CN109112215A - The target protein FUS of lncRNA H19 and its application - Google Patents

Abstract

The invention discloses application of the target protein FUS of lncRNA H19 in the drug for preparing and/or screening treatment tumour；Also disclose the application of lncRNA H19 and FUS being used in combination in the drug for preparing and/or screening treatment tumour.After the targeted inhibition agent for using FUS in the present invention, colony (colony) Forming ability of K562 cell is reduced, shows the proliferation that can effectively inhibit tumour by the expression of targeted inhibition FUS；Meanwhile after passing through promotion lncRNA H19 expression in the present invention, the growth of tumour can be inhibited, extend the time-to-live of the mouse with tumour, illustrate that the reagent of lncRNA H19 expression can be promoted to have the effect of inhibiting tumour.

Description

The target protein FUS of lncRNA H19 and its application

Technical field

The present invention relates to the target protein FUS of the treatment method of malignant tumour, especially lncRNA H19 and its applications.

Background technique

Chronic myelocytic leukemia (chronic myelocytic leukemia, CML) is a kind of occurs in multipotency hematopoiesis Pernicious bone marrow proliferative tumour on stem cell.CML patient often will appear characteristic chromosome translocation t (9:22) (q34: Q11) (i.e. Ph chromosome), on No. 9 chromosomes 2 transposition of C-ABL proto-oncogene to No. 22 chromosome long arms breaking point gathering Area (BCR) forms BCR-ABL fusion, and unconventionality expression BCR-ABL protein leads to the sustained activation of tyrosine kinases, and Cause the signal transduction pathway in downstream to be activated, to adjust the expression of cell factor, it is a large amount of to eventually lead to marrow stem/progenitor cells Hyperplasia, apoptosis reduce and the decline of the adhesiveness of marrow stromal cell, and a large amount of still immature myelocytes is caused to be discharged into peripheral blood, Lead to CML.According to the overall development process of disease, clinically its development process is divided into three periods, respectively slowly Property phase (Chronic phase, CP), accelerated period (Accelerated phase, AP) and rapid change period (Blastic phase, BP).Chronic phase disease symptoms slightly without specificity, are mainly shown as abdominal distension, abdominal mass, out of strength, low-heat, splenomegaly, see 90% patient, illness degree are different.

With the rapid development of genomic sequencing technique, it has been found that RNA can by 60%~70% genome sequence Transcribe, but wherein only less than 2% coding protein sequence, remaining is referred to as non-coding RNA.In fact, very Many years ago, many non-coding RNAs are just found to have a major impact vital movement, such as are related to the ribosomes of protein synthesis RNA (rRNAs), transfer RNA (tRNA) and small nuclear rna (snRNA), and these non-coding RNAs are commonly known as non-volume of running one's home Code RNA.Non-coding RNA attracts extensive attention in recent years, these RNA are primarily referred to as the non-coding RNA with life adjustment effect, The RNA (LncRNA) studied including microRNA and herein.Research has been found that LncRNA refers to that length is greater than 200 nucleotide And transcribed by RNA polymerase II and lacked or the RNA without open reading frame, it can between gene, enhancer original part, The antisense strand of gene, the other positions for including sub-district genome.

With deepening continuously to long-chain non-coding RNA research, it is found that it is many important LncRNA has the function of, takes part in The important biological process of body, the occurrence and development etc. including cell differentiation, aging, increment, apoptosis and tumour.A variety of LncRNA Such as H19, X chromosome specificity inactive transcription object (Xist) participate in Genomic Imprinting.

Pachnis etc., although can be transcribed by RNA polymerase II, can be sheared simultaneously tailing in discovery h19 genes in 1984, But its product for transcribing out can not translate into the protein of biological function, but be sent out in the form of non-coding RNA The effect of waving.Since H19 product length has been more than 200nt, therefore it is defined as LncRNA.H19 and IGF2 exists in Mice Body In No. seven chromosome, then it is located at 11p15.5 in human body, at a distance of 90kb, this belongs to first pair and is proved to be with the marking the two The gene of characteristic.H19 is presented as the paternal marking and maternal expression, and IGF2 is then presented as paternal expression and the maternal marking.The two Marking characteristic all by the methylation differential modified region (DMR) of H19 promoter upstream 4kb or marking regulatory region (ICR) Regulation.On spore, H19 has highly conserved feature, the high expression within three periods, when being embryonic development respectively It is several in hetero-organization other than heart and skeletal muscle in entoderm, mesoderm and its tissue that differentiates, but after being born It does not express, unless expression can be reactivated under three circumstances: when tissue damage reparation and stress situation and tumour occur.

H19 overall length 2.3kb by 35 small open reading frame, and has 5 ' end cap minor structures and the 3 ' ends of similar mRNA Poly-A tail fawns on structure, theoretically a kind of protein comprising 256 amino acid moleculars of codified, but LncRNA is only It plays a role in mRNA level in-site, and any protein molecule can not be encoded.It compiles in highly conserved region in first promoter of H19 The miRNA-675 molecule of code can control the growth of placenta in latter half of gestation by adjusting the expression of IGFLR gene.The study found that H19 largely assembles in human body placenta element and some embryonic tissues, indicates that H19 may rise very in embry ogenesis and growth and development stage Important role.After fetal birth, H19 expression is lowered, but maintenance basal expression is thrown away in mammary gland, adrenal gland and uterine tissue Amount.It has been reported that and shows in the nephroblastoma, embryonal rhabdomyosarcoma at present, H19 plays tumor suppressor gene, however many In solid tumor (including breast cancer, liver cancer, bladder cancer etc.), H19 is an oncogene.This shows that H19 passes through difference in human body Mechanism participate in body tumour formation and progress.Need further to be ground there are many more unknown link in these different mechanism Study carefully discovery.

Summary of the invention

Based on the above issues, present inventor participates in the formation of body tumour and the specific machine of progress in research H19 During system, it has unexpectedly been found that by promoting the expression of lncRNA H19 or inhibiting the target protein FUS of lncRNA H19, The proliferation of tumour cell can be effectively inhibited.Specifically, technical solution of the present invention includes the following aspects:

In the first aspect, the present invention provides the target protein FUS of lncRNA H19 to prepare and/or screen treatment tumour Drug in application.

Preferably, the tumour is leukaemia.

In the second aspect, the present invention provides being used in combination for lncRNA H19 and FUS to prepare and/or screen treatment Application in the drug of tumour.Wherein, the overexpression of lncRNA H19 can inhibit the proliferation of tumour cell, the low expression of FUS It can inhibit the Colony forming ability (colony) of tumour cell.

Preferably, the tumour is malignant tumour.

In the third aspect, the present invention provides a kind of drug for treating tumour, the inhibition containing FUS in the drug Agent.

Preferably, the inhibitor is the targeting SiRNA of PCBP1.

Preferably, also containing the reagent or carrier that can promote lncRNA H19 expression in the drug.

Preferably, shown in the base sequence of the inhibitor such as PCBP1-SiRNA-03 (i.e. SEQ ID NO.8).

It is highly preferred that the tumour is leukemia.

In conclusion the invention has the benefit that

After the targeted inhibition agent for using FUS in the present invention, the colony Forming ability of K562 cell is reduced, shows to pass through The expression of targeted inhibition FUS can effectively inhibit the proliferation of tumour；Meanwhile by promoting lncRNA H19 expression in the present invention After level, the growth of tumour can be inhibited, extend the time-to-live of the mouse with tumour, explanation can promote lncRNA H19 to express Horizontal reagent has the effect of inhibiting tumour.

