CN108642181A - The application of long-chain non-coding RNA SLC25A25-AS1 - Google Patents
The application of long-chain non-coding RNA SLC25A25-AS1 Download PDFInfo
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Abstract
The present invention relates to biotechnology, disclose SLC25A25 AS1 multiple fields new opplication.The present invention provides SLC25A25 AS1 the multiple fields based on diagnosing and treating cervical carcinoma field new opplication, to provide new treatment means and approach for the disease of related field, the achievement in research of long-chain non-coding RNA SLC25A25 AS1 is also supplemented.
Description
Technical field
The present invention relates to biotechnologies, and in particular to the application of long-chain non-coding RNA SLC25A25-AS1.
Background technology
In recent years, the research of long-chain non-coding RNAs arouses enough attention.Numerous research starts to report
LncRNA institute's roles in life process, these researchs are also to understand lncRNA played in neoplastic process
Function provides new angle.For researchers by high-throughput means, tissue and control to tumor patient have carried out large sample
Chip expression spectrum detection, to look for a variety of levels, horizontal differential expression spectrum.Urgently researchers are more for the data of magnanimity
For abundant and effective excavation.
SLC25A25-AS1 is the CRC gene expression profiles cohort study data-downloaded based on GEO databases
GSE39582 detection collection (N=585) is established and is found in the spectrum of the lncRNA differential expressions based on extensive colorectal cancer patients
, and confirm in Colorectal Carcinoma and serum and found in genetic chip as a result, being studied by biological function
SLC25A25-AS1 plays cancer suppressing action (Decreased expression of LncRNA in colorectal cancer occurrence and development
SLC25A25-AS1 promotes proliferation,chemoresistance,and EMT in colorectal
Cancer cells, Li et al., 2016).Currently, in addition to the functional study in Colon and rectum, there are no other about
Research reports of the SLC25A25-AS1 in tumour.
Invention content
In view of this, the purpose of the present invention is to provide SLC25A25-AS1 multiple fields new opplication.
SLC25A25-AS1 is the CRC gene expression profiles cohort study data-downloaded based on GEO databases
GSE39582 detection collection (N=585) is established and is found in the spectrum of the lncRNA differential expressions based on extensive colorectal cancer patients
, (sequence expression general lncRNA carrys out table to sequence in this field with the sequence of DNA as shown in SEQ ID No.1
Show)；Current result of study only shows that it can inhibit colorectal cancer, but for the effect in other cancers do not have into
The research conclusion of one step.
The present invention has detected contents of the SLC25A25-AS1 in cervical cancer patient and normal control's serum, finds serum
Middle SLC25A25-AS1 contents, which are presented from normal healthy controls person-epithelium of cervix uteri sample tumor, becomes becoming for patient-cervical cancer patient gradient reduction
Gesture, this demonstrate the tumor markers that SLC25A25-AS1 can be used as diagnosing cervical, by that can detect SLC25A25-AS1
Diagnostic reagent of expression quantity, such as primer, probe etc. or complete kit, such as amplification system, primer, probe, enzyme etc., can
Cervical carcinoma is diagnosed.Therefore, the present invention proposes SLC25A25-AS1 and is preparing uterine neck as diagnosis of cervical cancer marker
Application in cancer diagnostic reagent and/or kit.
Phenotype result of study is shown, in the Cervical Cancer HeLa Cells system of SLC25A25-AS1 high expression, cell Proliferation,
Clonality, migration and invasive ability are significantly inhibited；Nude mice by subcutaneous tumor formation experiment confirms that SLC25A25-AS1 exists
Inhibiting effect is formed with to the tumour of cervical carcinoma in vivo；The detection of signal path Activation finds to be overexpressed SLC25A25-AS1 then
It can promote Suppressor p53 and the activation of p21.Every test result shows that SLC25A25-AS1 can have cervical carcinoma notable suppression
It makes and uses.Therefore, the present invention proposes applications of the SLC25A25-AS1 in the drug for preparing treatment cervical carcinoma.
SLC25A25-AS1 expresses decline in drug resistant HeLa cells, this implies that SLC25A25-AS1 may be with drug
It is related to be resistant to situation.For this purpose, the present invention further has detected its reaction to cis-platinum in SLC25A25-AS1 overexpressing cells
Situation.Using MTT detection methods, experimental group is added corresponding drug, and control group is added isometric DMSO, 12 hours and 24 hours
Detect OD values, and the ratio of calculating survivaling cell respectively afterwards.The results show that after SLC25A25-AS1 overexpressions, chemotherapeutics
The lethal effect of cis-platinum significantly increases, this result prompt SLC25A25-AS1 can improve the sensitivity that tumour cell corresponds to chemotherapy
Property.Therefore, the present invention proposes SLC25A25-AS1 in preparing the cell killing drug of enhancing chemotherapeutic drugs on cervical cancer
Application.Wherein, the chemotherapeutics is preferably cis-platinum.
By above technical scheme it is found that the present invention provides SLC25A25-AS1 to be with diagnosing and treating cervical carcinoma field
The new opplication of main multiple fields also supplements length to provide new treatment means and approach for the disease of related field
The achievement in research of chain non-coding RNA SLC25A25-AS1.
