CN107446927A - Activate microRNA, screening technique and its application that LRIG1 is expressed in glioma U251 cells - Google Patents

Activate microRNA, screening technique and its application that LRIG1 is expressed in glioma U251 cells Download PDF

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CN107446927A
CN107446927A CN201710833623.0A CN201710833623A CN107446927A CN 107446927 A CN107446927 A CN 107446927A CN 201710833623 A CN201710833623 A CN 201710833623A CN 107446927 A CN107446927 A CN 107446927A
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黄益玲
胡火军
孙雨沛
李格
尤程程
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China Three Gorges University CTGU
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Abstract

The small activator RNA of double-strand that tumor suppressor gene LRIG1 is expressed in glioma U251 cells is activated the invention discloses a kind of（dsLRIG‑712）And its application.DsLRIG 712 is made up of the positive-sense strand and antisense strand of 21 nucleotides, and 3 ' ends of every chain hang prominent two dTdT nucleotides, and remaining 19 nucleotides is mutually paired.The expression of the saRNA molecule energy specific activation LRIG1 genes, target gene mRNA and protein expression level have rise, and suppress the propagation and locomitivity of brain glioblastoma cell, reach the purpose for suppressing tumour growth, the treatment available for glioma.

Description

Activate the microRNA, screening technique that LRIG1 is expressed in glioma U251 cells And its application
Technical field
The present invention provides a kind of small activation of double-strand for activating tumor suppressor gene LRIG1 expression in glioma U251 cells RNA (dsLRIG-712), and by dsLRIG-712 be used for glioma treatment.
Background technology
RNA activation (RNA activation, RNAa) is from small molecule doulbe-chain RNA target to acting on gene promoter area Domain, in the phenomenon of transcriptional level inducible gene expression.And the small dsRNA with activation function is referred to as small activator RNA (small activating RNA,saRNA).Early in 1969, Britten etc. had found that the RNA that Genome noncoding regions are transcribed out can The expression of a big genoid is activated, and proposed activator RNA (activator RNAs, RNAa) concept, but does not obtain confirmation. Until 2006, saRNA was just formally had found and named by Li etc., and Li etc. sticks plain gene promoter region in application targeting E- calcium SiRNA (dsEcad-302, dsEcad-215) has been surprisingly found that target gene does not have when transfecting prostate cancer PC-3 and DU-145 Expression silencing expected from generation, 14 times and 3.8 times are raised respectively on the contrary.Afterwards, Janowski etc. will target PgR DsRNA transfection breast cancer T47D and MCF7 the cell discoveries of (proges-terone receptor, PR) gene, PR expression 18 times have been raised compared with control group.Huang etc. had found by research to cercopithecus aethiops, gorilla and mouse, rat, SaRNAa has conservative in mammalian cell, and this research makes to establish animal model and implements saRNA drug therapies Preclinical laboratory becomes possibility.
Glioma is most common central nervous system primary tumor, has the feature of invasive growth, serious prestige Coerce human health.Effective treatment method is there is no to glioma at present, becomes field of neurosurgery research emphasis. LRIG1 is newfound tumor suppressor gene in recent years, there is expression, but the table in brain tissue in normal human respectively organizes Up to horizontal highest.Research finds LRIG1 expression and the cancer pathology rank of glioma is in high negative correlation, LRIG1 Expression it is lower, the grade malignancy of glioma is higher.Therefore, the expression by activating LRIG1 genes is expected to reach suppression Purpose with development occurs for glioma processed.