Detailed description of the invention

Fig. 1 is slow virus carrier structure chart；

Fig. 2 is the testing result figure of Normal group and the expression of K562 and KCL-22 cell H19；

Fig. 3 is the selection result figure of the Real-time PCR to H19-SiRNA ordered sequence；

Fig. 4 is the testing result figure of the expression of the H19 in K562-H19 LentiV and K562 empty LentiV；

Fig. 5 is the colony number testing result figure of K562-H19 Lenti and K562-empty LentiV group, wherein A For colony figure (40 ×)；B is opposite colony number；

Fig. 6 is influence result figure of the H19 overexpression to BALB/c nude mouse；Wherein,

After A shows that injection is overexpressed the P210 cell of H19, the size of mouse tumor；

B shows that injection is overexpressed influence to mouse survival after the P210 cell of H19；

After C shows that injection is overexpressed the P210 cell of H19, the statistical result of mouse tumor volume；

Fig. 7 is that NOD/SCID mouse tail vein transplants K562-NEG-LentiV and K562-H19 Lenti V, living imaging Mouse invasion rate is observed, the result figure of mouse survival rate is recorded.

Fig. 8 is the qualification result of PCR product；Wherein A is the agarose gel electrophoresis figure after PCR product.B is PCR product (sequence has taken T7 promoter) result is sequenced；

Fig. 9 is the lncRNA H19 agarose gel electrophoresis figure after being transcribed in vitro；

Figure 10 is that K562 cell pyrolysis liquid and RNA are incubated for the result figure for carrying out RNA pull-down, wherein using 10%SDS- Albumen in PAGE isolated complex, coomassie brilliant blue staining；

Figure 11 is the result figure of 2.0 software prediction of catRAPID software and starBase v；Wherein it is shown in the non-volume of long-chain In code RNAH19 pull-down product, there are PCBP1 and FUS albumen, 2.0 software of catRAPID software and starBase v Predicting long-chain non-coding RNA H19 and PCPB1 and FUS albumen has interaction；

Figure 12 is the result that PCBP1 primary antibody and FUS primary antibody verify the expression of two kinds of albumen in RNA pull-down product Figure；

Figure 13 is the selection result figure (* * P < 0.01, * P < 0.05, the FUS-siRNA transfection group of FUS-SiRNA ordered sequence Compared with blank group and SCR control group)；

Figure 14 is influence of the Imatinib of various concentration to relative activity after K562 cell transfecting SCR and FUS-siRNA Result figure (* * P < 0.01, * P < 0.05, FUS-siRNA transfection group are compared with blank group and SCR control group)；

Figure 15 shows the colony result of K562, SCR and FUS-siRNA group；Wherein A is colony figure (40 ×), and B is phase To colony number (* * P < 0.01, FUS-siRNA group is compared with K562, SCR group).

Specific embodiment

To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair The present invention is described further.The embodiment of the present invention mainly includes three parts, reagent, consumptive material, the Yi Jiqi being directed to Its test article can be obtained from market or other public's channels.

First part: the Function Identification of lncRNA H19

1.H19-SiRNA sequence and H19 Lentiviral

1.1 H19 SiRNA sequences

1.2 H19 Lentivirals

H19 Lentiviral is implemented in Guangzhou FulenGen Co., Ltd..

H19 Lentiviral: CS-GS3160-LV201；

Empty Lentiviral: CS-NEG-LV-201

2. cell strain

K562 cell strain, human chronic myelogenous leukemia system, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences

3. main agents are prepared

3.1 contain the cell culture medium of 10% serum

Australia fetal calf serum is placed in high-pressure sterilizing pot 56 DEG C, is sub-packed in the healthy and free from worry centrifuge tube of 10mL after 30min fire extinguishing, Serum after packing saves in -20 DEG C of refrigerators.RPMI-1640 culture medium is sub-packed in 200mL wide-mouth bottle, and every bottle of 180mL is put in It is saved in 4 DEG C of refrigerators.Fetal calf serum 20mL in Australia is added in every 180mL culture medium, prepares so that blood in RPMI-1640 culture medium Clear content is 10%.

3.2 pH7.2 phosphate buffers (PBS)

Said components are weighed respectively, dissolve the above-mentioned component weighed up using distilled water and are settled to 1000mL, and high pressure is steamed Vapour sterilizing 30min, takes out, 4 DEG C of preservations.

3.3 prepare 2.7% methylcellulose semisolid culturemedium

2.7g methylcellulose powder is weighed in wide-mouth bottle, 50mL boiling water is added, dissolves while stirring, 30min takes Out, to its cooling, sterile 2 × 1640 culture medium of 50mL is added in superclean bench, stirring is stored in 4 DEG C until dissolution Refrigerator is stand-by.

1 K562 cell culture of embodiment

CML cell K562 is inoculated in RPMI-1640 culture medium, the content of serum is 10% in RPMI-1640, will be inoculated with It is 37 DEG C, CO that good K562 cell, which is placed in temperature,₂It is cultivated in the cell incubator that volume fraction is 5%.

The screening of 2 H19-SiRNA ordered sequence of embodiment

2.2.1 H19-SiRNA transfection K 562 cell

(1) experimental group are as follows: blank control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19- SiRNA-03 group, the concentration of SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03 are 100nM.

(2) logarithmic growth phase cell, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension It is adjusted to 30 × 10⁴/ mL, the cell suspension inoculation of every hole 1.5ml is in 6 orifice plates.

(3) Lipofectamine is prepared^TMThe compound of 2000 and RNA: according to Lipofectamine^TM2000 specifications, The siRNA amount and Lipofectamine that each takes what he needs^TM2000 amounts are diluted with Optimal Medium Opti-MEM respectively.

(4)Lipofectamine^TMAfter the completion of 2000 dilutions, it is stored at room temperature 5min.

(5) dilute and stand the Lipofectamine of 5min^TM2000 with diluted SiRNA and mixed according to the ratio of 1:1, It is stored at room temperature after 20min to get Lipofectamine is arrived^TM2000-RNA compound.

(6) by Lipofectamine needed for each group^TM2000-RNA compound is slowly added drop-wise to the cell suspension in orifice plate In, so that the final volume in every hole is 2mL.6 orifice plates are placed in 37 DEG C, CO₂In the cell incubator that volume fraction is 5%.

2.2.2 Real-time PCR detects the relative expression levels of H19mRNA in group of cells

2.2.2.1 Total RNAs extraction

Experimental group: blank control group (BLANK), SCR, H19-SiRNA-01, H19-SiRNA-02, H19-SiRNA-03

(1) group of cells of logarithmic growth phase, 1500rpm are centrifuged 5min, abandon supernatant.

(2) PBS washes cell precipitation one time, and 1500rpm is centrifuged 5min, abandons supernatant.

(3) trizol of 1mL is added into cell precipitation, cracks 5min on ice.

(4) chloroform of 200 μ L is added into cell pyrolysis liquid, vibrates 45s, stands 10min on ice.

(5) 12000rpm, is centrifuged 15min by 4 DEG C.Careful supernatant of drawing is in a clean EP pipe, minus 20 DEG C of placements 20min。

(6) 12000rpm, is centrifuged 10min by 4 DEG C.Carefully discard supernatant.

(7) 70% alcohol washes RNA precipitate, and 12000rpm, is centrifuged 5min, in triplicate by 4 DEG C.

(8) precipitating is dried in draught cupboard, 2-3min.The RNA of 30 μ L is added in precipitating without enzyme water.RNA is placed in minus 80 DEG C save.

2.2.2.2 reverse transcription reaction (RT)

(1) PCR pipe for taking RNA sample 1 μ g of the A260/A280 between 1.8-2.0 and nuclease free to pollute, each group RNA It is placed in spare on ice.

(2) according to the specification of the All-in-One cDNA Synthesis SuperMix of bimake company, in nucleic acid Reverse transcription reaction (operating on ice) is carried out without following components in enzyme pipe, is added:

(3) after adding well according to said components, brief centrifugation 10s.

(4) PCR pipe is put into PCR instrument, the standard reaction program progress reverse transcription reaction provided according to kit: 42 DEG C 15min；85℃2min.

2.2.2.3 Real-time PCR

(1) each group is detected according to 2 × SYBR Green qPCR Master Mix kit of bimake company The relative expression levels of H19mRNA, GAPDH are internal reference, and 1 μ L cDNA is taken to carry out Real-time PCR amplification.