Description of the drawings
Fig. 1 show SLC25A25-AS1 and becomes (CIN) and uterine neck in Normal group (Normal), epithelium of cervix uteri sample tumor
Expression in cancer (Cancer) patients serum；
Fig. 2 show influence results of the MTT detections SLC25A25-AS1 to HeLa ability of cell proliferation；
Fig. 3 show the result for the influence that SLC25A25-AS1 forms HeLa cell colonies；
Fig. 4, which is shown, is overexpressed the result that SLC25A25-AS1 inhibits cervical cancer tumer line HeLa cell cycle progressions；Its
In, each cylindricality represents G2/M, S and G0/G1 period successively from top to bottom in column diagram；
Fig. 5 show the result of influences of the SLC25A25-AS1 to HeLa drug sensitivity testings；
Fig. 6 show the result for being overexpressed the transfer ability that SLC25A25-AS1 inhibits HeLa；
Fig. 7 show the result for being overexpressed the invasive ability that SLC25A25-AS1 inhibits HeLa；
Fig. 8 show the result for being overexpressed the one-tenth knurl ability in nude mice that SLC25A25-AS1 inhibits HeLa.
Specific implementation mode
The invention discloses the application of long-chain non-coding RNA SLC25A25-AS1, those skilled in the art can use for reference this
Literary content is suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to art technology
It is it will be apparent that they are considered as being included in the present invention for personnel.Application of the present invention is by preferably implementing
Example is described, and related personnel can obviously not depart from the content of present invention, carried out to application described herein in spirit and scope
It changes or suitably changes and combine, to realize and apply the technology of the present invention.
The Lentiviral that the present invention expresses SLC25A25-AS1 by structure transfects 293T cells acquisition virus liquid
Correlation test is carried out, wherein involved step is as follows：
One, artificial synthesized (JaRa bioengineering Co., Ltd, Shanghai) obtains the cDNA of coding SLC25A25-AS1 overall lengths
Sequence
1, the extraction of the collection of serum and total serum IgE
About 5ml venous blood is collected with serum collection pipe, after the static 1h of room temperature, is centrifuged 10 minutes with 4 DEG C of centrifuge 820g.
After upper serum to be transferred to new collecting pipe, 4 DEG C, 16,000g centrifugations 10 minutes, to remove any cell fragment, -80 DEG C of guarantors
It deposits.
250 μ l serum are taken, 750ul Trizol LS are added, with the thorough mixing of rifle, lysis at room temperature 5 minutes.
200 μ l chloroforms are added, the separation of RNA phases, this step is accelerated to rock EP pipe 15s, static 10 points of room temperature back and forth
Zhong Hou, 12000rpm are centrifuged 10 minutes.
It takes 400 μ l of supernatant fluid that isometric isopropanol is added, is placed on -20 DEG C of refrigerator overnights, 12000rpm centrifuges 10 points
Clock.
Precipitation is washed with 75% ethyl alcohol, 8000rpm is centrifuged after five minutes, and room temperature is dried.
The water dissolution of 26 μ l RNA-free is added to precipitate, -80 DEG C of preservations.
2, the extraction of cell total rna
In the 6 orifice plates for covering with cell, 1mL Trizol are added, with the thorough mixing of rifle, lysis at room temperature 5 minutes.
200 μ l chloroforms are added, the separation of RNA phases, this step is accelerated to rock EP pipe 15s, static 10 points of room temperature back and forth
Zhong Hou, 12000rpm are centrifuged 10 minutes.
It takes 400 μ l of supernatant fluid that isometric isopropanol is added, is stored at room temperature after ten minutes, 12000rpm is centrifuged 10 minutes.
Precipitation is washed with 75% ethyl alcohol, 8000rpm is centrifuged after five minutes, and room temperature is dried.
The water dissolution of 30 μ l RNA-free is added to precipitate, -80 DEG C of preservations or immediately reverse transcription are cDNA.
3, cDNA is synthesized
The synthesis of first chain of cDNA
It is carried out according to ReverseTranscriptaseM-MLVforFirstStrandcDNA kit specifications.Specifically
Operation is as follows：
(1) PCR pipe for taking RNase free sequentially adds following reagent：
DNA-free Total RNA 12μL
Random primer(25μm)2μL
Mixing simultaneously centrifuges.
Rapid chilling 2min or more on ice after (2) 70 DEG C of heat preservation 10min, centrifuges the several seconds.
(3) following inverse transcription reaction liquid is prepared in above-mentioned PCR pipe：
30 DEG C are incubated after ten minutes, and 42 DEG C keep the temperature one hour.
Cooled on ice after (4) 70 DEG C of heat preservation 15min, inactivates reverse transcriptase.
(5) RNaseFreeddH is added into cDNA products2O complements to 100ul, is stored in -20 DEG C or -80 DEG C of refrigerators
In, it is spare.2ul dilutions are taken to carry out next step Real time PCR reactions.
4, Real-time PCR Analysis
Using above-mentioned cDNA as template, usePremix Ex TaqTMKit (Takara), in ABI ViiA 7
It is detected on fluorescence real-time quantitative PCR instrument (Applied Biosystems).Using GAPDH as internal reference, quantitative approach choosing
With 2- Δ Δ Ct methods.Concrete operations are as follows：
(1) according to gene transcripts sequence, across introne primer is designed；
(2) reaction system：
(3) reaction condition：95 DEG C of pre-degenerations 30 seconds, then 95 DEG C 15 seconds, 60 DEG C 30 seconds, 45 cycles；
(4) melt curve analysis reacts：It is above-mentioned after circulation terminates, carry out melt curve analysis drafting, condition be 95 DEG C 15 seconds, 60 DEG C
1 minute, 95 DEG C 15 seconds.Each sample, which is repeated three times, to be averaged, quantitative accurate to ensure.
