effectively.The method and principle of the present invention is
It is shown and is illustrated in embodiment, without departing substantially from the principle, embodiment can make arbitrary modification.So
Present invention comprises all variant embodiments based on claim spirit and right.
Sequence table
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<120>Applications of the SNHG5 in breast cancer diagnosis and assessment and outcome prediction
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Claims (7)
1. long-chain non-coding RNA SNHG5 is being prepared for breast cancer diagnosis, assessment, outcome prediction, is being answered as biomarker
Purposes in the preparation that hair and transfer prevent.
2. purposes as described in claim 1, which is characterized in that the reagent of long-chain non-coding RNA SNHG5 expression can be detected
Purposes in preparing the preparation for breast cancer diagnosis, assessment or outcome prediction.
3. purposes as claimed in claim 2, which is characterized in that the reagent includes that can quantify detection long-chain non-coding RNA
The real-time quantitative PCR detection reagent of SNHG5 expressions.
4. purposes as claimed in claim 3, which is characterized in that the reagent includes to the long-chain non-coding RNA SNHG5
PCR primer with detection specificity.
5. purposes as claimed in claim 4, which is characterized in that the nucleotide sequence of the PCR primer is as follows：
SNHG5 sense primers such as SEQ ID NO:Shown in 2；
SNHG5 downstream primers such as SEQ ID NO:Shown in 3.
6. purposes as described in claim 1, which is characterized in that long-chain non-coding RNA SNHG5 can be interfered by specificity
Gene and establish the siRNA of the breast cancer cell model of SNHG5 low expressions prepare it is pre- for breast cancer relapse and transfer
Purposes in anti-preparation.
7. purposes as claimed in claim 6, which is characterized in that the siRNA is selected from si-SNHG5-Homo-83, si-
SNHG5-Homo-208 or si-SNHG5-Homo-306, sequence are as follows：
Si-SNHG5-Homo-83 sense strand sequences are as shown in SEQ ID NO.6, and antisense strand sequence is as shown in SEQ ID NO.7.
Si-SNHG5-Homo-208 sense strand sequences are as shown in SEQ ID NO.8, and antisense strand sequence is as shown in SEQ ID NO.9.
Si-SNHG5-Homo-306 sense strand sequences are as shown in SEQ ID NO.10, antisense strand sequence such as SEQ ID NO.11 institutes
Show.
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| CN112899309B (en) * | 2021-02-08 | 2022-02-11 | 中国人民解放军军事科学院军事医学研究院 | Cardiomyocyte-specific overexpression Snhg5 vector and method for constructing transgenic animal by using same |
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