(2) reaction system is as follows:

The relative expression levels of H19 and GAPDH are with 2^-△△CTIt calculates.

Embodiment 3 establishes the K562 cell strain for stablizing expression H19

3.3.1 H19 Lentiviral and empty Lentiviral transfection K 562 cell

(1) logarithmic growth phase cell, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension It is adjusted to 30 × 10⁴/ mL, every hole 1.75mL cell suspension inoculation is in 6 orifice plates.

(2) Lipofectamine is prepared^TM3000 in the compound of DNA: according to Lipofectamine^TM3000 specifications It prepares, every hole takes the amount of DNA of 2.5 μ g and the Lipofectamine of 3.75 μ L^TM3000 amounts, respectively with the Opti-MEM of 250 μ L Dilution.

(3) DNA and Lipofectamine^TM3000 dilutions are completed, and dilution are uniformly mixed according to the ratio of 1:1, room temperature After standing 5min, Lipofectamine is obtained^TM3000-DNA compound.

(4) by Lipofectamine^TM3000-DNA compound is slowly dropped in the cell suspension in orifice plate, and every hole is most Final volume is 2mL.It is 37 DEG C, CO that six orifice plates, which are placed in temperature,₂It cultivates in the cell incubator that volume fraction is 5%, after 6h, adds Enter the RPMI-1640 culture medium that 2mL is 20% containing serum.

3.3.2 the K562 cell strain of expression H19 is stablized in puromycin screening

After H19 slow virus carrier transfection K 562 cell, puromycin is added in cultivating system, keeps the end of puromycin dense Degree was 1mg/mL, every 2 days replacement culture solutions.

4 Real-time PCR of embodiment detects the relative expression levels of intracellular H19 mRNA

4.4.1 Total RNAs extraction

According to 2.2.2.1.

4.4.2 reverse transcription reaction (RT)

According to 2.2.2.2.

4.4.3 Real-time PCR

According to 2.2.2.3.

The influence that 5 H19 of embodiment forms K562 cell colony

5.5.1 experimental group

1.K562 blank control group

2.K562-H19 LentiV group (the K562 cell for stablizing expression H19)

3.K562-empty LentiV group (containing the K562 cell for being free slow virus carrier)

5.5.2 COLONY is tested

Experiment is divided into two groups: stablizing the K562 cell of expression H19 and has transfected the K562 cell of empty Lentiviral. The cell of each experimental group of logarithmic growth phase dilutes K562 cell with serum free medium, so that its density is 1 × 10³/ ML takes the K562 cell suspension of 450 μ L, is added to 550 μ L containing 20% fetal calf serum, 5 μm of ol/L beta -mercaptoethanols, 2mmol/L L-Glutamine and 0.9% methylcellulose semisolid culturemedium in, cell suspension and semisolid culturemedium is sufficiently mixed After even, every hole 1mL is inoculated in 24 orifice plates, and each experiment repeats three multiple holes, and it is 37 DEG C, CO that 24 orifice plates, which are placed in temperature,₂Body It is cultivated 7-14 days in the cell incubator that fraction is 5%.It is placed under inverted microscope and observes COLONY size and form, with The cell mass of approximately greater than 40 cells is 1 COLONY, and preservation of taking pictures, experiment is in triplicate.

Functional study of 6 H19 of embodiment in CML mouse model

6.6.1 the tumor formation of Balb/c nude mice by subcutaneous is tested

H19 slow virus carrier and empty slow virus carrier are transfected in P210 cell, and it is thin to construct the stable P210 for being overexpressed H19 Density is 1 × 10 by born of the same parents⁷200 μ L to the BALB/c nude mouse left lower extremity of P210-H19 and P210-NEG cell inoculation of/mL Its tumor formation situation is observed in oxter, after mouse tumor formation, the daily variation for measuring mouse tumor size.

6.6.2 NOD/SCID mouse tail vein is tested

H19 slow virus carrier and empty slow virus carrier are transfected in K562 cell, and it is thin to construct the stable K562 for being overexpressed H19 Density is 1 × 10 by born of the same parents⁷The 200 μ L of K562-H19 and K562-NEG cell transplantation of/mL, tail vein injection K562-H19 cell With K562-NEG cell, living imaging observes and records the morbidity and survival condition of mouse.

Experimental result:

Influence of the 1.H19 to K562 cell progression

The detection of 1.1 H19 expression quantity

The blood of normal person is taken, RNA is extracted, while to K562 cell, KCL-22 cell, 293T cell extraction RNA, fluorescence is fixed The expression quantity for measuring PCR detection each group H19, as a result as shown in Fig. 2, compared with Normal group, the table of H19 in K562, KCL-22 It is lower up to measuring.

The screening of 1.2 H19-SiRNA ordered sequences

Concentration is SCR (serum creatinine), the H19-siRNA transfection K 562 cell of 100nm, after normal culture 48h, extracts each group The total serum IgE of cell, Real-time PCR detect the relative expression levels of each group H19mRNA, MTT detection transfection H19-SiRNA Afterwards, the cell viability of each group K562 cell.As a result as shown in Figure 3.

The Efficiency testing of 1.3 H19 slow virus carrier transfection K 562 cells

H19 slow virus carrier (CS-GS3160-LV201) used and empty slow virus carrier (CS-NEG-LV-201) be by Provided by Guangzhou FulenGen Co., Ltd..H19 slow virus carrier screens after transfection K 562 cell through puromycin, obtains To the K562 cell for stablizing expression H19.The each group K562 cell of logarithmic growth phase is collected, the phase of total serum IgE detection H19mRNA is extracted To expression.The relative expression levels of the intracellular H19 of each group K562 are as shown in figure 4, relative to empty slow virus carrier (Empty LentiV), the expression of the H19mRNA of H19 slow virus carrier group (H19LentiV) is significantly raised.

1.4 H19 are overexpressed the influence formed to K562 cell colony

The competence for added value that method can be used to detect single tumor cell is formed by the colony of carrier of methylcellulose.It takes K562, K562-empty LentiV and K562-H19LentiV of logarithmic growth phase carry out colony experiment, as a result such as Fig. 5 institute Show, relative to K562 group and K562-empty LentiV group, the colony number and size of K562-H19LentiV group reduce (P <0.05)。

2. being overexpressed influence of the H19 to CML model mouse Balb/c nude mice

Influence of the H19 overexpression to BALB/c nude mouse tumor size is probed into nude mice by subcutaneous tumor formation, to BALB/c The influence of nude mouse survival, in order to probe into influence of the H19 to BALB/c nude mouse P210 tumor size, in P210 cell Middle transfection H19 slow virus carrier and empty slow virus carrier, construct the P210 cell stablized and be overexpressed H19, are 1 × 10 by density⁷/ 200 μ L to the BALB/c nude mouse left lower extremity oxter of P210-H19 and P210-NEG cell inoculation of mL, observes its tumor formation feelings Condition, after mouse tumor formation, the daily variation for measuring mouse tumor size.As can be seen from Figure 6 the tumour ratio of H19 overexpression group Control group it is small.

3. being overexpressed influence of the H19 to CML model mice NOD/SCID

Mouse tail vein injection K562-H19 cell and K562-empty cell, living imaging observe and record the morbidity of mouse And survival condition.As a result as shown in Figure 7.