Two, Lentiviral pLVX-Puro SLC25A25-AS1 are built
Artificial synthesized (JaRa bioengineering Co., Ltd, Shanghai) obtains the cDNA sequences of coding SLC25A25-AS1 overall lengths
Row, and its 5 ' end and 3 ' ends respectively contain restriction enzyme BamHI and XbaI enzyme cutting site sequence.Segment after synthesis is through limit
Property restriction endonuclease BamHI and XbaI (Fermentas, USA) double digestion processed is connected to the expression of pLVX-Puro slow virus and carries after purification
On body, it is named as pLVX-PuroSLC25A25-AS1.Sequence verification recombinant slow virus expression vector Insert Fragment it is correct
Property.
Three, slow virus prepares and surely turns the structure of cell line
1, the culture of 293T
(1) recovery 293T cells：Conventionally recovery 293T cells, when cell confluency degree reaches 80% or so into
Row passage.
(2) passage of 293T cells：Old culture medium is sucked, it is adherent to be carefully added into sterile PBS phosphate buffers, 293T
Cell line is adherent loosely, is easy to fall off, and is sure not directly to be added dropwise on cell.After gently shaking Tissue Culture Dish, PBS bufferings are discarded
Liquid.It is adherent be carefully added into the trypsase of 1ml pre-temperatures after, 37 DEG C are incubated about 2 minutes, and complete medium is added, and blow and beat adherent thin
Cell suspension is transferred in 15ml centrifuge tubes after born of the same parents, 800 revs/min, centrifuges 3 minutes, discard supernatant liquid.
(3) after the complete medium resuspension cell of pre-temperature is added, according to 1:3-1:5 ratios reach in new culture dish.Turn
Microscopically observation cell before dye, degrees of fusion should be of about 80%, and cell state will be got well, pollution-free, clean bright.
2, the transfection of 293T cells
(1) it transfects：Two sterile 1.5ml EP pipes are taken, are respectively labeled as overexpression group and control group, and in every pipe
DMEM culture mediums, 500 μ l/ pipes are added.PLVX-Puro SLC25A25-AS1 recombinant lentiviral diseases are sequentially added in overexpression group pipe
Malicious expression plasmid (5 μ g) and slow virus packaging plasmid pMD2.G (2.5 μ g), psPAX2 (5 μ g).It is sequentially added in control group pipe
PLVX-Puro zero loads are overexpressed plasmid (5 μ g, be abbreviated as pLVX-Puro vector) and slow virus packaging plasmid pMD2.G
(2.5 μ g), psPAX2 (5 μ g) vibrate mixing 30s-1min, are stored at room temperature.40 μ g PEI transfection reagents are often separately added into pipe,
Mixing 30s-1min is vibrated, is stored at room temperature 20 minutes.Period is with the not antibiotic DMEM culture mediums containing 10%FBS to 293T
Cell carries out changing liquid, the holes 10ml/.The rotaring redyeing system of preparation is slowly added in culture medium, even culture medium is gently shaken.
(2) liquid is changed：Liquid is changed after 12 hours, gimmick is soft, avoids blowing and beating in cell.Transfection efficiency has reached most at this time
Height changes the toxic effect that liquid can remove PEI to cell.
(3) virus is received：Virus liquid is collected for the first time after 48 hours, supernatant is fitted into the centrifuge tube of 15ml, is placed on 4 DEG C of ice
Case preserves, and new culture medium is added and continues to cultivate, and virus liquid is collected second after 72 hours, draws supernatant to new 15ml centrifugations
Guan Zhong.After inciting somebody to action supernatant merging twice, 3,000 × g is centrifuged 5 minutes and is removed residual cells, is reused 0.45 μ l filters and was carried out
Filter obtains the virus for being overexpressed pLVX-Puro SLC25A25-AS1 and control group virus, after virus liquid is dispensed, -80 DEG C of guarantors
It deposits spare.
3, slow-virus infection aim cell system
(1) bed board：It is one day before infection, normal by being pressed in exponential phase, cervical cancer tumer line HeLa in good condition
Rule method carries out pancreatin digestion, and uniform cell suspension is resuspended and be made using complete medium, and cell is passaged in six orifice plates,
37 DEG C, 5%CO2 overnight incubations, cell density reaches 20-30% or so and is preferred after 24 hours.
(2) infection aim cell system：Cell is carried out to change liquid, 1ml virus liquids are added in 1ml culture mediums, another that 8 μ g/ml are added
Polybrene (Sigma-Aldrich, Shanghai, China) enhance infect efficiency, gently shake even culture medium, 2500rpm
After centrifugation 30 minutes, it is put into be incubated 24 hours in 37 DEG C of cell incubators and changes liquid.
(3) puromycin screens：Cell continues culture 48 hours, and visual cell's stand density is passed if cell has been expired
Generation, if cell density can directly change liquid less than 80%, using 2 μ g/ml puromycins (Sigma-Aldrich, Shanghai,
China it) is screened, daily timely observation cell state, visual cell's death condition, the purine-containing mycin of replacement in every 2 days
Complete medium can carry out rinse when changing liquid with PBS buffer solution, broken with the cell after the dead cell of removal and cell death
Piece.
(4) efficiency of infection is verified：After puromycin screens 7 days, total serum IgE is extracted, is verified using RT-PCR and is overexpressed effect
Rate.It obtains surely turning clone after about 1 week of continuous culture, is enlarged culture and identification.