The content of the invention
The present invention provides a kind of small molecule of the treatment people's middle and advanced stage glioma obtained through screening regarding to the issue above RNA medicines.It is specific as follows：
The candidate dsRNA molecules of chemical synthesis targeting LRIG1 gene promoters used in the present invention and dsControl purchases From Shanghai JiMa pharmacy Technology Co., Ltd.Human glioma cell's (U251 cells) is purchased from Chinese Academy of Sciences's cell bank, makes With DMEM nutrient solutions the limited public affairs of Lanzhou lark biotechnology are purchased from purchased from Ji Nuo biological medicine technologies Co., Ltd, hyclone Department, transfection reagent turbofect are purchased from Thermo companies of the U.S., and Trizol is purchased from Invitrogen companies of the U.S., reversal agents Box is purchased from Thermo companies of the U.S., and PCR Mix are purchased from Tiangeng biochemical technology Co., Ltd, and holoprotein extracts kit is purchased from north Capital Puli's lema gene Technology Co., Ltd., BCA kits are purchased from Wuhan Ke Rui Bioisystech Co., Ltd, and Anti-LRIG1 is purchased from Santa Cruz companies of the U.S., horseradish peroxidase (HRP) sheep anti mouse, HRP sheep anti mouses have purchased from the auspicious biotechnology of Wuhan section Limit company, ECL chemical luminescence reagent kits are purchased from green skies Bioisystech Co., Ltd.
A kind of screening technique with the saRNA for activating LRIG1 gene expressions, comprises the following steps：
(1) dsRNA design：DsLRIG1-712 is for LRIG1 gene promoter areas relative transcript starting point -712 It is the dsRNA molecules of 21 nucleotide bases pair with this section of promoter complementary pairing, length nearby to design, and particular sequence is： dsLRIG1-712：Sense：UUU CCC ACC CAA GGU GUG A[dT] [dT]；Antisense：UCA CAC CUU GGG UGG GAA A[dT][dT]。
(2) culture and transfection of cell：U251 cell growths in the DMEM nutrient solutions containing 10% hyclone, 37 DEG C, 5%CO2Incubator culture, dsRNA is transfected into U251 cells with transfection reagent；
(3) extraction of total serum IgE and Semi quantitative PCR analysis：Cell is collected after transfection 72h, is extracted with Trizol reagents total RNA, with 1 μ g RNA templates, 1 μ l Oligo dT are added in PCR pipe successively, 12 μ l is added to DEPC water, reaction solution is put It is immediately placed on after 70 DEG C, 5 minutes 1 minute on ice, is separately added into μ l, the 20U/ μ l ribonuclease inhibitors 1 of 5 × buffer 4 μ l, 10mM dNTP mix are the 2 μ l and μ l of reverse transcriptase 1, are well mixed, brief centrifugation, mixture are placed in PCR instrument and completed Subsequent reactions：37 DEG C of 5min, 42 DEG C of 60min, 70 DEG C of 10min, reverse transcription cDNA.Using cDNA as template, amplifying target genes LRIG1 and GAPDH.Primer sequence：- the ATCATCACCCAGCCAGAAAC-3 ' of LRIG1 upstreams 5 ', downstream 5 '- CTACCGTGGTCCCATCCTT-3 '；- AACGGATTT the GGTCGTATTGGG-3 ' of GAPDH upstreams 5 ', downstream 5 '- CCTGGAAGATGGTGATGGGAT-3 ',
Reaction system：
PCR reaction systems
Response parameter：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 45sec, 35 Individual circulation, 72 DEG C of fully extension 10min.Detected through gel electrophoresis PCR primer fragment, it is 892bp that LRIG1, which is expected size, and GAPGH is 230bp。
