It is a kind of be capable of specificity inhibit ZBED1 gene siRNA construction method and its applicationTechnical field
The invention belongs to molecular biology and oncologies, and being specifically related to one kind being capable of specificity inhibition ZBED1The construction method of the siRNA of gene and its application.
Background technique
Zinc finger BED type forgives 1 (zinc finger, BED-type containing 1, ZBED1) of son also known as DNA replication dnaCoherent element binding factor belongs to zinc finger protein ZBED family, is made of 694 amino acid residues, molecular weight about 78kDa,Coding albumen is considered as the key turn of a kind of regulating DNA duplication, chromatin Structure remodeling, protein synthesis, cell cycle etc.Record the factor.Interestingly, research of the ZBED1 in tumour is not reported so far.Previous work discovery uses immune groupChange detection ZBED1 to express in human lung cancer tissue height, and by stages significant related to tumour T.Thus it prompts, ZBED1 is human lung cancerSpecial cance high-expression gene may play a significant role in lung cancer growth.
Summary of the invention
It is an object of the invention to be directed to the present Research of ZBED1 gene, its expression in lung cancer is illustrated, is proposedIt is capable of construction method and its application of the siRNA of specificity inhibition ZBED1 gene.
The purpose of the present invention is achieved through the following technical solutions.
1. detecting expression of the ZBED1 albumen in clinical lung cancer, comprising the following steps:
(1) collection of clinical cancerous lung tissue sample and specimen information: in January, 2009 is collected in June, 2016 in Yunnan ProvinceBy the cancerous tissue and cancer of 122 I-III A phase patients with lung adenocarcinoma of tumour hospital's thoracic surgery row pulmonary primary tumor radical operationLung tissue sample.Wherein, male 63, women 59；Patient's average age 54.4 years old, the range of age 27 to 78 years old；
(2) haematoxylin-Yihong (HE) dyeing carries out pathological diagnosis；
(3) expression of the immunohistochemistry detection ZBED1 albumen in clinical lung cancer cancerous tissue and cancer beside organism；
(4) differential expression of the statistical analysis ZBED1 albumen in clinical lung cancer cancerous tissue and cancer beside organism；
2. a kind of construction method for the siRNA for capableing of specificity inhibition ZBED1 gene, comprising the following steps:
(1) NCBI (https: //www.ncbi.nlm.nih.gov/gene) database is utilized, people ZBED1 gene is obtainedTranscript and the area coding (CDS) information (area transcript 1:NM_001171136, CDS: 1283-2367, particular sequence are shown in sequenceTable)；
(2) according to siRNA sequence design principle, for the code area of people's ZBED1 gene, design 2 can specificity strike and subtractThe target sequence of people's ZEBD1 gene；
| 5’——3’ |
| RNAi-1-F | GGAAGUACAUCUCUACCAA |
| RNAi-1-R | CCUUCAUGUAGAGAUGGUUdTdT |
| RNAi-2-F | GCAAACUGGUGGAGUACUU |
| RNAi-2-R | CGUUUGACCACCUCAUGAAdTdT |
(3) chemical synthesis target sequence；
3. siRNA inhibits the method for efficiency in verifying 2, it is characterised in that directly detect ZBED1 egg using WesternblotWhite expression, internal reference albumen are GAPDH；
4. detecting influence and the specific method of ZBED1 gene pairs lung carcinoma cell biological function, comprising:
(1) cell-proliferation activity detection (MTS method): by 103In 96 orifice plates, every hole adds a good cell inoculation of growth conditionsEnter 150ul complete medium, is placed in 5%CO2After cultivating 1 day in 37 DEG C of incubators, every hole adds MTS solution (5mg/ml) 30ul,Continue to be incubated for 2 hours；Selection 490nm wavelength measures the absorbance in each hole, continuous detection 5 days；
(2) the 2 × 10 of logarithmic growth phase will fluidic cell cycle detection: be in6A cell is fixed in 70% ethyl alcohol,Rinsing, is collected by centrifugation cell, 0.2M citric acid/sodium citrate high osmotic treatment 30 minutes, 1 × PBS washes 2 times, 0.1%Triton(PBS preparation) is washed 1 time, then with 500 μ g/mlPI and 0.1%RNAase A, and room temperature is protected from light incubation 30 minutes, flow cytometer inspectionSurvey analysis.
