CN103272221A - Applications of WSB-1 protein in preparing drugs for treating osteosarcoma - Google Patents
Applications of WSB-1 protein in preparing drugs for treating osteosarcoma Download PDFInfo
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- CN103272221A CN103272221A CN2013102127002A CN201310212700A CN103272221A CN 103272221 A CN103272221 A CN 103272221A CN 2013102127002 A CN2013102127002 A CN 2013102127002A CN 201310212700 A CN201310212700 A CN 201310212700A CN 103272221 A CN103272221 A CN 103272221A
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Abstract
The invention discloses applications of WSB-1 protein in preparing drugs for treating osteosarcoma or products for diagnosing osteosarcoma and predicting osteosarcoma metastasis. The amino acid sequence of the WSB-1 protein is shown as SEQ ID No:1. The drugs for treating osteosarcoma comprise double-stranded ribonucleic acid (RNA) capable of inhibiting expression of WSB-1 gene through RNA interference, or protein for inhibiting the activity of the WSB-1 protein, or a small-molecular compound for inhibiting the functions of the WSB-1 protein. The products for diagnosing osteosarcoma and predicting the osteosarcoma metastasis include products for diagnosing osteosarcoma and predicting the osteosarcoma metastasis through RT-PCR (reverse transcription-polymerase chain reaction), real-time quantitative PCR, immunodetection, in-situ hybridization or a gene chip. By applying the WSB-1 protein, the accuracy for diagnosing osteosarcoma and predicting the osteosarcoma metastasis can be improved, so that the osteosarcoma diagnosis is more accurate and faster. The WSB-1 protein provides a new therapeutic target and a new effective drug for preventing and treating the osteosarcoma.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to the application of a kind of WSB-1 albumen in preparation treatment osteosarcoma medicine.
Background technology
Osteosarcoma is one of modal pernicious skeletonization tumor, and epidemiological study finds that this disease sends out the teenager in year in 15-25 well, and male's sickness rate is higher than the women.The osteosarcoma predilection site is the metaphysis of long bone, this malignancy of tumor degree height, the prognosis extreme difference, 5 years survival rates of patient are lower than 20% after the simple amputation, another distinguishing feature of this tumor is that pulmonary shifts early in addition, make osteosarcoma when diagnosis clinical, wherein the small transfer of lung has taken place in major part.Therefore, efficient diagnosis and to prevent and treat osteosarcoma be emphasis and difficult point in the present bone tumor research.
People WSB-1 albumen is by WSB-1 gene (Genebank No.NM 015626.8) coding, is one of WD albumen subfamily member.People WSB-1 albumen and mice and chicken WSB-1 albumen height homology, the SOCS box structure domain of being held by several WD repetitives and C constitutes.The function of this albumen is not verified as yet fully at present, only knows that 2 type iodate first gland amino acids of this albumen degradable thyroid excitement take off the iodine enzyme; Idol has the prognosis of report WSB-1 albumen and neuroblastoma that certain relation is arranged aspect tumor, but still has nothing to do in the research report of WSB-1 albumen in osteosarcoma.
Summary of the invention
The purpose of this invention is to provide the application of a kind of WSB-1 albumen in the osteosarcomatous medicine of preparation treatment.The aminoacid sequence of this WSB-1 albumen is shown in SEQ ID No:1.The osteosarcomatous medicine of described treatment comprises: disturb the double stranded RNA that suppresses WSB-1 gene expression by RNA, or be used for suppressing the protein of WSB-1 protein active, or be used for suppressing the micromolecular compound of WSB-1 protein function.
Another object of the present invention provides the application of a kind of WSB-1 albumen in the product of preparation diagnosis osteosarcoma and the transfer of prediction osteosarcoma.The product that described diagnosis osteosarcoma and prediction osteosarcoma shift comprises: the product of using RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or gene chip diagnosis osteosarcoma and prediction osteosarcoma to shift.
The wherein said product that shifts with immune detection diagnosis osteosarcoma and prediction osteosarcoma comprises: the antibody of being combined with the WSB-1 protein-specific.