(4)Westernblot：The analysis that cell is used for protein expression is collected after dsRNA transfectional cells 72h, it is clear with PBS Wash cell, add protein lysate, be collected by centrifugation supernatant after cracking on ice, the OD values of BCA kit measurement protein solutions, The protein concentration of sample is adjusted, the protein content for making each sample is 20ug, and actin is internal reference albumen, and the albumen of extraction is entered Row SDS-PAGE gel electrophoresis, after albumen transferring film, add the closing of skimmed milk power confining liquid, wash film, by 1：1000 dilution proportion LRIG1 Primary antibody (volume ratio, 5ul LRIG1 antibody is added in 5ml TBS), and 1:(volume ratio, will for 2000 dilution proportion actin primary antibodies 2.5ul actin antibody is added in 5ml TBS), pvdf membrane is respectively put into the solution containing respective primary antibody, 4 DEG C of incubations of primary antibody Overnight, film is washed.According to 1:The secondary antibody (volume ratio) of 2000 dilution proportion HRP marks, pvdf membrane is added the secondary antibody diluted, room Temperature, which is shaken, is incubated 1h, washes film；Developed using ECL chemical luminescence reagent kits, egg after detection dsLRIG1-712 transfection U251 cells The change expressed in vain.
In described step (2), the final concentration of 20nmol/L of dsRNA, specific transfection process：Add 4ul dsRNA extremely 100ul serum-free DMEM nutrient solutions, add 6ul turbofect transfection reagents to 100ul serum-free DMEM nutrient solutions, both Mixing, places 15-20min, adds dsRNA-turbofect compounds into 6 orifice plates at room temperature, the control of 6 orifice plate cell confluency degree 50%, nutrient solution final volume is 4ml.
In described step (4), described protein lysate is：RIPA lysates, composition are：50mM Tris-HCl (pH 7.4), 150mM NaCl, 1%NP-40,0.1%SDS.
The dsLRIG1-712 that the present invention obtains can significantly activate LRIG1 expression compared with control group, wherein, LRIG1mRNA relative expression quantity is more than 2 times of control group, and the expression quantity of LRIG1 albumen is also more than 2 times of control group, It is feature saRNA to show dsLRIG1-712, has the function of LRIG1 genes in specific activation U251 cells.U251 is thin Born of the same parents belong to human glioma cell, by raising the expression of tumor suppressor gene LRIG1 in the cell, can suppress U251 cell growths And migration, the treatment available for middle and advanced stage glioma.
Brief description of the drawings
Fig. 1 is that dsLRIG1-712 transfects mRNA expressions after U251 cells 72h.
Fig. 2 is that dsLRIG1-712 transfects protein expression level after U251 cells 72h.
Embodiment
Embodiment 1
DsRNA designs dsLRIG1-712 is nearby to be set for LRIG1 gene promoter areas relative transcript starting point -712 Meter is the dsRNA molecules of 21 nucleotide bases pair with this section of promoter complementary pairing, length.Control group dsRNA (dsControl) non-homogeneous with known human genome sequence, length is similarly the dsRNA sequences of 21 nucleotide bases pair.
Using Genebank databases, LRIG1 gene promoter region sequences are found, for the sequence of promoter upstream -712 The template as design dsRNA is arranged, particular sequence is：dsLRIG1-712：Sense：UUU CCC ACC CAA GGU GUG A [dT][dT]；Antisense：UCA CAC CUU GGG UGG GAA A[dT][dT].
Cell culture is with transfection U251 cell growths in the DMEM nutrient solutions containing 10% hyclone, 37 DEG C, 5%CO2 Incubator culture.Experiment is divided into 3 groups：Blank group, control group (transfection dsControl) and experimental group (transfection candidate dsRNA), The dsRNA concentration of each experimental group is 20nmol/L.DsLRIG1-712, dsControl are each separately transfected into transfection reagent U251 cells.Cell confluency degree control about 50%, transfection reagent turbofect, dsRNAs, serum-free DMEM are put down during transfection Weigh to room temperature；Appropriate dsRNAs is diluted with serum-free DMEM nutrient solutions, dilutes transfection reagent with same nutrient solution turbofect；After both fully mix, 20min is stored at room temperature to form dsRNA-turbofect compounds, by dsRNA- Turbofect complexes drop-wises add to place in culture plate to be continued to cultivate into cell culture incubator.
