阅读说明：本技术 一种yap抑制剂及其筛选方法与应用 (YAP inhibitor and screening method and application thereof ) 是由 张还添 查振刚 桂涛 黎贞燕 于 2019-10-14 设计创作，主要内容包括：本发明公开了一种YAP抑制剂的筛选方法,该方法利用各带一半荧光基团的TAZ与YAP重组质粒转染细胞,构建得到异源二聚体为筛选剂从而可以通过荧光强度来筛选YAP的抑制剂。筛选得到的YAP抑制剂将有助于推动相关抗肿瘤药物的制备,提高骨及软骨肉瘤相关药物的疗效,并为新药物研发提供线索。(The invention discloses a screening method of a YAP inhibitor, which utilizes TAZ and YAP recombinant plasmids with half of fluorescent groups to transfect cells, constructs heterodimers as a screening agent and screens the YAP inhibitor through fluorescence intensity. The screened YAP inhibitor is helpful to promote the preparation of related antitumor drugs, improve the curative effect of bone and chondrosarcoma related drugs, and provide clues for the research and development of new drugs.)

1. A method for screening YAP inhibitors, comprising:

designing a primer: designing a primer pair for amplifying the YAP gene and a primer pair for amplifying the TAZ gene;

preparing a recombinant plasmid: constructing a recombinant plasmid for expressing YAP genes and a recombinant plasmid for expressing TAZ genes; wherein, the recombinant plasmid for expressing YAP gene and the recombinant plasmid for expressing TAZ gene have half of fluorescent groups respectively;

fusing: transfecting a recombinant plasmid for expressing YAP genes and a recombinant plasmid for expressing TAZ genes into cells through lipo2000, and carrying out induced expression to obtain a heterodimer;

constructing a screening model: adding the inhibitor to be screened into the culture medium of the cells expressing fluorescence, treating for 24-48h, and if the fluorescence intensity is weakened, the inhibitor is an effective inhibitor of YAP.

2. The screening method of claim 1, wherein in the step of designing primers, the upstream primer of the TAZ is shown as SEQ ID No.1 and the downstream primer of the TAZ is shown as SEQ ID No. 2.

3. The screening method of claim 1, wherein in the step of designing primers, the YAP upstream primer is shown as SEQ ID No.3 and the YAP downstream primer is shown as SEQ ID No. 4.

4. The screening method according to claim 1, wherein in the step of preparing recombinant plasmids, the vector plasmids for expressing the YAP and TAZ genes are each a bimolecular fluorescent complementary vector plasmid.

5. The screening method of claim 1, wherein in the step of preparing a recombinant plasmid, the vector plasmid expressing YAP gene is pCMV-Myc-VN 155-Linker-MCS.

6. The screening method according to claim 1, wherein in the step of preparing a recombinant plasmid, the vector plasmid for expressing the TAZ gene is pCMV-HA-VC 155-Linker-MCS.

7. The screening method according to claim 1, wherein in the step of preparing the recombinant plasmid, human TAZ and YAP genes are amplified using human 293T cell cDNA as a template.

8. An inhibitor of YAP obtained by the screening method of any one of claims 1 to 7.

9. The use of a YAP inhibitor as defined in claim 8 in the preparation of a bone tumor medicament.

Technical Field

The invention relates to the technical field of medicines, in particular to a screening method and application of a YAP inhibitor.

Background

Osteosarcoma is a common primary malignant tumor for children and adolescents, has extremely poor clinical prognosis, seriously threatens the life of a patient, and brings heavy economic and mental burden to families and society.

At present, the molecular mechanism of osteosarcoma pathogenesis is still unclear, mainly comprising:

1) abnormalities in osteoblast development-related signaling pathways such as Hedghog, RUNX2, Hippo/YAP, BMP, Wnt/β -catenin, PKC, etc.;

2) abnormalities in metastasis associated signaling pathways such as Notch, Fas/FasL, Ezrin, CXCR4, VEGF, MMP-2, and MMP-9, and the like.

3) Mutation or inactivation of a key gene. Aiming at the current pathogenesis of osteosarcoma, comprehensive treatment such as early surgical excision combined with new adjuvant chemotherapy is mainly adopted at present.

However, according to the current treatment scheme, the 5-year survival rate of patients with recurrent osteosarcoma and metastatic osteosarcoma is not significantly improved, and the use of clinical high-dose chemotherapeutic drugs such as methotrexate, adriamycin and cisplatin has great side effects.

Chondrosarcoma (Chondrosarcoma) originates from cartilage connective tissue or cartilage, and the incidence rate of the Chondrosarcoma is second to that of osteosarcoma in malignant bone tumor, and accounts for about 10-20% of bone malignant tumor. Chondrosarcoma is extremely insensitive to traditional chemotherapy and radiation therapy, and surgical resection remains the primary treatment for current therapy. However, some patients still relapse after surgical resection due to the lack of effective adjuvant therapy. In addition, the clinical prognosis of chondrosarcoma is very poor, seriously threatens the life of patients and brings heavy economic and mental burden to families and society.

The Hippo-YAP pathway is a signal pathway which is discovered in recent years and has the functions of regulating organ volume and maintaining cell proliferation and apoptosis balance, and is closely related to uncontrolled proliferation of tumor cells. The major function of the Hippo-YAP pathway in mammals is to inhibit the activity of the transcriptional regulators YAP and TAZ to negatively regulate tumor progression, and thus the Hippo pathway is also considered to be a cancer suppressor pathway. Specifically, the core members of this pathway include the serine/threonine kinases MST1, MST2, LATS1 and LATS2, the scaffold proteins SAV1 (binding to MST1 and MST 2), MOB1 (binding to LATS1 and LATS 2), the transcriptional co-activator YAP, and the transcription factor TEAD, which comprises a TEA binding domain. Briefly, when the Hippo pathway is activated, MST1/2 kinase phosphorylation activates LATS1/2, activated LATS1/2 in turn phosphorylates YAP, phosphorylated YAP is inactivated and subsequently nucleated, and YAP within the cytoplasm is degraded by proteasomes.