Beneficial effects of the present invention:
1. the high expression in clinical cancerous lung tissue that the invention discloses ZBED1 albumen.
2. can the specific expression for inhibiting endogenous ZBED1 in lung cancer cell line with the siRNA that the present invention develops.
3.MTS method detection cell Proliferation shows compared with transfection control RNA, transfects two siRNA energy that the present invention developsThe proliferative capacity for inhibiting lung carcinoma cell, illustrates that ZBED1 gene can promote the proliferation of lung carcinoma cell.
4. the Flow cytometry lung carcinoma cell period shows compared with transfection control RNA, transfection two developed of the inventionA siRNA can inhibit the cell cycle progression of lung carcinoma cell, make its retardance in the G1 phase, illustrate that ZBED1 gene can promote lung cancer thinBorn of the same parents' period converts from the G1 phase to the S phase.
5. function assessment result prompts, the siRNA that the present invention develops may have the targeted therapy of lung cancer highly importantApplication value.
Detailed description of the invention
Fig. 1 is the high expression in clinical cancerous lung tissue of ZBED1 albumen；
Fig. 2 is that two kinds of siRNA can the specific expression for inhibiting endogenous ZBED1 in lung cancer cell line；
Fig. 3 is inhibition situation of two kinds of siRNA to proliferation of lung cancer cells ability；
Fig. 4 is retardance situation of two kinds of siRNA to the lung carcinoma cell cell cycle；
Fig. 5, which is 2, specific to strike the target sequence for subtracting people's ZBED1 gene.
The present invention that the following detailed description will be further explained with reference to the above drawings.
Specific embodiment
1. detecting expression of the ZBED1 albumen in clinical lung cancer, comprising the following steps:
(1) collection of clinical cancerous lung tissue sample and specimen information: in January, 2009 is collected in June, 2016 in Yunnan ProvinceBy the cancerous tissue and cancer of 122 I-III A phase patients with lung adenocarcinoma of tumour hospital's thoracic surgery row pulmonary primary tumor radical operationLung tissue sample.Wherein, male 63, women 59；Patient's average age 54.4 years old, the range of age 27 to 78 years old；
(2) haematoxylin-Yihong (HE) dyeing carries out pathological diagnosis；
(3) expression of the immunohistochemistry detection ZBED1 albumen in clinical lung cancer cancerous tissue and cancer beside organism；
(4) differential expression of the statistical analysis ZBED1 albumen in clinical lung cancer cancerous tissue and cancer beside organism；
As shown in Figure 1, be expression of the ZBED1 albumen in clinical lung cancer cancerous tissue and cancer beside organism, it can be with from Fig. 1Find out, ZBED1 is expressed in 70.50% cancerous lung tissue, is expressed in 46.70% cancer beside organism, illustrates ZBED1 albumen in lung cancerExpression in tissue is high compared with by cancer.