Meanwhile specific test method is as follows：
1, mtt assay measures cell growth curve
By the cell of exponential phase, after being counted with trypsin digestion, configured with the culture medium containing 10% fetal calf serum
At the cell suspension of suitable concentration.
It seeds cells into 96 well culture plates, 1000 cells is inoculated with per hole, if 6 parallel holes, 37 DEG C, 5%CO2,
Culture 24 hours.
Its 492nm optical density (OD) value is detected with mtt assay at 1,2,3,4,5 day respectively：Liquid is discarded supernatant before detection, is used
PBS cleans cell 1 time, and 110 μ L MTT dye liquors (the 20 μ L 5mg/mL MTT+90 μ L free serum cultures of Fresh are added per hole
Liquid), 37 DEG C, 5%CO2 is cultivated 4 hours.
Most supernatant is carefully abandoned, 150 μ L DMSO dissolving MTT formazans are added and precipitate, after mixing, in wavelength in microplate reader
OD values are measured at 492nm.
To measure number of days as abscissa, growth curve is drawn by ordinate of absorbance.
2, plate clone forms experiment
Logarithmic growth phase cell, conventional digestion prepare single cell suspension and count.
Inoculating cell gently shakes culture dish in six orifice plates, the holes 1000/2ml/ with ten word directions, keeps cell dispersion equal
It is even, 37 DEG C, 5%CO2Middle routine culture 10-14 days.
When occurring the visible clone of naked eyes in culture dish, culture is terminated, culture solution is abandoned, PBS is carefully cleaned 2 times, and air is dry
It is dry.Methanol fixes 15min, is air-dried after abandoning methanol.10 min are dyed with Giemsa stain, flowing water slowly washes away dye liquor, empty
Gas is dried.It takes pictures, count.
3, the flow cytometer detection cell cycle tests
Cell cycle (cell cycle), which refers to cycling cell, to be terminated from a mitosis to there is silk point next time
It splits and terminates undergone whole process.In this process, cellular genetic material is replicated and is doubled, and average at the end of division
It is assigned in two daughter cells.Cell cycle can be divided into a phase (interphase) and m period (Mphase) again, carefully
The intercellular phase is often divided into rest period (G0) again, DNA pre-synthesis phases (G1), and DNA synthesizes the phase (S), DNA post-synthesis phases (G2), entirely
Period is represented by G1 → S → G2 → M.DNA cycle detections can be used to the situation of each phase in reacting cells period, i.e. cell increases
Grow situation.The characteristic that can be combined with fluorescent dye (such as propidium iodide PI) using intracellular DNA, cell its DNA of each period
Fluorescent dye of the content difference to combine is different, and the fluorescence intensity of flow cytomery is also different.
Cell is fixed：Pretreated cell is carefully digested to single cell suspension, takes in the EP pipes of 1.5mL, 1000g
5min is centrifuged, cell is collected into tube bottom.After ice-cold PBS is washed one time, cell is softly resuspended in 70% ethyl alcohol
In, -20 DEG C of fixations are overnight.
Dyeing：The cell fixed is centrifuged into 1000g, 5min.After discarding ethyl alcohol, ice-cold PBS is washed one time,
Centrifugation, discards PBS, be added PI dye liquors to final concentration of 50 μ g/mL, TritonX-100 final concentration 0.25% (volume ratio) and
The RNase of final concentration of 100 μ g/mL.
Room temperature is protected from light effect 30min or so, upper machine testing.
4, Cell migration assay
Collect cell：Conventionally after vitellophag, serum free medium washs twice, with the training of 1mL serum-frees
Base weight is supported to hang cell and carry out cell count.
DMEM culture solutions of the 600 μ L containing 10%FBS is added in room under Transwell, and it is thin that upper chamber adds 200 μ L DMEM to be resuspended
Born of the same parents (8 × 104~10 × 104A/well), pay attention to：Mixing cell suspension is blown and beaten before being added dropwise, and is vacantly added dropwise above cell
In addition cell ensures cell film and liquid contact surface bubble-free.37 DEG C, 5%CO2 cultures 12~for 24 hours.
It is careful to take out cell, the liquid on cell upper layer is sucked, after dipping PBS with cotton swab, cleans cell upper layer 5 times,
It is careful not to exert oneself very much, to prevent making polycarbonate membrane deform, ice-cold methanol room temperature fixes 20min.
Dyeing：0.5% violet staining liquid is taken to be added in 24 orifice plates, per 600 μ L of hole.By the cell after fixation from methanol
It takes out, carefully softly wipes cell upper layer with cotton swab, then put it into violet staining liquid and dye 10 minutes, take out small
Room, cell is softly rinsed for several times with flowing water, and the dyeing liquor that do not rinse well on cell upper layer is carefully wiped with cotton swab and is not wiped
After clean cell, it is air-dried.
Film is set on glass slide, film bottom surface upward, randomly selects 5 visuals field and takes pictures and count under mirror.
5, cell invasion is tested
Cell invasion experiment uses 24 well of chamber marigel invasion, BD biocoat.
Pipette tips, centrifuge tube, the cells Matrigel and Transwell are placed in 4 DEG C of precoolings overnight.
Next day, by the DMEM culture solutions 1 of Matrigel serum-frees:After 8 dilutions, 25 μ L/well are added to small interior, room
Temperature solidification one hour after abandoning dilution, after being cleaned twice with the DMEM culture solutions of 37 DEG C of preheatings, is used for Matrigel.