Cell is collected after the extraction of total serum IgE and Semi quantitative PCR analysis transfection 72h, total serum IgE is extracted with Trizol reagents, with 1 μ g RNA templates, 1 μ l Oligo dT are added in PCR pipe successively, 12 μ l are added to DEPC water, place reaction liquid into 70 DEG C, 5 It is immediately placed on 1 minute on ice after minute, is separately added into 5 × buffer, 4 μ l, 20U/ μ l ribonuclease inhibitors 1 μ l, 10mM DNTP mix are the 2 μ l and μ l of reverse transcriptase 1, are well mixed, brief centrifugation, mixture are placed in PCR instrument and completes subsequent reactions： 37 DEG C of 5min, 42 DEG C of 60min, 70 DEG C of 10min, reverse transcription cDNA.Using cDNA as template, amplifying target genes LRIG1 and GAPDH.Primer sequence：- the ATCATCACCCAGCCAGAAAC-3 ' of LRIG1 upstreams 5 ', downstream 5 '- CTACCGTGGTCCCATCCTT-3 '；- AACGGATTT the GGTCGTATTGGG-3 ' of GAPDH upstreams 5 ', downstream 5 '- CCTGGAAGATGGTGATGGGAT-3 ', response parameter：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 45sec, 35 circulations, 72 DEG C of fully extension 10min.Detected through gel electrophoresis PCR primer fragment, LRIG1 It is expected that size is 892bp, GAPGH 230bp.
The analysis that cell is used for protein expression is collected after Westernblot detection dsRNA transfectional cells 72h, it is clear with PBS Wash cell, add protein lysate, be collected by centrifugation supernatant after cracking on ice, the OD values of BCA kit measurement protein solutions, The protein concentration of sample is adjusted, the protein content for making each sample is 20ug, and actin is internal reference albumen, and the albumen of extraction is entered Row SDS-PAGE gel electrophoresis, after albumen transferring film, add the closing of skimmed milk power confining liquid, wash film, by 1：1000 dilution proportion LRIG1 Primary antibody (volume ratio, 5ul LRIG1 antibody is added in 5ml TBS), and 1:(volume ratio, will for 2000 dilution proportion actin primary antibodies 2.5ul actin antibody is added in 5ml TBS), pvdf membrane is respectively put into the solution containing respective primary antibody, 4 DEG C of incubations of primary antibody Overnight, film is washed.According to 1:The secondary antibody (volume ratio) of 2000 dilution proportion HRP marks, pvdf membrane is added the secondary antibody diluted, room Temperature, which is shaken, is incubated 1h, washes film；Developed using ECL chemical luminescence reagent kits, egg after detection dsLRIG1-712 transfection U251 cells The change expressed in vain.
Influence horizontal to U251 cell target genes LRIG1mRNA dsRNA
The activation expressed for clear and definite dsLRIG1-712 LRIG1, this research detect dsRNA by semiquantitive PCR method Target gene LRIG1mRNA expression after transfection.Feature saRNA is filtered out according to expression of target gene level.dsLRIG1- After 712 with 20nmol/L concentration transfection U251 cells 72h, LRIG1 mRNA expression significantly raises compared with control group, sees Fig. 1.
Influences of the dsRNA to U251 cell target gene LRIG1 protein levels is clear and definite dsLRIG1-712 to LRIG1 eggs DsLRIG1-712 is transfected into U251 cells and detected by Westernblot and transfected by the activation expressed in vain, this research The intracellular LRIG1 protein expressions situation of each group after 72h.As a result show, dsLRIG1-712 group LRIG1 protein expression levels are more right According to group significantly rise, for more than 2 times of control group, Fig. 2 is seen.