2. developing the siRNA that energy specificity inhibits ZBED1 gene, comprising the following steps:
(1) NCBI (https: //www.ncbi.nlm.nih.gov/gene) database is utilized, people ZBED1 gene is obtainedTranscript (transcript 1:NM_001171136.1) and coding the area (CDS) information (particular sequence is shown in sequence table)；
Transcript 1 (variant 1, mRNA):
CGCGGGCGGCGCGGAAGCGGCGGCGGCGCGGCCGGGGCAGCCATGTCGCCATTGTCTGCGGCGCGGGCGGCCCTGCGGGTCTACGCGGTAGGCGCCGCGGTGATCCTGGCGCAGCTGCTGCGGCGCTGCCGCGGGGGCTTCCTGGAGCCAGCAGGTCCCCACGCATGAGCAGCCGTCCAGCAGGCAGGCTCCGGTGGAGAAGCAATGGAG
AATAAAAGCCTGGAGAGCTCCCAGACAGACCTGAAGCTGGTGGCCCACCCCCGCGCCAAGAGCAAGGTGTGGAAGTATTTCGGCTTCGACACCAACGCCGAGGGATGCATCCTGCAGTGGAAGAAAATCTACTGCCGCATCTGCATGGCCCAGATCGCCTACTCCGGAAACACCTCCAACCTGTCCTACCACCTGGAGAAGAACCACCCCGAGGAATTCTGCGAGTTCGTCAAGAGCAACACGGAGCAGATGCGTGAAGCCTTCGCCACCGCCTTCTCCAAGCTGAAGCCCGAGTCGTCCCAGCAGCCCGGGCAGGACGCGCTGGCCGTCAAGGCCGGCCACGGCTACGACAGCAAGAAGCAGCAGGAGCTGACGGCCGCCGTGCTGGGCCTCATCTGCGAGGGGCTGTACCCAGCCTCCATCGTGGACGAGCCCACCTTCAAGGTGCTGCTGAAGACGGCCGACCCCCGGTATGAGCTGCCCAGCCGGAAGTACATCTCTACCAAGGCCATCCCTGAGAAGTACGGGGCCGTCCGGGAGGTGATCCTGAAGGAGCTGGCCGAGGCCACCTGGTGTGGCATCTCCACCGACATGTGGAGGAGTGAGAATCAGAACCGCGCCTACGTCACGCTGGCCGCCCACTTCCTGGGCCTGGGCGCCCCCAACTGCCTGTCCATGGGCTCCCGCTGCCTGAAGACCTTCGAGGTGCCCGAAGAGAACACGGCGGAGACCATCACGCGAGTGCTCTATGAGGTCTTCATCGAGTGGGGCATCAGCGCCAAGGTCTTCGGGGCCACCACCAACTATGGCAAGGACATCGTGAAGGCGTGCTCCCTGCTGGACGTCGCAGTGCACATGCCCTGCCTGGGCCACACCTTCAATGCCGGCATCCAGCAGGCCTTCCAGCTCCCGAAGCTGGGGGCGCTGCTGTCGCGCTGCCGCAAACTGGTGGAGTACTTCCAGCAGTCTGCCGTGGCCATGTACATGCTCTATGAGAAGCAGAAGCAGCAGAACGTGGCCCACTGCATGCTGGTGAGCAACCGCGTCTCCTGGTGGGGGAGCACGCTGGCCATGCTGCAGCGCCTCAAGGAGCAGCAGTTCGTCATCGCCGGGGTCTTGGTGGAGGACAGCAACAACCACCACCTCATGCTGGAGGCCAGCGAGTGGGCCACCATCGAGGGGCTGGTGGAGCTCCTGCAGCCCTTCAAGCAGGTGGCCGAGATGCTGTCGGCCTCCAGGTACCCCACCATCAGCATGGTGAAGCCGCTGCTGCACATGCTCCTGAACACCACGCTCAACATCAAGGAGACCGACTCCAAGGAGCTCAGCATGGCCAAGGAGGTCATCGCCAAGGAGCTTTCCAAGACCTACCAGGAGACGCCCGAGATCGACATGTTTCTCAACGTGGCCACCTTCCTGGACCCCCGCTACAAGAGGCTGCCCTTCCTCTCCGCCTTCGAGCGGCAGCAGGTGGAGAATCGCGTGGTGGAAGAGGCCAAGGGCCTGCTGGACAAGGTCAAAGACGGCGGCTACCGGCCGGCTGAGGACAAGATCTTCCCGGTGCCCGAGGAGCCTCCCGTCAAGAAGCTCATGCGGACATCCACGCCGCCGCCCGCCAGCGTCATCAACAACATGCTGGCCGAGATCTTCTGCCAGACAGGCGGCGTGGAGGACCAGGAAGAGTGGCATGCCCAGGTGGTGGAGGAGCTGAGCAACTTCAAGTCCCAGAAGGTGCTTGGCCTCAACGAAGACCCCCTCAAGTGGTGGTCAGACCGCCTGGCCCTCTTCCCCCTGCTGCCCAAGGTGCTGCAGAAGTACTGGTGCGTGACGGCCACGCGCGTCGCCCCTGAGCGTCTCTTCGGATCCGCCGCCAACGTGGTCAGCGCCAAGAGGAACCGGCTGGCTCCCGCGCACGTGGACGAGCAGGTGTTTCTGTATGAGAACGCCCGGAGTGGGGCAGAGGCGGAACCCGAGGACCAGGACGAGGGGGAGTGGGGCCTGGACCAGGAGCAGGTGTTCTCCTTGGGGGATGGCGTCAGCGGCGGTTTCTTTGGCATTAGGGACAGCAGCTTCCTGTAGCGAGGAAGCGTGTTGTCTTACAAGTCATCCCCGCAGCAGCCCATTGGATGCTTTGCTGTAAATACTTACCCGGTCAGCTTGGTTTTGAACCTCAGAGACCATCCACTGTCTTTGACACCTAGAAGGTGGAAAAAGGAAAGAGATTTGAGAAGTGAGAGAGGGTCGGGGGCGGTGGCTCCTGTCTATAATCGCAGCACTTTGGGAGGCCGAGGTGGGCAGATCAGCTGAGGTCAGGAGATCGAGACCAGCCTGGCCAACATGGCGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGACTGATGGCATGTGCCTGTAATCCCAGCTTCTGGAGGCTGAGGCCAGAGAATCGCTTGAACCTGGGAGGTGGAGGTTGCAGTAAGCTGAGATCGCTGCACTCCAGCCTGGGCGACAAGAGCGAGACTCTCTCAAAAAGAAAAAAGAAGACACAAGAGAGGTGGCTTTGAGTGGGTTTCCTTTCCTCCCCTATTCCCGGGGCCCGGACGACTTCTGCTTGGGAACTGCCAACGCTTCTGCTTGGGAATTGCGTGCAGCAGAGCCTAGAGGAGCTGTTCCTTCCTTCACAGATACTTAAGACCTCCACCATGTCTGATTCGAGTTCTCCCTGGGAGGGTTTAGAAGAAACGCAGGAACATTCTGGGTGGCGTCGAAGGAGCCTTTCCCGATCATCATGTGTGACTTCTGCGAGGTCCAGATCACTGAATTCATGTTTACATTCTCGTGCAAGCAGGGACCTCTTGCTTCTGAGAAATGGGGAAGAGACCTTTTAGCCAAAATGCCCTTCTTAAAAAGAGAGACCTTTTTTTTTAATGTTCGGTTAAAAATGTGACAGATGAGTACAAAAATGCAGACACTTAACAAAAAGCAAACAGAAAAAAAAGTGTGGAATGTGTTGTATTTTCGACAGGTTGCTGCCAGAGAGCCTGCTTCCTGCTGCCTGCCGAAATTTCACTTTGCGGAGTTGGTCCTTAAAACTGGGCGGTGGCCGGGCGTGGTGGCTCACGCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACCAACATGGTGAAACCTCGTGTCTACTAAAAATACAAGAAACTAGCCAGATGCGGTGGCACCCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCGGAAGATTCGCTTGAGCCCAGGAGGTGGAGGTTGCAGTGAGCCAAGACTACGCCACTGCACTCCAGCCTGGAGGCCAGAGTGAGACTCCGTCTCAAAACAAAACAAAAGTGGGTGGCTCACGGGTCCGGAGGTTATGTCTTCGGTGTCTCAGCCCTAAAAGTCCAGTTCCCCCGTGCGGCCAGCTTTTCCACATAAGGTGTTTTTGATTTGATTACCGGAAAGGACTCTTGATTCTTCTCTTTTAAACTGAATACCATAGGGGAAATGAATTTTAAAATATTGCCCCCGGAGGGGTTTTCCGTGGCTGGATTCCTGCGAGTTGCTTTCAGTCATTCAGGGAAACAGAAAGACGTTTTCCAACATGTAGAACTGCTTTTTAACTGGAGGAAAAATACTTCAGGAGGCTTAGCATATTGCTTGGATTCTACGTGCAGCGGGTTCTCTGCCTCCGTGAAGACAAGCTGGGCTGGGGAGAACGGTGTCTAGGAGGGATGACCCCACTCAGCTCCAGGCAGTGTTCTGCCGAGACCCCAAGAACTCGGGGTGTCAGAGGGCAAAGGAACTACCTGCCTTTCACGGCTGCTGACTTCTCAGGGCTGCAAGCAGCACAGAATGTTATCCTTACGTCCTGAGCCGGTTTAAGTCTGTGGAAAAGGAAGCACGGGAGAAATCCACGTAACCTTTGCTTTCTTTTTAAGGGAAGCGGTTCCGCCGTGAACTTGGAACCCTCAGCTCCGGGTGTTCTCGGCAGAAGGGCAGCTGGAAGGGACACAGTGGGGCAGGCTTTGGGGTTGCTCCCTGTTCTGCCCCGAGGCCGGGGACGCAGGGCAGCCCACGCCTCCGTGGGCTCCATTCTGTAGCATTGCCAGCGTTCTCTTCACGTCTCTAACAATCCTTCGCTTTTCCTCACTCACGTGGAAATGTGAACTGTCCCGGCTCTGTCTTCATTTTTATTTTTAAGCCATCGTTCCCCTCCTGAACGGTTGCCCCTTATTTAATGCTGTAAAGTTGGACTGTTGTTCAATAAACCAGAGCAATGCATTAGCTCCTCATTCATTTGTCAAAAAAAAAAAAAAAAAA
The area CDS (X/Y chromosome: 2488635-2490719):
ATGGAGAATAAAAGCCTGGAGAGCTCCCAGACAGACCTGAAGCTGGTGGCCCACCCCCGCGCCAAGAGCAAGGTGTGGAAGTATTTCGGCTTCGACACCAACGCCGAGGGATGCATCCTGCAGTGGAAGAAAATCTACTGCCGCATCTGCATGGCCCAGATCGCCTACTCCGGAAACACCTCCAACCTGTCCTACCACCTGGAGAAGAACCACCCCGAGGAATTCTGCGAGTTCGTCAAGAGCAACACGGAGCAGATGCGTGAAGCCTTCGCCACCGCCTTCTCCAAGCTGAAGCCCGAGTCGTCCCAGCAGCCCGGGCAGGACGCGCTGGCCGTCAAGGCCGGCCACGGCTACGACAGCAAGAAGCAGCAGGAGCTGACGGCCGCCGTGCTGGGCCTCATCTGCGAGGGGCTGTACCCAGCCTCCATCGTGGACGAGCCCACCTTCAAGGTGCTGCTGAAGACGGCCGACCCCCGGTATGAGCTGCCCAGCCGGAAGTACATCTCTACCAAGGCCATCCCTGAGAAGTACGGGGCCGTCCGGGAGGTGATCCTGAAGGAGCTGGCCGAGGCCACCTGGTGTGGCATCTCCACCGACATGTGGAGGAGTGAGAATCAGAACCGCGCCTACGTCACGCTGGCCGCCCACTTCCTGGGCCTGGGCGCCCCCAACTGCCTGTCCATGGGCTCCCGCTGCCTGAAGACCTTCGAGGTGCCCGAAGAGAACACGGCGGAGACCATCACGCGAGTGCTCTATGAGGTCTTCATCGAGTGGGGCATCAGCGCCAAGGTCTTCGGGGCCACCACCAACTATGGCAAGGACATCGTGAAGGCGTGCTCCCTGCTGGACGTCGCAGTGCACATGCCCTGCCTGGGCCACACCTTCAATGCCGGCATCCAGCAGGCCTTCCAGCTCCCGAAGCTGGGGGCGCTGCTGTCGCGCTGCCGCAAACTGGTGGAGTACTTCCAGCAGTCTGCCGTGGCCATGTACATGCTCTATGAGAAGCAGAAGCAGCAGAACGTGGCCCACTGCATGCTGGTGAGCAACCGCGTCTCCTGGTGGGGGAGCACGCTGGCCATGCTGCAGCGCCTCAAGGAGCAGCAGTTCGTCATCGCCGGGGTCTTGGTGGAGGACAGCAACAACCACCACCTCATGCTGGAGGCCAGCGAGTGGGCCACCATCGAGGGGCTGGTGGAGCTCCTGCAGCCCTTCAAGCAGGTGGCCGAGATGCTGTCGGCCTCCAGGTACCCCACCATCAGCATGGTGAAGCCGCTGCTGCACATGCTCCTGAACACCACGCTCAACATCAAGGAGACCGACTCCAAGGAGCTCAGCATGGCCAAGGAGGTCATCGCCAAGGAGCTTTCCAAGACCTACCAGGAGACGCCCGAGATCGACATGTTTCTCAACGTGGCCACCTTCCTGGACCCCCGCTACAAGAGGCTGCCCTTCCTCTCCGCCTTCGAGCGGCAGCAGGTGGAGAATCGCGTGGTGGAAGAGGCCAAGGGCCTGCTGGACAAGGTCAAAGACGGCGGCTACCGGCCGGCTGAGGACAAGATCTTCCCGGTGCCCGAGGAGCCTCCCGTCAAGAAGCTCATGCGGACATCCACGCCGCCGCCCGCCAGCGTCATCAACAACATGCTGGCCGAGATCTTCTGCCAGACAGGCGGCGTGGAGGACCAGGAAGAGTGGCATGCCCAGGTGGTGGAGGAGCTGAGCAACTTCAAGTCCCAGAAGGTGCTTGGCCTCAACGAAGACCCCCTCAAGTGGTGGTCAGACCGCCTGGCCCTCTTCCCCCTGCTGCCCAAGGTGCTGCAGAAGTACTGGTGCGTGACGGCCACGCGCGTCGCCCCTGAGCGTCTCTTCGGATCCGCCGCCAACGTGGTCAGCGCCAAGAGGAACCGGCTGGCTCCCGCGCACGTGGACGAGCAGGTGTTTCTGTATGAGAACGCCCGGAGTGGGGCAGAGGCGGAACCCGAGGACCAGGACGAGGGGGAGTGGGGCCTGGACCAGGAGCAGGTGTTCTCCTTGGGGGATGGCGTCAGCGGCGGTTTCTTTGGCATTAGGGACAGCAGCTTCCTGTAGATGGAGAATAAAAGCCTGGAGAGCTCCCAGACAGACCTGAAGCTGGTGGCCCACCCCCGCGCCAAGAGCAAGGTGTGGAAGTATTTCGGCTTCGACACCAACGCCGAGGGATGCATCCTGCAGTGGAAGAAAATCTACTGCCGCATCTGCATGGCCCAGATCGCCTACTCCGGAAACACCTCCAACCTGTCCTACCACCTGGAGAAGAACCACCCCGAGGAATTCTGCGAGTTCGTCAAGAGCAACACGGAGCAGATGCGTGAAGCCTTCGCCACCGCCTTCTCCAAGCTGAAGCCCGAGTCGTCCCAGCAGCCCGGGCAGGACGCGCTGGCCGTCAAGGCCGGCCACGGCTACGACAGCAAGAAGCAGCAGGAGCTGACGGCCGCCGTGCTGGGCCTCATCTGCGAGGGGCTGTACCCAGCCTCCATCGTGGACGAGCCCACCTTCAAGGTGCTGCTGAAGACGGCCGACCCCCGGTATGAGCTGCCCAGCCGGAAGTACATCTCTACCAAGGCCATCCCTGAGAAGTACGGGGCCGTCCGGGAGGTGATCCTGAAGGAGCTGGCCGAGGCCACCTGGTGTGGCATCTCCACCGACATGTGGAGGAGTGAGAATCAGAACCGCGCCTACGTCACGCTGGCCGCCCACTTCCTGGGCCTGGGCGCCCCCAACTGCCTGTCCATGGGCTCCCGCTGCCTGAAGACCTTCGAGGTGCCCGAAGAGAACACGGCGGAGACCATCACGCGAGTGCTCTATGAGGTCTTCATCGAGTGGGGCATCAGCGCCAAGGTCTTCGGGGCCACCACCAACTATGGCAAGGACATCGTGAAGGCGTGCTCCCTGCTGGACGTCGCAGTGCACATGCCCTGCCTGGGCCACACCTTCAATGCCGGCATCCAGCAGGCCTTCCAGCTCCCGAAGCTGGGGGCGCTGCTGTCGCGCTGCCGCAAACTGGTGGAGTACTTCCAGCAGTCTGCCGTGGCCATGTACATGCTCTATGAGAAGCAGAAGCAGCAGAACGTGGCCCACTGCATGCTGGTGAGCAACCGCGTCTCCTGGTGGGGGAGCACGCTGGCCATGCTGCAGCGCCTCAAGGAGCAGCAGTTCGTCATCGCCGGGGTCTTGGTGGAGGACAGCAACAACCACCACCTCATGCTGGAGGCCAGCGAGTGGGCCACCATCGAGGGGCTGGTGGAGCTCCTGCAGCCCTTCAAGCAGGTGGCCGAGATGCTGTCGGCCTCCAGGTACCCCACCATCAGCATGGTGAAGCCGCTGCTGCACATGCTCCTGAACACCACGCTCAACATCAAGGAGACCGACTCCAAGGAGCTCAGCATGGCCAAGGAGGTCATCGCCAAGGAGCTTTCCAAGACCTACCAGGAGACGCCCGAGATCGACATGTTTCTCAACGTGGCCACCTTCCTGGACCCCCGCTACAAGAGGCTGCCCTTCCTCTCCGCCTTCGAGCGGCAGCAGGTGGAGAATCGCGTGGTGGAAGAGGCCAAGGGCCTGCTGGACAAGGTCAAAGACGGCGGCTACCGGCCGGCTGAGGACAAGATCTTCCCGGTGCCCGAGGAGCCTCCCGTCAAGAAGCTCATGCGGACATCCACGCCGCCGCCCGCCAGCGTCATCAACAACATGCTGGCCGAGATCTTCTGCCAGACAGGCGGCGTGGAGGACCAGGAAGAGTGGCATGCCCAGGTGGTGGAGGAGCTGAGCAACTTCAAGTCCCAGAAGGTGCTTGGCCTCAACGAAGACCCCCTCAAGTGGTGGTCAGACCGCCTGGCCCTCTTCCCCCTGCTGCCCAAGGTGCTGCAGAAGTACTGGTGCGTGACGGCCACGCGCGTCGCCCCTGAGCGTCTCTTCGGATCCGCCGCCAACGTGGTCAGCGCCAAGAGGAACCGGCTGGCTCCCGCGCACGTGGACGAGCAGGTGTTTCTGTATGAGAACGCCCGGAGTGGGGCAGAGGCGGAACCCGAGGACCAGGACGAGGGGGAGTGGGGCCTGGACCAGGAGCAGGTGTTCTCCTTGGGGGATGGCGTCAGCGGCGGTTTCTTTGGCATTAGGGACAGCAGCTTCCTGTAG
(2) according to siRNA sequence design principle, for the code area of people's ZBED1 gene, design 2 can specificity strike and subtractThe target sequence of people's ZBED1 gene；
| 5’——3’ |
| RNAi-1-F | GGAAGUACAUCUCUACCAA |
| RNAi-1-R | CCUUCAUGUAGAGAUGGUUdTdT |
| RNAi-2-F | GCAAACUGGUGGAGUACUU |
| RNAi-2-R | CGUUUGACCACCUCAUGAAdTdT |
(3) chemical synthesis target sequence；
3. siRNA inhibits the method for efficiency in verifying 2, it is characterised in that directly detect ZBED1 egg using WesternblotWhite expression, internal reference albumen are GAPDH；
As shown in Fig. 2, verifying siRNA to the inhibition situation of ZEBD1 in A549 lung carcinoma cell, from Fig. 2 for WesternblotAs can be seen that 2 siRNA that the present invention designs can make ZBED1 egg in A549 lung carcinoma cell compared with negative control group siRNAWhite expression reduces, and illustrates that 2 target sequences of the invention designed can obviously inhibit the expression of people's ZBED1 gene, can be used for subsequent grindStudy carefully.
4. detecting influence and the specific method of ZBED1 gene pairs lung carcinoma cell biological function, comprising:
(1) cell-proliferation activity detection (MTS method): by 103In 96 orifice plates, every hole adds a good cell inoculation of growth conditionsEnter 150ul culture medium, is placed in 5%CO2After cultivating 1 day in 37 DEG C of incubators, every hole adds MTS solution (5mg/ml) 30ul, continuesIt is incubated for 2 hours；Selection 490nm wavelength measures the absorbance in each hole, continuous detection 4 days；