Subsequent step is same to migrate experiment.
6, nude mice by subcutaneous tumor formation is tested
Experimental animal in this research selects the Female nude mice of BALB/cA-nu no-special pathogen grades, from Beijing China Fukang
It buys biotech inc.No-special pathogen refers in nude mice body without specific pathogenic microorganism and parasite
In the presence of, but allow that there are unspecific microorganism and parasites.BALB/cA-nu nude mices are by CRJ (Charles River
Japan) company is by BALB/cABon-nu and BALB/cAnNCrj-nu mate and can hand over to obtain the small of the strain
Mouse, is mainly characterized by that whole body is hairless, and hypoevolutism, itself is lower for the resistance, especially lower with female mice resistance, is easy to happen
The infection of serious mortality, thymus gland congenital absence is the recessive inheritance of o.11 chromosome, T cell immune deficiency, but B
Cellular immunity is normal, innately has higher NK cells (natural killer cells) vigor, contactless sensibility, no graft rejection
Reaction.
This research uses BALB/cA-nu nude mices, 4-5 week old, weight in 16g between 18g, all females, raising in
Cancer Hospital of Chinese Academy of Medical Sciences animal experimental center.Between 25 DEG C to 27 DEG C of steady temperature, constant humidity 45% to
50%, fresh aseptic filtration air, dust and bacteria removing without being raised under the conditions of special pathogen receptacle.By animal be put into through
In the organic glass raising box of disinfection, each raise raises 5 nude mices in box, and raising box number is then emitted on ultra-clean life again
On object laminar-flow rack, the water and feed that are handled by high pressure sterilization are freely absorbed for nude mice, are replaced by autoclave sterilization every 3 days
Feed and bedding and padding, cage for rearing poultry and drinking bottle will pass through disinfection by ultraviolet light processing once every three days, feed per treatment and feeding
To ensure to strictly observe sterile working when supporting cage tool.Experimental animal will again carry out in animal feeding room by 7 days laundering period
The inoculation of tumour cell.
While nude mice carries out adaptation experimental situation, amplification in vitro is overexpressed HeLa and surely turns cell line and control group
Cell.
When degrees of fusion of the cell in culture dish reaches 80-90% or more, using trypsin digestion cell, full culture is used
Cell suspension is made in base, 800 revs/min, centrifuges 3 minutes, discards supernatant, and washing cell then is resuspended using sterile PBS solution,
It 800 revs/min, centrifuges 3 minutes, discards supernatant, reuse PBS buffer solution and prepare single cell suspension, made using cell counter
The concentration of cell suspension reaches 2.5 × 107/ml, and the cell suspension after the completion of counting is put on ice for preserving, prepare to
Mouse bare subcutaneous injection implantation tumor cell.
It before experiment, is numbered first using ear nail to experimental animal, carries out grouped record, avoid nude mice in injection tumor formation
Shi Fasheng dies unexpectedly.
Using capacity for 1ml skin test needle as pumping needle, mix well cell before suction of cells suspension, then draw
In 0.5ml to syringe tube, the bubble at syringe needle is discharged, with syringe needle gently blunt separation armpit skin, cell is concentrated and is injected
To mouse back back upper place position, and 100 μ L are slowly injected, cell suspension is made to focus on specific tumor formation position.
The animation and tumor formation situation of routine observation mouse.
Method of craning one after being inoculated with 25 days puts to death experimental animal, puts to death animal and carefully completely strips out subcutaneous tumor tissue later,
With the wide diameter of major diameter at electronics vernier caliper measurement tumor formation position, the volume of tumor is calculated with 1/2 (major diameter × wide diameter × wide diameter).PBS
Tissue after taking pictures, is immersed formalin and fixed in case paraffin embedding by the structural bloodstain of buffer solution for cleaning.
In addition, in contrast test in a particular embodiment, other than the due difference of each group, other experimental conditions are protected
Card is consistent, it is ensured that the comparability of experimental result；Statistical method using IBM SPSS STATISTICS V20_32bit softwares into
Row analysis.When analyzing continuous variable, (Student's t test) is examined using t between two groups of inspection results, three groups of inspection results
Between using one-way analysis of variance discontinuous variable when, examined using Chi-Square.P<0.05 is considered to have statistics meaning
Justice.Real time PCR experimental results use paired-sample t test, non-matching sample t-test, Mann-Whimey inspection parties
Method, Spearman sum of ranks correlation analyses etc. are for statistical analysis.Using GraphPad Prism 6.0 and PhotoShop C5 into
Row experimental result is drawn.
The application with regard to long-chain non-coding RNA SLC25A25-AS1 provided by the present invention is described further below.
Embodiment 1：SLC25A25-AS1 becomes the expression in (CIN) patients serum in cervical cancer patient and epithelium of cervix uteri sample tumor
The present invention has detected SLC25A25-AS1 and becomes patient and 76 in 76 cervical cancer patients, 67 epithelium of cervix uteri sample tumors
Expression in normal healthy controls person's peripheral blood (demographic data of serum donors is shown in Table 1).Result of study is found, in serum
SLC25A25-AS1 contents, which are presented from normal healthy controls person-epithelium of cervix uteri sample tumor, becomes the trend that patient-cervical cancer patient gradient reduces
(Fig. 1).