Interpretation of result:

The present invention first detects the expression quantity of H19, takes the blood and K562 cell and KCL-22 cell of normal person, RNA is extracted, H19 expression quantity is carried out and is detected.Such as Fig. 2, it can be seen that compared with Normal group, in K562 and KCL-22 The expression quantity of H19 is relatively low.Secondly, having carried out the screening of ordered sequence, H19-siRNA and SCR synthesis to the SiRNA of H19 In the sharp rich biological Co., Ltd in Guangzhou.It is screened through Real-time PCR, H19-SiRNA-02 and H19-SiRNA-03 two sequences Effectively, unobvious to the effect of vigor of K562 cell such as Fig. 3, but after targeted inhibition H19.Therefore, inventor is sick slowly using H19 Poisonous carrier transfection K 562 cell, puromycin screens positive cell, so that H19 overexpression, stablizes strain and be named as K562-H19 LentiV, research H19 are overexpressed the effect being in progress to K562 malignant, and such as Fig. 4, H19 overexpression inhibit K562 cell Clone formation.Lentiviral CS-GS3160-LV201 and CS-NEG-LV-201 building and technical support are by Guangzhou reactivation Genome company provides.CS-GS3160-LV201 contains H19 and eGFP target gene fragment, and CS-NEG-LV-201 is contained only EGFP can tentatively judge transfection efficiency, according on carrier so observing the positive cell of GFP albumen after transfection is completed The resistant maker gene having carries out preliminary screening using puromycin.Strain is stablized to the K562 after transfection slow virus and extracts RNA, The relative expression levels of Real-time PCR detection H19.Fig. 5 shows compared with K562-empty LentiV groups of cells, The expression of K562-H19 LentiV intracellular H19 mRNA is significantly raised, the above result shows that, stablize expression H19's K562 cell is successfully established, this provides experimental model for function of the research H19 in CML.

Clonal hyperplasia is one important feature of CML K562 cell strain, therefore, to K562 cell clonal formation energy Power, which carries out research, can prove influence of the H19 to CML cell progression.Colony forms experiment and refers to individual cells especially tumour Upgrowth situation of the cell under methylcellulose semisolid culturemedium condition of culture, by inverted microscope observe cell number with And colony form, it can be used to evaluate the proliferative capacity of individual cells.Experiment is formed by colony to learn, is overexpressed H19 The colony size and number of K562 cell are reduced afterwards, and the proliferative capacity of the expression and K562 cell of H19 is closely related.

Chronic myelocytic leukemia has characteristic BCR/ABL fusion and its product BCR/ABL fusion protein (P210), the albumen have strong tyrosine kinase activity, can abnormal activation many A signal pathways, cause haemocyte pernicious Conversion.BaF-P210 cell, abbreviation P210 are the cell strains for stablizing expression BCR/ABL.Therefore, this part is in animal water The flat influence for having visited influence and survival that H19 is overexpressed to BALB/c nude mouse tumor formation.Inventor stablized constructing Density is 1 × 10 by the P210 cell for expressing H19⁷200 μ L to the BALB/c of P210-H19 and P210-NEG cell inoculation of/mL From the mouse left lower extremity oxter nude is injected, the tumor formation situation and survival condition of mouse are observed.Fig. 6 can be seen that with it is right It is compared according to group, the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse.Meanwhile inventor is constructing Stablize the K562 cell for being overexpressed H19, is 1 × 10 by density⁷K562-H19 the and K562-NEG cell of/mL is infused by tail vein It penetrates, by 200 μ L to NOD/SCID Mice Body of cell transplantation, living imaging observes the incidence and survival condition of mouse, From figure 7 it can be seen that compared with the control group, the overexpression of H19 inhibits the growth of tumour and extends the survival of mouse.

The functional study of the target protein FUS of second part lncRNA H19

1. with T7 promoter sequence h19 gene primer sequence it is as shown in the table, primer by give birth to work bioengineering (on Sea) limited liability company designs and synthesizes.

2. cell strain

K562 cell strain, human chronic myelogenous leukemia system, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.

3. main agents are prepared

3.1 prepare 10 × TAE electrophoretic buffer

Said components are weighed in beaker, 800mL distilled water, stirring, so that it is sufficiently dissolved are added into beaker；It is added 11.4mL glacial acetic acid, is sufficiently stirred；1L is settled to distilled water, room temperature preservation, for use.

3.2 prepare 0.8% agarose gel

It weighs 0.4g agarose and is dissolved in 50mL distilled water, stir evenly, and microwave stove heating, until boiling, takes out, to Temperature drops to 60 DEG C or so, and the GeneRed of 3 μ L is added, is sufficiently stirred, pours into stick mixing tank, is inserted into comb, standby to its solidification With.

3.3 LB culture mediums are prepared

Above-mentioned each component is weighed in beaker, distilled water is added to stir and is settled to 1L, is placed in high-pressure steam sterilizing pan sterilizing, It is spare.

3.4 LB solid mediums are prepared

Other than ingredient each in aforesaid liquid culture medium, then plus 15g agar, equally plus distilled water constant volume arrive 1L, sterilizing greatly It after about 30min, takes out, drops to 60 DEG C or so to temperature, be poured into the culture dish for having added antibiotic, shake up, to solidifying Gu after, 4 DEG C of preservations are spare.

3.5 prepare the sodium chloride of 5M

292.5g sodium chloride is weighed, distilled water is added to stir, until dissolution, and is settled to 1L.It is spare.

3.6 10 × TBS buffer (pH=7.6)

Said components are weighed, appropriate distilled water dissolution, being adjusted to pH with HCL is 7.6, and distilled water is added to be settled to 1L, room temperature It saves.10 × TBS is diluted to 1 × TBS when use.

3.7 prepare 1 × TBST buffer

100mL1 × TBS solution is measured, 1mLTween-20 is added, appropriate distilled water is added and is settled to 1L, as 1 × TBST buffer.

3.8 prepare 1 × transferring film buffer (pH=8.5)

Said components are weighed, appropriate distilled water dissolution is added, is 8.5 with HCL adjustment pH, is settled to 1L, room with distilled water Temperature saves.

3.9 prepare confining liquid

5g skimmed milk power is weighed, is dissolved in 100mLTBST, matching while using.

3.10 preparing antibody diluent

5g skim milk is dissolved in 50mLTBST, 5% skim milk is made into, first with now matching.

3.11 5 × SDS-PAGE electrophoretic buffer

Add appropriate distilled water, stirring and dissolving adds distilled water to be settled to 1L.Room temperature preservation.Distilled water is added to be diluted to 1 when use ×SDS-PAGE。

3.12 1.5mol/L pH=8.8Tris-HCL buffers

Add appropriate distilled water to dissolve, and is settled to 100mL.4 DEG C of preservations.

3.13 30% acrylamide

Weigh said components, distilled water dissolution and constant volume value 100mL.4 DEG C of preservations.

3.14 10% separation gel

3.15 5% concentration glue

Embodiment 7 obtains the template being transcribed in vitro

1) preparation of competent E.coli

(1) the one minus 70 DEG C conservation bacterium solution bacillus coli DH 5 alphas frozen are taken, 5 μ L bacterium solutions and 5mL LB Liquid Culture are drawn In base, 37 DEG C, the activation of 250rpm shaking table is overnight.

(2) 50 μ L are taken from the bacterium solution of step (1), are inoculated into the fresh LB liquid medium of 5mL, 37 DEG C, 250rpm Shaking table culture 3h takes survey OD value in the EP pipe of appropriate bacterium solution and 1.5mL (0.3-0.4OD value can be tested).

(3) take the bacterium solution of 1mL in the sterile EP tube of 1.5mL in super-clean bench, 5000rpm is centrifuged 5min, removes supernatant.It is added The CaCl of the 0.1mol/l of 1mL pre-cooling₂Resuspended bacterium solution places 30min on ice.

(4) 5000rpm, is centrifuged 5min, removes supernatant by 4 DEG C.

(5) CaCl of the 0.1mol/l of 50 μ L pre-cooling is added₂Resuspended bacterium solution, as competent cell (are placed in minus 80 DEG C of ice Case saves).

2) H19 Lentiviral converts Escherichia coli

(1) competent cell is placed on ice to melt, after it melts, it is sick slowly that H19 is added into competent cell suspension Malicious over-express vector DNA1 μ L, mixing, ice bath 30min are gently blown and beaten with pipettor.

Centrifuge tube, is transferred in ice bath rapidly, stands 3min on ice by (2) 42 DEG C of thermal shock 45s.

(3) the sterile not antibiotic LB culture medium of 450 μ L is added into centrifuge tube, mixes, is placed in 37 DEG C of shaking tables, 150rpm shaken cultivation 45min, so that thallus is recovered.