The described product that shifts with RT-PCR diagnosis osteosarcoma and prediction osteosarcoma comprises the primer of a pair of specific amplification WSB-1 gene: forward primer: 5 '-GATGGTTCATACTTTGCTTGGT-3 ' (SEQ ID No:2) at least; Reverse primer: 5 '-CCACTTCAGTATGATCTACCAAG-3 ' (SEQ ID No:3).
The described product that shifts with real-time quantitative PCR diagnosis osteosarcoma and prediction osteosarcoma comprises the primer of a pair of specific amplification WSB-1 gene: forward primer: 5 '-GAATATGGATAAATACACCATGATAC-3 ' (SEQ ID No:4) at least; Reverse primer: 5 '-TAATGCTCCATCAGGAGAAAAGTCA-3 ' (SEQ ID No:5).
The described product that shifts with in situ hybridization diagnosis osteosarcoma and prediction osteosarcoma comprises: with the probe of the nucleic acid array hybridizing of WSB-1 gene: 5 '-GAATTGATGAGGATTATCCAGTGCAAG-3 ' (SEQ ID No:6).
The described product that shifts with gene chip diagnosis osteosarcoma and prediction osteosarcoma comprises: with the probe of the nucleic acid array hybridizing of WSB-1 gene: 5 '-GCTCCAGATGGTTCATACTTTGCTTG-3 ' (SEQ ID No:7).
In the present invention, can use a series of methods known in the art to prepare at the special antibody of WSB-1 albumen.For example, people WSB-1 albumen or its antigen fragment with purification is injected in the animal body to produce polyclonal antibody.Equally, the cell of expressing human WSB-1 albumen or its antigen fragment also can be used for animal is caused immunity and produces antibody.Antibody prepared in accordance with the present invention also can be monoclonal antibody, and these monoclonal antibodies can prepare with hybridoma technology.Antibody of the present invention comprises the antibody that can prevent the WSB-1 function, also can be the antibody that does not influence people WSB-1 protein function.Each antibody-like can produce by the segment of people WSB-1 albumen or functional domain are caused immunity, and people WSB-1 protein product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the protein bound antibody of the WSB-1 of non-modified forms, can utilize the gene outcome that for example produces among the E. coli at prokaryotic cell to come immune animal and obtain.With the antibody that post translational modification form such as glycosylation or phosphorylation WSB-1 albumen or polypeptide are combined, can utilize the gene outcome that in eukaryotic cell such as yeast or insect cell, produces to come immune animal and obtain.
In the present invention, described probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivants.The length of described probe without limits, as long as can finish specific hybrid, be combined with purpose nucleotide sequence specificity, any length can.The length of described probe may be as little to 25,20,15 or 10 base length.Equally, the length of described probe can be grown to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different influences to hybridization efficiency, signal specificity, the length of described probe is 14 base pairs usually at least, the longlyest generally be no more than 30 base pairs, with the length of purpose nucleotide sequence complementation with 15-25 base pair the best.Described probe self complementary series is most preferably less than 4 base pairs, in order to avoid influence hybridization efficiency.
People WSB-1 albumen is by WSB-1 gene (Genebank No.NM 015626.8) coding, only knows that this albumen can take off the iodine enzyme as 2 type iodate first gland amino acids of albumen ubiquitin E3 ligase degraded thyroid excitement at present; But still have nothing to do in the research report of WSB-1 albumen in osteosarcoma.The present invention experimental results show that the expression of WSB-1 albumen in osteosarcoma tissue apparently higher than cancer beside organism, and closely related with the transfer of osteosarcoma lung and mortality rate; Express the external invasive ability that WSB-1 albumen can significantly increase osteosarcoma cell and in osteosarcoma cell, cross.Therefore, WSB-1 albumen can be used as the specificity marker protein of diagnosis and prediction transfer, the accuracy that the raising osteosarcoma is diagnosed and the prediction osteosarcoma shifts, and it is accurate more, quick that osteosarcoma is diagnosed.WSB-1 albumen of the present invention provides new treatment target and effective new drug for preventing and treating osteosarcoma.