1 normal healthy controls of table, CIN and cervical cancer patient population statistics
Embodiment 2：SLC25A25-AS1 inhibits the proliferative capacity of cervical cancer tumer line HeLa
In order to study the influence of SLC25A25-AS1 cell proliferations, the present invention is aforementioned steady using MTT colorimetric determinations
Turn the proliferative capacity of cell line.Experimental result shows that up-regulated expression SLC25A25-AS1 significantly reduces the proliferation of HeLa cells
Ability (Fig. 2).
Embodiment 3：Influences of the SLC25A25-AS1 to cervical cancer tumer line HeLa Colony formings
In order to reflect that the proliferative capacity of individual cells, the present invention have carried out plate clone and formed experiment again.It plants in the orifice plate
It plants seldom cell and makes individual cells, the cell that can form visible clone must be cell that is adherent and having proliferative capacity.
Experimental result is shown, compared with cellular control unit, up-regulated expression SLC25A25-AS1 significantly inhibits cervical cancer cell HeLa
Colony forming (Fig. 3).The result prompts SLC25A25-AS1 to have the function of significantly inhibiting to individual cells proliferation.
Embodiment 4：It is overexpressed SLC25A25-AS1 and inhibits cervical cancer tumer line HeLa cell cycle progressions
Aforementioned MTT and plate clone experiment all prove that the pernicious of tumour cell can be inhibited by being overexpressed SLC25A25-AS1
Proliferation.The reason of being overexpressed SLC25A25-AS1 Inhibit proliferatons for research, the present invention by SLC25A25-AS1 stablize high expression and
After cellular control unit system expands culture, pancreatin digests and collects cell, using flow cytomery cell cycle situation of change
(Fig. 4).Experimental result is shown, compared with cellular control unit, up-regulated expression SLC25A25-AS1 causes thin in the G0/G1 phases
Born of the same parents' ratio dramatically increases, and G0/G1 phases cell proportion rises to 59.3% by 51.4%, and the cell proportion in the S phases is then reduced, by
16.7% is reduced to 15.8%, and the cell proportion in the G2/M phases also substantially reduces, and is reduced to 24.8% by 31.8%.These grind
Study carefully result prompt, height expression SLC25A25-AS1 causes the HeLa cells generation G0/G1 phases to be blocked, causes cell cannot positive normal open
Spend the cell cycle.Data all have statistical significance (P through t inspections<0.05).
Embodiment 5：It is overexpressed the influence that SLC25A25-AS1 is resistant to cervical cancer cell cisplatin chemotherapy situation
Before this invention in the research of phase, to cervical cancer cell HeLa carry out cisplatin treated 12h, control group without drug at
Reason.After extracting RNA, situation of change is expressed using Realtime PCR detections SLC25A25-AS1.The results show that at by cis-platinum
The expression of SLC25A25-AS1 is significantly lowered compared to the control group in the HeLa cells of reason, shows SLC25A25-AS1 in tumour
In expression decline may be related with Chemoresistance.
Since the above results show that SLC25A25-AS1 expresses decline in drug resistant HeLa cells, this hint
It is related that SLC25A25-AS1 may be resistant to situation to cis-platinum.For this purpose, the present invention is further in SLC25A25-AS1 overexpressing cells
In have detected its response situation to cis-platinum.Using MTT detection methods, suitable cis-platinum, the bodies such as control group addition are added in experimental group
Long-pending DMSO detects OD values, and ratio (the experimental group OD values/control group of calculating survivaling cell respectively after 12 hours and 24 hours
OD values).The results show that after SLC25A25-AS1 overexpressions, the lethal effect of chemotherapeutic drugs Cisplatin significantly increases (Fig. 5).
Embodiment 6：It is overexpressed the transfer ability that SLC25A25-AS1 inhibits HeLa
Present invention employs Transwell experiments to detect after SLC25A25-AS1 is overexpressed to HeLa cell line migration energies
The influence of power.Detection cell migration ability common method has cut healing method and the cells Transwell method etc..Transwell is real
It tests in the way of Serum-induced, so that the cell in serum free medium is passed through the micropore on the cells transwell film, reach film
In the rich blood serum medium of lower layer, the locomitivity of cell is compared using the cell number across microporous barrier.Transwell this
One method is more accurate compared with cut healing method, is also more easy to carry out data analysis.The present invention will compare pLVX-Puro vector with
It is overexpressed pLVX-Puro SLC25A25-AS1 and surely turns HeLa cell lines, carry out pancreatin digestion and count, be inoculated with 8 × 10 respectively4
A/well fixed dyeing, cell that microscopically observation cell lower layer passes through behind the cells Transwell, 18h randomly select 5
A different visual field take pictures and counting statistics.The ability that tumour cell passes through 8 μm of micropores to enter bottom chamber from upper chamber is anti-
The external transfer ability of cell is answered.The results show that the cell number of pLVX-Puro vector cell migrations to film lower layer is
140.0 ± 10.0, and the cell number of pLVX-Puro SLC25A25-AS1 cell migrations to film lower layer is 27.0 ± 3.0,
In the state of being overexpressed SLC25A25-AS1, the transfer ability of HeLa cells reduces 5.2 times, and t examines display, the difference
With statistical significance (P=0.0084) (Fig. 6).The result illustrates, SLC25A25- is overexpressed in cervical cancer tumer line HeLa
AS1 genes inhibit the transfer ability of cell.