(4) 100 μ L bacterium solutions are taken, are coated on the LB plate containing ampicillin, will uniformly be applied using sterile spreading rod It opens, plate is placed in 37 DEG C and is absorbed to liquid, be inverted culture, 37 DEG C of overnight incubations.

(5) prepare the centrifuge tube of a 10mL the next morning, add 5mLLB culture solution, then plus the concentration of 2.5 μ L be ammonia benzyl Penicillin, picking single bacterium colony are inoculated in the LB culture medium for having added ampicillin, 37 DEG C of shaken cultivations, 10 left sides h It is right.

3) extraction of Plasmid DNA

Extracting the reagent used see the table below:

(1) bacterium solution for taking 5mL to be incubated overnight is added in centrifuge tube, and 13000rpm is centrifuged 1min and collects bacterial sediment, carefully Suck supernatant.

(2) 250 μ L Buffer P1 are added into the centrifuge tube containing precipitating, are mixed well using pipettor, it is heavy to suspend It forms sediment.

(3) 250 μ L Buffer P2 are added into centrifuge tube, mixing 10 times of mildly turning upside down cracks thallus sufficiently, It is placed at room temperature for 5min.Solution should become limpid sticky at this time.

(4) 250 μ L Buffer E3 are added into centrifuge tube, white wadding occurs at this time in mixing 10 times of turning upside down immediately Shape precipitating is placed at room temperature for 5min, and 13000rpm is centrifuged 5min, draws supernatant, and filtering note (Endo-Remover is added in supernatant FM in), 13000rpm is centrifuged 1min filtering, and filtrate is collected in centrifuge tube.

(5) 225 μ L isopropanols, mixing of turning upside down are added into filtrate.

(6) 200 μ L Buffer column equilibration: are added into the adsorption column (Spin Col μm ns DM) for having been charged into collecting pipe PS, 13000rpm are centrifuged 1min, outwell the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

(7) mixed liquor of filtrate and isopropanol in step (5) is transferred to the adsorption column balanced and (has been charged into collection Pipe) in.

(8) 13000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.(absorption The maximum volume of column is 750 μ L, if sample volume is greater than 750 μ L and can be added portionwise.)

(9) Buffer PW, the 13000rpm centrifugation that 750 μ L have been added to dehydrated alcohol in advance is added into adsorption column Min outwells the waste liquid in collecting pipe.

(10) adsorption column is placed back in collecting pipe, 13000rpm is centrifuged 1min.(note: the purpose of this step be by Remaining ethyl alcohol removes in adsorption column, and ethyl alcohol residual will affect subsequent enzymatic reaction.)

(11) adsorption column is placed in a new collecting pipe, 50-100 μ L Edno- is added to the intermediate position of adsorbed film Free Buffer EB, is placed at room temperature for 5min, and 13000rpm is centrifuged 2min, plasmid solution is collected into centrifuge tube.Minus 20 DEG C Save plasmid.

4) Plasmid DNA is quantitative

Plasmid DNA is quantitative: being quantified using nucleic acid quantification instrument, is quantitatively averaged three times.

5) PCR amplification

According to the Thermo Scientific Phusion High-Fidelity of thermo scientific company DNA Polymerase carries out PCR amplification

(1) EP of the EXYGEN of one 200 μ L is taken to manage.

(2) amplification system see the table below:

(3) after mixing well, upper machine carries out PCR amplification.

(4) it is expanded according to following procedure

6) agarose gel electrophoresis carries out the identification of pcr amplification product

(1) take the PCR product of 10 μ L in the EP pipe of a 200 new μ L.

(2) 5 × Loading buffer of 2 μ L is added into EP pipe, is mixed with micro sample adding appliance.

(3) the fresh electrophoretic buffer of 1 × TAE is added into electrophoresis tank, comb is pulled out, so that electrophoresis liquid there was not gel.

(4) successively loading: the mixed liquor of DNA Ladder of 5 μ L, PCR product and Loading buffer.Purpose PCR is produced 14 multiple holes on object, so that the enough subsequent experimentals of amount of DNA of recycling.

(5) condition is set, and the electricity is at 110 volts of pressure for adjusting, time 30min.Carry out agarose gel electrophoresis.

(6) electrophoresis terminate under ultraviolet lamp observe pillar location it is whether correct, preservation of taking pictures.

7) purification and recovery gel DNA

The reagent and other compositions that recycling DNA is used see the table below:

(1) single goal DNA band is cut into (excision redundance as far as possible) from Ago-Gel and is put into clean centrifugation Guan Zhong weighs weight.

(2) the Buffer NJ of 3 times of volumes is added: if gel weight is 0.1g, then its volume is 0.1mL in 55-65 DEG C of water Melt completely in bath to gel.(note: after gel is completely dissolved, observation mixture color is then wanted if it is pink It is 3M, the sodium acetate that pH is 5.2, to turn down its pH value that 5 μ L concentration, which are added,.Through overregulating, the color of mixture should be pale yellow Color.)

(3) the resulting solution of previous step is added on GBC adsorption column to (note: absorption column volume is 800 μ L, if sample Volume is greater than 800 μ L.It is added can be centrifuged in batches after).After placing 2min at room temperature, 1000 × g is centrifuged 60s.It abandons in collecting pipe and filters Pillar is recovered 2mL collecting pipe by liquid.

(4) filtrate in collecting pipe is abandoned, pillar is recovered into 2mL collecting pipe.600 μ LDNA Wash Buffer are added to pillar In.10000 × g is centrifuged 30-60s at room temperature.

(5) it repeats to wash pillar with DNA Wash Buffer.10000 × g is centrifuged 30-60s at room temperature.

(6) filtrate in collecting pipe is abandoned, pillar recovers 2mL collecting pipe.At room temperature, 13000 × g is centrifuged 2min to dry pillar Matrix residual liquid.

(7) pillar on the centrifuge tube of a clean 1.5mL, 15-25 μ L is added and (it is dense to be specifically dependent upon final product Degree) on Elution Buffer to base for post matter, it is placed at room temperature for 1min, 12000 × g is centrifuged 1min with eluted dna.

Embodiment 8 obtains the lncRNA H19 of desthiobiotin label

1) PCR product of purification and recovery is transcribed in vitro as template

(1) thaw each reagent: RNA Polymerase Enzyme Mix is placed on ice；

(2) by 10 × Reaction Buffer and 4 kinds of Ribonucleotide solutions (ATP, CTP, GTP and UTP) vortex gently vibrates, until dissolution.After dissolution, 10 × Reaction Buffer is placed in room temperature, by four kinds Ribonucleotide solutions is placed on ice.

(3) responsive transcription is carried out in room temperature.

1. taking the EP pipe without RNA enzyme of a 200 μ L

2 μ LATP solution, 2 μ L CTP solution, 2 μ LGTP are sequentially added using micro sample adding appliance Solution, 2 μ LUTP solution, 2 μ L 10 × Reaction Buffer, 1 μ g of template DNA, add the Enzyme of 2 μ L Mix, finally using nucleic acid without 20 μ L of enzyme water polishing.

2. being mixed gently up and down using micro sample adding appliance.

3. 37 DEG C of overnight incubations.

It completes, takes out 4. being incubated for, the Dnase of 1 μ L is added inside reaction system, mix, 37 DEG C of incubation 15min.Dnase's Purpose is the template DNA inside removal system.

5. terminating reaction, RNA is precipitated.In the reaction system plus the chlorine of the Nuclease-free Water of 30 μ L and 30 μ L Change lithium precipitating reagent.

6. thoroughly mixing, -20 DEG C of precipitating 30min and 30min or more.

7. 4 DEG C, maximum (top) speed is centrifuged 15min to precipitate RNA.

8. carefully removal supernatant.It is primary that precipitating is washed with the ethyl alcohol of 1mL70%.Purpose is farthest to remove impurity.4 DEG C, maximum (top) speed is centrifuged 15min.

9. the carefully ethyl alcohol of removal 70% is resuspended RNA without enzyme water with nucleic acid, quantifies to RNA, minus 70 DEG C of preservations.