Description of drawings
Fig. 1 is that the present invention passes through the expression figure of SABC method checking WSB-1 in human osteosarcoma tissue (A) and cancer beside organism (B).
Fig. 2 is that the present invention passes through WSB-1 protein level (A: tumor tissues, B: cancer beside organism) in the SABC method detection osteosarcoma.
Fig. 3 is the influence of adopting the plasmid transfection method to cross in osteosarcoma cell to express behind the WSB-1 albumen external osteosarcoma cell invasion and attack.
The specific embodiment
The present invention is further detailed explanation below in conjunction with drawings and Examples.Following examples only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1:
Choose osteosarcoma clinical sample 50 examples, fix 7 days with 10% formalin, prepare paraffin-embedded piece of tissue subsequently.It is 8 microns paraffin section that sample is prepared thickness, and adopts the SABC method to detect the expression of WSB-1 albumen (aminoacid sequence is shown in SEQ ID No:1), meanwhile chooses another section and carries out H﹠amp; E dyeing.At the expression of just putting microscopically observation WSB-1 albumen, and H﹠amp; The situation of E dyeing.Shown in Figure 1A, the expression of WSB-1 albumen is higher than nonneoplastic tissue in same the slice, thin piece in the sample cancerous tissue.
Choose 25 pairs of osteosarcoma clinical sample and cancer beside organisms, fix 7 days with 10% formalin, prepare paraffin-embedded piece of tissue subsequently.It is 8 microns paraffin section that sample is prepared thickness, and adopts the expression of SABC method detection WSB-1 albumen, at the expression of just putting microscopically observation WSB-1 albumen.As shown in Figure 1B, the expression of WSB-1 albumen is significantly higher than cancer beside organism in the cancerous tissue.
Embodiment 2:
Expression to WSB-1 albumen in the 25 pairs of osteosarcoma clinical samples in the previous embodiment 1 and the cancer beside organism is added up, and finds that strong positive is 12 examples in the cancerous tissue, and the positive is 8 examples, and the weak positive is 2 examples, and feminine gender is 3 examples (Fig. 2 A); And strong positive is 2 examples in cancer beside organism, and the positive is 5 examples, and the weak positive is 1 example, and feminine gender is 17 examples (Fig. 2 B).
Embodiment 3:
50 routine osteosarcoma in previous embodiment 1 and 2 are carried out retrospective investigation in 3 years, wherein have 28 examples that the detailed record of following up a case by regular visits to is arranged, so in this 28 routine sample, analyze the relation between WSB-1 protein expression and the osteosarcoma patient prognosis.Found that WSB-1 expresses more strong, osteosarcoma patient's the rate of transform is more high, and is accompanied by the decline (table 1) of survival rate.
Embodiment 4:
In go down to posterity the osteosarcoma cell KHOS that cultivates and U2OS, adopt slow virus method transfection WSB-1 gene (WSB-1 group) respectively, and blank (PCCL group) is set.Treat transfection after 72 hours, adopt the Transwell method, detect the external invasive ability of transfectional cell.Referring to Fig. 3, behind the expression WSB-1 gene, the external invasive ability of osteosarcoma cell KHOS and U2OS significantly increases excessively.
＜110〉Zhejiang University
＜120〉application of WSB-1 albumen in preparation treatment osteosarcoma medicine
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Claims (8)
1. the application of WSB-1 albumen in the osteosarcomatous medicine of preparation treatment, the aminoacid sequence of this WSB-1 albumen is shown in SEQ ID No:1.
2. the application of a kind of WSB-1 albumen according to claim 1 in the osteosarcomatous medicine of preparation treatment, it is characterized in that, the osteosarcomatous medicine of described treatment comprises: disturb the double stranded RNA that suppresses WSB-1 gene expression by RNA, or for the protein that suppresses the WSB-1 protein active, or for the micromolecular compound that suppresses the WSB-1 protein function.