As shown in figure 3, for the variation for interfering proliferation of lung cancer cells ability after people ZBED1 gene with siRNA.It can be with from Fig. 3Find out, compared with negative control group cell, after interfering ZBED1 gene, A549 proliferation of lung cancer cells ability obviously slows down, and illustrates thisTwo siRNA of design are invented, after inhibiting ZBED1 gene, can inhibit the proliferative capacity of lung carcinoma cell.
(2) the 2 × 10 of logarithmic growth phase will fluidic cell cycle detection: be in6A cell is fixed in 70% ethyl alcohol,Rinsing, is collected by centrifugation cell, 0.2M citric acid/sodium citrate high osmotic treatment 30 minutes, 1 × PBS washes 2 times, 0.1%Triton(PBS preparation) is washed 1 time, then with 500 μ g/mlPI and 0.1%RNAaseA, and room temperature is protected from light incubation 30 minutes, flow cytomeryAnalysis.
As shown in figure 4, for the variation in lung carcinoma cell period after interference people ZBED1 gene.From fig. 4, it can be seen that with feminine genderCellular control unit compares, and after interfering ZBED1 gene, A549 lung carcinoma cell is in the increase of G1 phase cell proportion, illustrates that the present invention is setTwo siRNA of meter can make the cell-cycle arrest of lung carcinoma cell in the G1 phase after inhibiting ZEBD1 gene.
It can be seen that the siRNA for capableing of specificity inhibition ZBED1 gene is inhibiting proliferation of lung cancer cells and preparation treatment to swellThere is good application in terms of tumor medicine.
Embodiment described above, the description thereof is more specific and detailed, and but it cannot be understood as to the invention patentThe limitation of range.It should be pointed out that for those of ordinary skill in the art, in the premise for not departing from present inventive conceptUnder, various modifications and improvements can be made, and these are all within the scope of protection of the present invention.Therefore, the protection of the invention patentRange should be determined by the appended claims.