2) RNA completed is transcribed in vitro and carries out desthiobiotin label

(1) DMSO is placed in and is thawed on ice；By 30% PEG (polyethylene glycol) in 37 DEG C of heating until it becomes flowable Liquid.Remaining reagent thaws on ice.

(2) prepare heater, be adjusted to 85 DEG C.

(3) it takes the EP of a 200 μ L to manage, the unmarked RNA (50pmol) of 5 μ L is added, adds the DMSO of 1.25 μ L. The effect of DMSO is:

(4) add reaction system according to following table:

(5) 16 DEG C of overnight incubations, reaction efficiency can be increased by being incubated overnight.

(6) reaction terminates, and in the reaction system plus the nucleic acid of 70 μ L is without enzyme water.It mixes well.

(7) chloroform:alcohol (24:1) of 100 μ L is added (to remove RNA therein in the reaction system Ligase), mix gently, maximum (top) speed is centrifuged 3min.It is careful to draw supernatant, it is transferred to the EP pipe of a new nuclease free In.

(8) concentration that 10 μ L are separately added into the supernatant into the 7th step is the glycogen and 300 μ of the Nacl of 5M, 1 μ L The cold ethyl alcohol of the 100% of L.Minus 20 DEG C of placements at least 1hour.

(9) 4 DEG C, 13000 × g is centrifuged 15min, carefully removes supernatant, is careful not to abandon precipitating.

(10) the cold ethyl alcohol pipette tips that 300 μ L 70% are added mix gently up and down, wash precipitating.4 DEG C, 13000 × g centrifugation 15min carefully removes supernatant, and precipitating is dried (no more than 5min).

(11) precipitating, minus 80 DEG C of preservations is resuspended with (nucleic acid is without enzyme water) the nuclease-free water of 20 μ L.

The RNA that the label of embodiment 9 is completed carries out RNA-Protien Pull Down experiment

1) prepare cell pyrolysis liquid:

(1) cell of logarithmic growth phase collects cell suspension in the centrifuge tube of 15mL, and 1500rmp, centrifugation 5min are collected Cell abandons supernatant.

(2) it is primary to wash cell by PBS, and piping and druming mixes, 1500rmp, and centrifugation 5min collects cell.

(3) according to cell precipitation amount, suitable cell pyrolysis liquid is added, mixes, cracks 30min on ice, cell is complete Cracking.

(4) 4 DEG C, 12000rmp, it is centrifuged 30min.

(5) supernatant is drawn to the EP pipe of a new and clean 1.5mL, is tried according to Coomassie brilliant blue determination of protein concentration The specification of agent box measures protein concentration.Protein concentration need to be greater than 20mg/mL in cell pyrolysis liquid.

(6) ready cell pyrolysis liquid is placed on ice, for use.

2) RNA marked is integrated on Streptavidin MagneSphere

In conjunction with RNA amount be 50pmol, steps are as follows:

(1) it takes the EP of a 1.5mL to manage, the Streptavidin MagneSphere of 50 μ L is added.

(2) EP pipe is placed in magnetic frame, collects magnetic bead in one end of EP pipe, removes supernatant.

(3) Tris (pH7.5) that the 20mM of isometric (50 μ L) is added is washed, and magnetic bead is resuspended using pipette tips.

2. and 3. (4) step is repeated.

(5) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, removes supernatant.

(6) 1 × RNA Capture Buffer of isometric (50 μ L) is added, magnetic bead is resuspended using pipette tips.

(7) the labeled RNA of 50pmol is added in magnetic bead, mixes gently.

(8) room temperature rotation mixes, and is incubated for 30min.

3) RNA-Binding Protein combines labeled lncRNA

(1) it will be incubated for the EP pipe for completing magnetic bead in above-mentioned (8) to remove, places it in magnetic frame, collects magnetic bead in EP pipe One end.Remove supernatant.

(2) Tris (pH7.5) that the 20mM of isometric (50 μ L) is added is washed, and is gently blown and beaten using pipette tips, is resuspended Magnetic bead.

(3) step (1) and (2) is repeated.

(4) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, removes supernatant.

(5) 10 × Protein-RNA Binding Buffer is diluted to 1 × (for each reaction, takes 1o μ L's The RNA of 90 μ L is added without enzyme water, mixing in 10 × Protein-RNA Binding Buffer), for use.

(6) 1 × Protein-RNA Binding Buffer of 100 good μ L of above-mentioned dilution is added in magnetic bead, sufficiently It mixes.

(7) prepare RNA-Protein Binding Reaction system, wherein each component is as follows:

(8) the EP pipe in (6) is placed in magnetic frame, collects magnetic bead in one end of EP pipe, removes supernatant.

(9) plus 100 μ L 7. in the Master Mix for preparing into magnetic bead, mix gently.

(10) 4 DEG C of rotations are incubated for 60min.

4) eluted rna-Binding Protein compound

(1) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, removes supernatant.

(2) compound is carried out with 1 × wash buffer of isometric (100 μ L) to wash.

(3) step (1) (2) are repeated twice.

(4) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, removes supernatant.

(5) plus the Elution Buffer of 50 μ L is in magnetic bead, mixes, 37 DEG C of incubation 30min.

(6) EP pipe is placed in magnetic frame, collects magnetic bead in the other end of EP pipe, collects supernatant.For subsequent analysis.

(7) plus 1 × sample-loading buffer is in compound.10min, minus 20 DEG C of preservations are boiled in boiling water.

The detection of 10 RNA-Binding Protein compound of embodiment

1) Western Blot electrophoresis detection is carried out to the RNA-Binding Protein compound that elution is completed and examined Mas bright blue dyeing detection

(1) electrophoresis: SDS-PAGE glue is prepared, 50 μ L compound loadings, 80V electrophoresis 30min, 120V electrophoresis 1h are taken.

(2) dye: electrophoresis terminates, and gel is taken out, and is put into the plate of 10cm, 100mL distilled water is added, on shaking table 5min is shaken, liquid is abandoned, is repeated two more times, is washed altogether three times.(purpose of this step is to wash away SDS in gel etc. Interfering substance)

(3) distilled water is abandoned, the Coomassie brilliant blue rapid dye liquor of proper volume is added, dyeing liquor is enabled to cover gel, It is dyed on horizontal shaker.

(4) according to the size of molecular weight of albumen, big band 30min of molecular weight of albumen or so occurs, most of band meeting Occur in 2h or so.

(5) dyeing is completed, and decolourizes: appropriate distilled water is added, decolourizes on shaking table, repeatedly adds distillation water washing, Compare clearly band until seeing, photographs to record.

(6) 4 DEG C of gel preservations.

2) gel after electrophoresis is analyzed by mass spectrometry identification

The gel that above-mentioned decoloration is terminated and saved carries out Mass Spectrometric Identification, and Mass Spectrometric Identification is completed by Guangzhou brightness fine horse biology.

11 Western Blot verifying purpose albumen of embodiment

1) acquisition of RNA-Protein biding compound

(1) be transcribed in vitro according to the method for front and RNA pulldown is tested, obtain RNA-Protein Biding compound.

(2) sample-loading buffer is proportionally added, boils 10min in boiling water bath.

2) Western Blot verifying purpose albumen

(1) electrophoresis.SDS-PAGE glue is prepared, wherein resolving gel concentration is 10%, and concentration gum concentration is 5%.After loading, 80V electrophoresis 40min or so, until albumen enters separation gel, there is clear band in MARKER, then 120V electrophoresis 1h makes MARKER It sufficiently runs away, stops electrophoresis.

(2) electrophoresis terminates, transferring film.Firstly, two an equal amount of dedicated filter paper of transferring film are put into transferring film liquid sufficiently It impregnates, according to the molecular weight of the position of MARKER and destination protein, determines the separation gel comprising destination protein, and the glue that will be cut out It is put in transferring film liquid and sufficiently impregnates, the pvdf membrane sheared is put activates 30s in methyl alcohol, pvdf membrane is taken out, and be put in transferring film liquid Middle immersion.Place pvdf membrane and glue in the following order in wet rotary device: positive (blank), foam-rubber cushion, filter paper, film (smooth surface Upward), glue, filter paper, foam-rubber cushion, cathode (blackboard), are put into transferring film slot, gently drive the gas between glue and film away with glass bar Bubble, finally closes device, 100V, 1h.