Embodiment 7：It is overexpressed the invasive ability that SLC25A25-AS1 inhibits HeLa
Influence of the present invention using the cells Transwell method detection SLC25A25-AS1 to cell invasion ability.
Matrigel is the basement membrane matrix extracted from the EHS mouse tumors rich in extracellular matrix protein, and main ingredient is viscous including layer
Even albumen, IV Collagen Type VIs, nestin, heparin sulfate glycoprotein etc..Under certain condition, polymerizable formed of Matrigel has life
The active three dimensional matrix of object, composition, structure, physical characteristic and the function of analogue body inner cell basilar memebrane.It is small in Transwell
Cup upper layer is covered with certain density Matrigel, and cell passes through secretory protein hydrolase, decomposes basilar memebrane, could pass through micropore into
Enter lower room.This process reflects the vitro invasion ability of cell.The results show that pLVX-Puro vector are invaded to lower layer
Number of cells be 157.5 ± 4.5, cell pLVX-Puro SLC25A25-AS1, which are invaded to the number of lower layer, is respectively
91.5 ± 3.5.After being overexpressed SLC25A25-AS1, the invasive ability of HeLa cells reduces 1.7 times, and t, which is examined, shows P=
0.0074, which has statistical significance.This inhibits HeLa cells the results show that being overexpressed SLC25A25-AS1 genes
Invasive ability (Fig. 7).
Embodiment 8：It is overexpressed SLC25A25-AS1 and inhibits one-tenth knurl abilities of the HeLa in nude mouse
It is overexpressed the influence grown to transplanted tumor in nude mice in order to study SLC25A25-AS1, the present invention utilizes pLVX-Puro
Vector and pLVX-Puro SLC25A25-AS1 surely turn HeLa cell lines structure Nude Mouse Model, use and crane one after 25 days
Execution method puts to death animal, and tumor tissues is taken to take pictures and weigh, and measures subcutaneous tumors major diameter and minor axis twice, with formula V=1/2 ×
(major diameter × minor axis × minor axis) calculates knurl product.Experimental result is shown, in the control group of injection pLVX-Puro vector, is swollen
The volume of tumor is 498.9 ± 101.3mm3, and inject in the experimental group of pLVX-Puro SLC25A25-AS1, gross tumor volume is
127.7±36.43mm3, hence it is evident that it is less than control group (P=0.017).In the control group for injecting pLVX-Puro vector, tumour
Weight be 334.0 ± 69.87mg, and inject in the experimental group of pLVX-Puro SLC25A25-AS1, tumor weight 127.1
± 28.00mg is lighter than control group (P=0.0413).This result illustrates, compared with empty over-express vector control group, up-regulated expression
SLC25A25-AS1 can significantly inhibit the growth of transplanted tumor in nude mice, consistent with Vitro Experimental Results (Fig. 8).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Affiliated Hospital of Zunyi Medical College
Cancer Hospital of Chinese Academy of Medical Sciences
<120>The application of long-chain non-coding RNA SLC25A25-AS1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3842
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
attctccatc cctgcattcc tccatcgcag catccctgca tgcctccgtt ctctgcatcc 60
ctccacccca gtatccctgc acctcttcat ccctccattc ctgcatccct ctatttttcc 120
atccctccat tcctgtatcc ctgtgccctc catcctccat cccagcatcc ctccatccct 180
gctccccact ctcacttccc ttcccttcac agacaggctt ttcctgccat cctcagaccc 240
cacccagggt tacctgatgc ctttccagct gcacacgggg actgactcac ctctctcttt 300
ctcagtccca aagtcccgaa agagagcatc tgatgtggtc agcttgtgac aaggcgtcca 360
ccttctgtcc atgacatgga ggccagggga aggtctcact gcccagcacc ccaccctgtg 420
ctcccaggcc ttttgaatgt tcctcctgct cagcccagtg actgcgggct gtggctcctc 480
ctccagcctc ccctcgaggt cctggtctta cttaggaggc cccgggtgta gatgccttcc 540
cacccaccag gcattgcccc ttttcctggc ttcacagact cgggaataaa gtttccttct 600
gtttcccctc ttgcagaagg agatccggtt ggcagctaaa ccgcgctggg aacaggggcc 660
tgagtcctgg actagggctc tttccccggg gctgctgcag atggggagga gcctacaccc 720
gcctcccgag tgctaatcag acctgacagg ctggagaatg gccagtcagc ctgaggccac 780
cgcgggacac cacctaggcc cagcttctcc ccccaccaga gggcgccaga gcccgccaag 840
cctcgtagga gatggcaaca gggcctgctg ctgccaccta gcggccaatt ccgggaatga 900
atctcggcac gctcattacc cagcgactct gcccatcttg accctttatg tgaagcagaa 960