3. the application of WSB-1 albumen in the product of preparation diagnosis osteosarcoma and the transfer of prediction osteosarcoma, the aminoacid sequence of this WSB-1 albumen is shown in SEQ ID No:1, it is characterized in that the product of the product that described diagnosis osteosarcoma and prediction osteosarcoma shift for shifting with RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or gene chip diagnosis osteosarcoma and prediction osteosarcoma.
4. the application of a kind of WSB-1 albumen according to claim 3 in the product of preparation diagnosis osteosarcoma and the transfer of prediction osteosarcoma, it is characterized in that the described product that shifts with immune detection diagnosis osteosarcoma and prediction osteosarcoma is the antibody of being combined with the WSB-1 protein-specific.
5. the application of a kind of WSB-1 albumen according to claim 3 in the product of preparation diagnosis osteosarcoma and the transfer of prediction osteosarcoma, it is characterized in that the described product that shifts with RT-PCR diagnosis osteosarcoma and prediction osteosarcoma comprises the primer of a pair of specific amplification WSB-1 gene at least: forward primer: 5 '-GATGGTTCATACTTTGCTTGGT-3 '; Reverse primer: 5 '-CCACTTCAGTATGATCTACCAAG-3 '.
6. the application of a kind of WSB-1 albumen according to claim 3 in the product of preparation diagnosis osteosarcoma and the transfer of prediction osteosarcoma, it is characterized in that the described product that shifts with real-time quantitative PCR diagnosis osteosarcoma and prediction osteosarcoma comprises the primer of a pair of specific amplification WSB-1 gene at least: forward primer: 5 '-GAATATGGATAAATACACCATGATAC-3 '; Reverse primer: 5 '-TAATGCTCCATCAGGAGAAAAGTCA-3 '.
7. the application of a kind of WSB-1 albumen according to claim 3 in the product of preparation diagnosis osteosarcoma and the transfer of prediction osteosarcoma, it is characterized in that the described product that shifts with in situ hybridization diagnosis osteosarcoma and prediction osteosarcoma comprises: with the probe of the nucleic acid array hybridizing of WSB-1 gene: 5 '-GAATTGATGAGGATTATCCAGTGCAAG-3 '.
8. the application of a kind of WSB-1 albumen according to claim 3 in the product of preparation diagnosis osteosarcoma and the transfer of prediction osteosarcoma, it is characterized in that the described product that shifts with gene chip diagnosis osteosarcoma and prediction osteosarcoma comprises: with the probe of the nucleic acid array hybridizing of WSB-1 gene: 5 '-GCTCCAGATGGTTCATACTTTGCTTG-3 '.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10093930B2 (en) | 2015-05-06 | 2018-10-09 | Mayo Foundation For Medical Education And Research | Targeting WSB1 and pVHL to treat cancer |
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Non-Patent Citations (3)
| Title |
|---|
| CENDRINE ARCHANGE ET AL.: "The WSB1 Gene Is Involved in Pancreatic Cancer Progression", 《PLOS ONE》, vol. 3, no. 6, 25 June 2008 (2008-06-25) * |
| DONG WOOK CHOI ET AL.: "Ubiquitination and Degradation of Homeodomain-interacting Protein Kinase 2 by WD40 Repeat/SOCS Box Protein WSB-1", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 283, no. 8, 2 February 2008 (2008-02-02), pages 4682 - 4689 * |
| QING-RONG CHEN ET AL.: "Increased WSB1 Copy Number Correlates with its Over-Expression Which Associates with Increased Survival in Neuroblastoma", 《GENES, CHROMOSOMES & CANCER》, no. 45, 31 December 2006 (2006-12-31), pages 856 - 862 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10093930B2 (en) | 2015-05-06 | 2018-10-09 | Mayo Foundation For Medical Education And Research | Targeting WSB1 and pVHL to treat cancer |
| US10526606B2 (en) | 2015-05-06 | 2020-01-07 | Mayo Foundation For Medical Education And Research | Targeting WSB1 and pVHL to treat cancer |
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