(3) it closes.After transferring film, film is placed in TBST immediately, decolorization swinging table washing, in triplicate, each 5min, After, it is put into the confining liquid of 5% skim milk, room temperature closes 2h.

(4) primary antibody is incubated for.Closing terminates, and film is placed in TBST, and decolorization swinging table washs three times, each 10min.According to anti- Body specification dilutes antibody to suitable concentration.Film is put into the incubation box of the primary antibody diluted, 4 DEG C, is incubated on shaking table Overnight.

(5) film is placed in TBST, is washed three times, each 10min.

(6) secondary antibody is incubated for.Secondary antibody is diluted according to secondary antibody specification.Film is put in the incubation box for having diluted secondary antibody, Room temperature, shaking table are incubated for 2h.

(7) film is placed in TBST, is washed three times, each 10min.

(8) it shines.Washing terminates, and water extra on film is blotted with filter paper, prepares ECL luminescent solution, A liquid and B liquid are pressed It mixes, is kept in dark place according to 1:1.Appropriate ECL luminescent solution is added drop-wise on film, is put into camera bellows, is carried out using gel imaging system It takes pictures preservation.

Bioinformatics software involved in the present invention includes the following:

1) RNA protein-interacting forecasting software: catRAPID:http: //service.tartaglialab.com/ update_submission/108643/f560944664

2) RNA protein-interacting forecasting software: http://starbase.sysu.edu.cn/

Experimental result:

1, the template of the in-vitro transcription using H19 slow virus carrier as template amplification with T7 promoter, as a result such as Fig. 8 institute Show.

2. the identification of product lncRNA H19 is transcribed in vitro

Using the DNA with T7 promoter as template, it is carried out according to kit specification in-vitro transcription, to the RNA after transcription Agarose gel electrophoresis is carried out, is compared according to the size of relative molecular mass and marker, electrophoresis result is as shown in Figure 9.

The identification of RNA-binding protein after 3.RNA pull-down

After the completion of in-vitro transcription, biotin labeling is carried out to RNA, the lysate of RNA and K562 cell after the completion of marking It is incubated with, obtains the compound of RNA albumen and Streptavidin MagneSphere, by elution, obtain the compound of RNA and albumen. This compound is subjected to polyacrylamide gel electrophoresis, it, will after carrying out coomassie brilliant blue staining and destainer decoloration to running gel Sample sends to Mass Spectrometric Identification.SDS-PAGE glue carries out full swimming lane proteomic image identification, the results showed that, long-chain non-coding RNA H19 A variety of specific proteins are identified with protein complex swimming lane.

The protein spectrum of 4.RNA pull-down product is identified

In order to verify with which the long-chain non-coding RNA H19 albumen to interact has, we in vitro to RNA into Row transcription, and marked using biotin, RNA and the K562 cell pyrolysis liquid of biotin labeling, which are incubated for, carries out RNA Pull-down, obtained compound product carry out electrophoresis, and coomassie brilliant blue staining, the results are shown in Figure 10.

The Mass Spectrometer Method and software prediction of 5.lncRNA H19pull-down existing protein molecule jointly

The albumen of catRAPID software and the interaction of 2.0 software prediction of starBase v and long-chain non-coding RNA H19, As a result as shown in figure 11, find in the protein of the protein predicted and Mass Spectrometric Identification there are two be it is common, i.e. PCBP1 and FUS。

The target protein PCBP1 and FUS of 6.Western Blot verifying long-chain non-coding RNA H19

There is interaction between long-chain non-coding RNA H19 and PCBP1 and FUS to further verify, inventor is to RNA The product of pull-down has carried out Western Blot verifying, as a result as shown in figure 12；RNA pull- as seen from Figure 9 In down product, it is able to detect that PCBP1 and FUS.

Interpretation of result:

The interaction type of LncRNA and albumen: the guidance molecule of first, lncRNA as albumen.Huart etc. is in p53 Non-coding RNA p21 (long intergenic between the long chain gene that discovery has p53 induction to generate in the research of signal path NcRNA p21, lincRNA-p21) the guidance molecule that can be used as hnRNP-K (hnRNP-K), it is located To the gene promoter area p21, p21 transcriptional expression is activated.Second: lncRNA as albumen scaffold molecule.LncRNA is alternatively arranged as The scaffold molecule of two or more protein, by these protein constitutive protein matter compound figures.Homeobox Between gene RNA (HOX antisense intergenic RNA, HOTAIR) be located at homeobox gene C (homeobox C, HOXC) gene cluster, and expressed in the cell of distal end after limbs.HOTAIR can inhibit homeobox by trans-acting The transcription of gene D (homeobox D, HOXD) gene cluster inhibits HOTAIR that can activate the expression of HOXD gene cluster.Third: Bait molecule of the lncRNA as albumen.IncRNA-DNA damage activation P21 correlative coding RNA (P21associated NcRNA DNA damage activiated, PANDA) frequently as transcription factor decoys molecule participation gene expression regulation. PANDA be located at cyclin-dependent kinase inhibiting factor 1A (cycin-dependent kinase inhibitor 1A, CDKN1A, i.e. p21) at upstream region of gene 5kb, structure is fawned on 5 ' caps and 3 ' poly-A tails of not clipped modification.When When DNA damage occurs, PANDA is transcribed, by combining nuclear factor Y α subunit (nuclear transcription Factor Y subunit α, NF-YA) itself and target gene interaction are prevented, and then the life span of cell is regulated and controled.

In this section in experiment, we obtain lncRNA H19 (Fig. 8) in vitro, then using the method being transcribed in vitro Using biotin labeling RNA, the compound of lncRNA H19 albumen in connection is obtained using RNA pull-down technology, together When obtain compound of the lncRNA H19 in conjunction with RNA.Protein Detection utilizes mass spectrum (MS), identifies multiple proteins. CatRAPID is a kind of on-line Algorithm, it is main according to RNA and Secondary structure, hydrogen bond and intermolecular force to To assess the tendency to interact between them, and then the interaction of prediction RNA and protein.Starbase V2.0 software It can predict miRNA-lncRNA interactions, miRNA-circRNA interactions, miRNA-sncRNA interactions、miRNA-mRNA interactions、Protein-lncRNA interactins、Protein-mRNA interactions、Protein-pseudogene interactions、Protein-sncRNA interactions。RNA By Mass Spectrometric Identification after pull-down, there are multiple protein molecules to be detected, catRAPID on-line Algorithm and Starbase V2.0 software prediction has 9 to the albumen with lncRNA H19 interaction, and there are two the albumen by western blot identification, i.e., PCBP1 and FUS.

The functional study of Part III lncRNA H19 target protein FUS

1, FUS-SiRNA sequence is designed and synthesized by Guangzhou Rui Bo company

2. cell strain:

K562 cell strain, human chronic myelogenous leukemia system, is purchased from Shanghai Cell Bank of the Chinese Academy of Sciences.

The screening of 12 FUS-SiRNA ordered sequence of embodiment

Test method is shown in embodiment 2.

The influence that 13 targeted inhibition FUS of embodiment forms K562 cell colony

Specific method is shown in 5.5.2 in embodiment 5.

Effect of the 14 targeted inhibition FUS of embodiment to K562 cell Imatinib drug susceptibility

1) FUS-siRNA and SCR transfection K 562 cell

(1) experimental group are as follows: blank control group (BLANK), random controls group, FUS-siRNA group, every group setting 3 multiple Hole, FUS-siRNA and SCR concentration are 100nM.

(2) logarithmic growth phase cell, with serum-free, antibiotic-free RPMI-1640 culture medium by concentration of cell suspension It is adjusted to 30 × 10⁴/ mL, for every 50 μ L cell suspension inoculation of hole in 96 orifice plates, each experimental group repeats three multiple holes.