cagccgcttc cgcagtgagc tgtcagaagg cgtcgtgcct gtcttgctag tggggaaact 1020
gaggctcaga gaggcagaag acttgcccaa gatcacacca ccgggaccca ggattcaagc 1080
gcaggcctgc ccaggctctc tgtggcctcc tggctgcgag gaggcagcca gggaccaggt 1140
gccacccttc tgagacacct gagatcccag gcccgagagg atgaaggcgg gattacctgg 1200
agcgtgtctg aatgctggag gaagaagggc agctgggaga tgaagctgtc aggatgggcc 1260
gcatcccatt tcctgcctcg tttcagttca actttccaac agacctccct ggctcgtctt 1320
gctcttctct aatggacaaa caaacaggct cagagaggtg gtgtgacttg cccaaggtca 1380
ctcagcttgg atgctatgga acagggacgt ccactgtccc agtctgttta tgggaagccg 1440
ctctgcaact gtcctgaccc accacatgcc ccaccgctgt ttctcttgcc ctgacccctt 1500
gttccctgga ccagggtggc acagctccag gctcttgggc ccttcccgag ggcaggcacc 1560
tgtgactgtg tccccaaaga cctgagtggc tgagggggcc ccacagagct tggacttcct 1620
ggaggacaag gaggggtctg ccagccaccc ccaccacgcc cgccccaggg ctcccctgga 1680
gcttccatgc cagccggact caggtgggtc tggaggagca ccgtgcctcc aatcagacct 1740
tgagatgtgc cccctgcccc cactgtgccc tcccctgccc aggagtctgg ttgcaaaccc 1800
tgattaaggg gattttatct ccaccagagg gccagtaggt gggaagtagc ttaaacaatg 1860
caggtttata atctcacagt tctggaggtc aagagtctga aatgggcctc atggggctaa 1920
aaccaaggtg tctgcagggc tgtgttcctt ctggaggctc cagggcagga aggggaggat 1980
ccacttctgt gcctttccag cttctagagg ctgcctgcgt tccttggctc gtggcccctt 2040
cctccacctt caagccagca gcggaggcct gagtccttct catgccatct ctctgttctc 2100
tctcctgcct cctcctccac actgaaggac ccctgtgatc acactggccc ccccaccgga 2160
tgacccagga taatccatct ccctgtttga aggtcggctg attagcaacc ttcattccat 2220
ctgcctcctt cattccccct ggccatgtaa tgggattcac agcttctggg gattaggaca 2280
tggacatctt gtggcggggg cataattctg tcgacgacac caagaaacac ttggatgtta 2340
aggattcacc gaacactgtt caggctgagg caggagaatt gcttgaacct gggaggcaga 2400
ggttgcagtg agccgagatc ataccattgc actccagcct gggcaacaag agtgaacctc 2460
catcacaaaa aacaaacaac aaaaaaaatc aggtataatt acatacagtg aaacatatgg 2520
tgactcaagt acacagttca gtgttttttt tgtttttgtt tttttttaag atggactctt 2580
gctctgtcgc ccaggctgga gttcagtggc gcgatcttgg ctcactgcaa cctctgtctt 2640
ccaggttcaa gcaattctgc ctcagcctcc caagtagctg ggactacaag tgcacgccac 2700
catgcctggc taatttttgt atttttaata gagatggggt ttttttttct ttcttttttt 2760
ttttttttga gacagggtct cactctgtca cccaggctgg agtgcagtgt cacaatctcg 2820
gctcactgca acctctgcct cttgggttca agcaattctt ctgcctcagc ctcctgagta 2880
gctggtagct gggactatag gcacgtgcca ccacacccag ttaatttttt gtatttttag 2940
tagagatggg gtttcaccgg gttagccagg atggtctcaa tctcttgacc tcgtgatctg 3000
cccgttttgg tctcccaaag tgatgggatt acaggcgtga gccaccgcgc caggctgaga 3060
tggggtttca ccacgttggc caggctggtc tggaactcct gacctcaggt gatccgcctg 3120
ccttggactc ccaaagtgct gggattacag acgtgagcca ccgtgcccgg ccagttctat 3180
gagttttgat gaatgtgtcc tgcttcttcc caggctccac catctcttcc cctaaaggca 3240
accaccattc tgatttctct caccaaagac tggaaaaata tcccaatacc tggatgtatg 3300
cctcacgtgt cccgctggcc ccagcaccca ggtatagtta gttactgctg tgcctggctt 3360
cactctcact tttttttttt ttttcttttt ttgagacata gtctcgctct gtagcccagg 3420
ctggagtgca gtgtggcaca atctcagctc actgcaacct tcacctccca ggttcaagcg 3480
attctcctgc ctcagcctct cgagtagctg cgactacagg tgcccaccac cacacctggc 3540
taatttttgt atttttagta gagatggggt ttcaccttgt tggccaggct ggtctcgaac 3600
tcctgacctc aagtgatcca cctgcctcag cctcccaagg tgctgggatt gcaggccggc 3660
cttacctggt atggttttta ggttcatgtt gatgcctgga gtttcagtaa ttccctccca 3720
ttcgttgctg agtagcactg caacacatgc aggcagctat ttggggatct tttcacctgt 3780
tgatgggtgt ctggactgtt tccagttttt ggctgttaca aatgaagcct ctgtgagcca 3840
cg 3842
Claims (7)
1.SLC25A25-AS1 as diagnosis of cervical cancer marker answering in preparing diagnosis of cervical cancer reagent and/or kit
With.
2. applying according to claim 1, which is characterized in that the SLC25A25-AS1 sequences are as shown in SEQ ID No.1.
Applications of the 3.SLC25A25-AS1 in the drug for preparing treatment cervical carcinoma.
4. applying according to claim 3, which is characterized in that the SLC25A25-AS1 sequences are as shown in SEQ ID No.1.
Applications of the 5.SLC25A25-AS1 in preparing the cell killing drug of enhancing chemotherapeutic drugs on cervical cancer.
6. applying according to claim 5, which is characterized in that the SLC25A25-AS1 sequences are as shown in SEQ ID No.1.
7. applying according to claim 5, which is characterized in that the chemotherapeutics is cis-platinum.
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