Claims (5)

1. a kind of activate the microRNA that LRIG1 is expressed in glioma U251 cells, it is characterised in that：MicroRNA (dsLRIG1-712) it is for the design nearby of LRIG1 gene promoter areas relative transcript starting point -712 and this section of promoter Complementary pairing, length are the dsRNA molecules of 21 nucleotide bases pair, and particular sequence is：dsLRIG1-712：Sense：UUU CCC ACC CAA GGU GUG A[dT][dT]；Antisense：UCA CAC CUU GGG UGG GAA A[dT][dT].
2. activate the screening technique of the microRNA that LRIG1 is expressed in glioma U251 cells, it is characterised in that including such as Lower step：
(1) culture and transfection of cell：U251 cell growths containing mass fraction be 10% hyclone DMEM nutrient solutions, 37 DEG C, 5%CO2Incubator culture, dsRNA is transfected into U251 cells with transfection reagent；
(2) extraction of total serum IgE and Semi quantitative PCR analysis：Cell is collected after transfection 72h, total serum IgE is extracted with Trizol reagents, with RNA template, Oligo dT are added in PCR pipe, graduation mark is added to DEPC water, are placed reaction liquid into 65-80 DEG C of water-bath, 5 It is immediately placed on 1 minute on ice after minute, is separately added into 5 × buffer, ribonuclease inhibitor, dNTP mix and reverse transcription Enzyme, it is well mixed, brief centrifugation, mixture is placed in PCR instrument and completes subsequent reactions：37 DEG C of 5min, 42 DEG C of 60min, 70 DEG C 10min, reverse transcription cDNA；
(3)Westernblot：The analysis that cell is used for protein expression is collected after dsRNA transfectional cells 72h, it is thin with PBS Born of the same parents, protein lysate is added, supernatant, the OD values of BCA kit measurement protein solutions, adjustment are collected by centrifugation after cracking on ice The protein concentration of sample, actin are internal reference albumen, carry out SDS-PAGE gel electrophoresis to the albumen of extraction, after albumen transferring film, are added The closing of skimmed milk power confining liquid, film is washed, by 1：1000 dilution proportion LRIG1 primary antibodies, 1：2000 dilution proportion actin primary antibodies, will Pvdf membrane is respectively put into the solution containing respective primary antibody, 4 DEG C of overnight incubations of primary antibody, film is washed, according to 1：2000 dilution proportion HRP are marked The secondary antibody of note, pvdf membrane is added to the secondary antibody diluted, room temperature, which is shaken, is incubated 1h, washes film；Shown using ECL chemical luminescence reagent kits Shadow, detection dsLRIG1-712 transfect the change of protein expression after U251 cells, you can completing screening has activation LRIG1 genes The saRNA of expression.
3. the screening technique for the microRNA that LRIG1 is expressed in the activation glioma U251 cells described in claim 2, its It is characterised by, using cDNA as template, amplifying target genes LRIG1 and GAPDH, primer sequence：LRIG1 upstreams 5 '- ATCATCACCCAGCCAGAAAC-3 ' ,-CTACCGTGGTCCCATCCTT-3 ' of downstream 5 '；GAPDH upstreams 5 '- AACGGATTTGGTCGTATTGGG-3 ' ,-CCTGGAAGATGGTGATGGGAT-3 ' of downstream 5 '.
4. the screening technique for the microRNA that LRIG1 is expressed in the activation glioma U251 cells described in claim 2, its It is characterised by, PCR reaction systems are as follows：
Response parameter：94 DEG C of pre-degeneration 5min, 94 DEG C denaturation 30sec, 55 DEG C annealing 30sec, 72 DEG C extension 45sec, 35 follow Ring, 72 DEG C of fully extension 10min, detected through gel electrophoresis PCR primer fragment, it is 892bp that LRIG1, which is expected size, and GAPGH is 230bp。
5. any one of the claim 1-4 obtained saRNA with activation LRIG1 gene expressions that screen are in human glioma Application in drug therapy.
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WO2019196887A1 (en) * 2018-04-10 2019-10-17 中美瑞康核酸技术(南通)研究院有限公司 Novel small activating rna
CN112912402A (en) * 2018-10-17 2021-06-04 古德T细胞有限公司 Specific binding molecules for LRIG-1 proteins and uses thereof
CN113528517A (en) * 2021-06-03 2021-10-22 三峡大学 Double-stranded saRNA molecule for activating LRIG1 gene expression in cervical cancer HeLa cells
WO2024001170A1 (en) * 2022-06-27 2024-01-04 Ractigen Therapeutics Small activating nucleic acid molecule and use thereof in treatment of hereditary angioedema

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019196887A1 (en) * 2018-04-10 2019-10-17 中美瑞康核酸技术(南通)研究院有限公司 Novel small activating rna
CN112912402A (en) * 2018-10-17 2021-06-04 古德T细胞有限公司 Specific binding molecules for LRIG-1 proteins and uses thereof
CN113528517A (en) * 2021-06-03 2021-10-22 三峡大学 Double-stranded saRNA molecule for activating LRIG1 gene expression in cervical cancer HeLa cells
CN113528517B (en) * 2021-06-03 2022-03-18 三峡大学 Double-stranded saRNA molecule for activating LRIG1 gene expression in cervical cancer HeLa cells
WO2024001170A1 (en) * 2022-06-27 2024-01-04 Ractigen Therapeutics Small activating nucleic acid molecule and use thereof in treatment of hereditary angioedema

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