(3) Lipofectamine is prepared^TMThe compound of 2000 and RNA: according to Lipofectamine^TM2000 specifications, The siRNA amount and Lipofectamine that each takes what he needs^TM2000 amounts are diluted with Optimal Medium Opti-MEM respectively.

(4)Lipofectamine^TMAfter the completion of 2000 dilutions, it is stored at room temperature 5min.

(5) dilute and stand the Lipofectamine of 5min^TM2000 with diluted siRNA and mixed according to the ratio of 1:1, It is stored at room temperature after 20min to get Lipofectamine is arrived^TM2000-RNA compound.

(6) by Lipofectamine needed for each group^TM2000-RN compound is slowly added drop-wise to the cell suspension in orifice plate In, so that the final volume in every hole is 100 μ L.96 orifice plates are placed in 37 DEG C, CO₂In the cell incubator that volume fraction is 5%.

2) Imatinib is acted on

Experimental group:

Blank control group: (K562)；

SCR, FUS-siRNA group of Imatinib effect.

Prepare Imatinib working solution: by RPMI- of the Imatinib containing 20% fetal calf serum that mother liquid concentration is 10mM 1640 are diluted to working concentration, and working concentration is successively are as follows: 0.05,0.1,0.15,0.2,0.25 μm.By it is above-mentioned transfected SCR, 96 orifice plates after FUS-siRNA 6h take out, and the Imatinib working solution diluted are slowly added in corresponding hole, every hole The volume of addition is 100 μ L, so that the final volume in each hole is 200 μ L, 100 μ L are added containing 20% serum in blank control group 96 orifice plates after mixing, are placed in 37 DEG C, CO by RPMI-1640 culture medium₂It is cultivated in the cell incubator that volume fraction is 5%.

3) CCK8 cytoactive detection kit is detected

The detection of Biotool Vita-Blue cytoactive detection kit is carried out after 48h

(1) the Vita-Blue cytoactive detection reagent of 20 μ L (1/10 volume) is added in cell culture fluid, it is sufficiently mixed It is even, it is not possible to the presence of bubble.

(2) cell 1-4h is cultivated, but need to avoid being directly exposed in light.Illustrate: when the sensitivity of detection is with culture Between extension and improve.For the cell of negligible amounts, it can cultivate and detect afterwards for 24 hours.

(3) when culture solution becomes pale pink from pure blue, fluorescence microscope or luminescent counter (exciting light are used 530nm emits light 590-620nm) record fluorescence reading.Since the experiment is living cells experiment, it should choose multiple time points With the optimal result of determination.But blue shows that incubation time is too long when completely disappearing.Illustrate: living using Vita-Blue cell There can be slight autofluorescence at 590nm when property detection reagent.This fluorescence Beijing is with culture medium, pH value, incubation time, sudden and violent It is exposed to the variation of the factors such as the time of light and changes.In order to correct background, it is necessary to prepare one or more cell-free cultures Liquid control wells calculate average fluorescent value, then deduct the value in the air from test.

(4) cell viability calculates:

Versus cell activity (%)=[A (drug+)-A (blank)]/[A (drug -)-A (blank)] × 100%

A (drug+): the absorbance in the hole with cell, Vita-Blue reagent and drug solution；

A (drug -): have cell, Vita-Blue reagent without the absorbance in the hole of drug solution

A (blank): have culture medium, Vita-Blue reagent without the absorbance in the hole of cell.

Bioinformatics software involved in the present invention includes:

RNA protein-interacting forecasting software:

catRAPID:http://service.tartaglialab.com/update_submission/108643/ f560944664

RNA protein-interacting forecasting software: http://starbase.sysu.edu.cn/

Signal path integrates website: http://kobas.cbi.pku.edu.cn/

Data processing in the present invention uses SPSS statistical software, and data are indicated with mean ± standard deviation, using single factor test Method of analysis of variance, (one-way ANOVA) analyze the conspicuousness of difference between each group of data, are to have significant difference with P ﹤ 0.05.

Experimental result:

The screening of 1.FUS-SiRNA ordered sequence

Concentration is SCR the and FUS-siRNA transfection K 562 cell of 100nm, and after cell normally cultivates 48h, it is thin to extract each group The total serum IgE of born of the same parents, Real-time PCR detects the relative expression levels (as shown in figure 13) of each group FUS mRNA, to filter out Ordered sequence is FUS-SiRNA-02 and FUS-SiRNA-03.The sequence that subsequent functional experiment uses are as follows: FUS-SiRNA-03.

The research that 2.FUS is in progress in K562 malignant

2.1 to the influence of K562 malignant progress after targeted inhibition FUS

2.1.1 influence of the detection to K562 cell Imatinib Pharmaceutical sensibility after targeted inhibition FUS

After concentration is SCR the and FUS-siRNA transfection K 562 cell of 100nm, then at the Imatinib of various concentration Cell after the completion of reason transfection, cell detect Cell relative activity after normally cultivating 48h, as a result as shown in figure 14, find FUS- SiRNA transfection group cell it is significantly raised to the drug susceptibility of Imatinib (there were significant differences compared with blank group and SCR group P < 0.05), Cell relative activity is reduced with the increase of Imatinib Pharmaceutical concentration.

2.1.2 influence K562 cell colony formed after targeted inhibition FUS

Colony, which is carried out, after transfection concentrations are 100nm in K562 cell SCR-siRNA and FUS-siRNA forms reality It tests, as a result as shown in figure 15, compared with blank group and SCR group, finds the colony Forming ability of FUS-siRNA transfection group cell Weaken, has statistical significance (P < 0.05).

Interpretation of result:

This part experiment is broadly divided into two parts, and first part tests to study the function of the target protein of lncRNA, Inventor has designed and synthesized FUS-siRNA, after making its low expression first, studies influence of the FUS to K562 cell.FUS- SiRNA and SCR synthesis is in the sharp rich biological Co., Ltd in Guangzhou.FUS is a DNA/RNA binding protein, participates in gene expression Multiple steps in.In prostate cancer, FUS is the activity factor of androgen receptor.In acute myeloid leukemia of children In, FUS and ERG generate FUS-ERG fusion, encode the fusion protein F US-ERG albumen of 497 amino acid.

Although the means for the treatment of CML have very much, first generation protein tyrosine kinase inhibitor Imatinib still makees extensively For the first-line drug of CML treatment.Firstly, inventor transfects SCR and FUS-siRNA in K562 cell, filter out effective SiRNA sequence, FUS-SiRNA-03 effect preferably (Figure 13) use Biotool then by ordered sequence transfection K 562 cell Vita-Blue cytoactive detection reagent has detected group of cells to Imatinib Pharmaceutical Susceptible change situation, such as Figure 14 institute Show, after targeted inhibition FUS, K562 cell enhances the drug susceptibility of Imatinib.Such as Figure 15, to transfection SCR and FUS- The K562 cell of siRNA has carried out colony experiment, and after finding targeted inhibition FUS, the colony for reducing K562 cell is formed Ability.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that, It can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the spirit and scope of technical solution of the present invention.

Claims (9)

1.lncRNA application of the target protein FUS of H19 in the drug for preparing and/or screening treatment tumour.

2. application according to claim 1, which is characterized in that the tumour is leukaemia.

The application of 3.lncRNA H19 and FUS being used in combination in the drug for preparing and/or screening treatment tumour.

4. application according to claim 3, which is characterized in that the tumour is malignant tumour.

5. a kind of drug for treating tumour, which is characterized in that contain the inhibitor of FUS in the drug.

6. drug according to claim 5, which is characterized in that the inhibitor is the SiRNA of FUS.

7. drug according to claim 5, which is characterized in that also containing in the drug can promote lncRNA H19 to express Reagent or carrier.

8. drug according to claim 6, which is characterized in that the base sequence of the inhibitor such as FUS-SiRNA-03 institute Show.

9. drug according to claim 5, which is characterized in that the tumour is leukemia.