Research has shown that the Hippo pathway is involved in the progression of various tumors, such as lung, colon, ovarian, prostate, liver, and the like. However, in human tumors, mutations in genes of the Hippo pathway occur less frequently. Based on this, it was concluded that the deregulation of the Hippo pathway in human tumors is due to cross-talk between other aberrantly expressed proteins or signaling pathways within the tumor cell and the Hippo pathway, in addition to mutations derived from key proteins of the Hippo pathway itself. In addition, the role of the Hippo signaling pathway in tumors is closely related to the nuclear translocation of YAP/TAZ. Recently, YAP was found to be highly expressed in various tumors, which was associated with high pathological grade, late TNM stage, lymph node metastasis, etc., and there was a phenomenon of nuclear localization.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a screening method of YAP inhibitor based on fluorescence detection. The method can rapidly and effectively screen out the YAP inhibitor through the change of the intensity of a fluorescence signal by utilizing the heterodimer induced and expressed by the YAP gene expression recombinant plasmid and the TAZ gene expression recombinant plasmid with half of fluorescent groups.

Another object of the present invention is to provide a YAP inhibitor obtained by the screening method.

The invention also aims to provide the application of the YAP inhibitor in preparing bone tumor medicaments.

One of the purposes of the invention is realized by adopting the following technical scheme:

a method of screening for a YAP inhibitor comprising:

designing a primer: designing a primer pair for amplifying the YAP gene and a primer pair for amplifying the TAZ gene;

preparing a recombinant plasmid: constructing a recombinant plasmid for expressing YAP genes and a recombinant plasmid for expressing TAZ genes; wherein, the recombinant plasmid for expressing YAP gene and the recombinant plasmid for expressing TAZ gene have half of fluorescent groups respectively;

fusing: transfecting a recombinant plasmid for expressing YAP genes and a recombinant plasmid for expressing TAZ genes into cells through 6 mu L lipo2000, and carrying out induced expression to obtain a heterodimer;

constructing a screening model: adding the inhibitor to be screened into the culture medium of the cells expressing fluorescence, treating for 24-48h, and if the fluorescence intensity is weakened, the inhibitor is an effective inhibitor of YAP.

Specifically, the heterodimer was obtained by transfecting 1. mu.g of a recombinant plasmid expressing YAP gene and 1. mu.g of a recombinant plasmid expressing TAZ gene into cells through 6. mu.L of lipo2000 and inducing expression.

Further, in the step of designing the primer, the upstream primer of the TAZ is shown as SEQ ID No.1, and the downstream primer of the TAZ is shown as SEQ ID No. 2.

Further, in the step of designing the primers, the upstream primer of the YAP is shown as SEQ ID No.3, and the downstream primer of the YAP is shown as SEQ ID No. 4.

Furthermore, in the step of preparing recombinant plasmids, the carrier plasmids for expressing YAP and TAZ genes are bimolecular fluorescence complementary carrier plasmids.

Furthermore, in the step of preparing the recombinant plasmid, the vector plasmid for expressing YAP gene is pCMV-Myc-VN 155-Linker-MCS.

Furthermore, in the step of preparing the recombinant plasmid, the carrier plasmid for expressing the TAZ gene is pCMV-HA-VC 155-Linker-MCS.

Furthermore, in the step of preparing recombinant plasmids, human TAZ and YAP genes are amplified by taking human 293T cell cDNA as a template.

The second purpose of the invention is realized by adopting the following technical scheme:

YAP inhibitors obtained by the screening method described above.

The third purpose of the invention is realized by adopting the following technical scheme:

the YAP inhibitor is applied to preparing bone tumor medicaments.

Compared with the prior art, the invention has the beneficial effects that:

the invention utilizes TAZ and YAP recombinant plasmids constructed by a fluorescence complementary vector to transfect cells, constructs to obtain heterodimer as a screening agent, and detects the inhibition degree of an inhibitor on the expression of YAP genes and induced cell cycle retardation through the change of fluorescence intensity; the screened YAP inhibitor has larger application potential in preparing bone tumor medicaments.

Drawings

FIG. 1 is a graph of the fluorescence localization of YAP in osteosarcoma tissues of varying degrees of differentiation;

FIG. 2 is a graph of the fluorescence localization of YAP in chondrosarcoma tissues of varying degrees of differentiation;

FIG. 3 is a graph of the fluorescence localization of YAP in chondrocyte lines and chondrosarcoma cells;

FIG. 4 is a graph of the effect of siRNA knockdown YAP on chondrosarcoma cells SW 1353;

FIG. 5 is a graph of the effect of shRNA knockdown of YAP on chondrosarcoma cells SW 1353;

FIG. 6 is a graph showing the effect of suppressing the growth of transplanted tumors in nude mice by knocking-down YAP;

FIG. 7 is a map of a recombinant plasmid expressing the TAZ gene;

FIG. 8 is a map of a recombinant plasmid expressing YAP gene;

FIG. 9 is a graph of the effect of JQ1 on YAP expression in SW 1353 and Hs819.T cells;

FIG. 10 is a graph of the effect of 17-AAG on YAP expression in U2 OS and Saos-2 cells;

FIG. 11 is a graph of 17-AGG induced cell cycle arrest with U2 OS and Saos-2;

FIG. 12 is a graph showing the effect of 5-AZA on YAP expression in U2 OS and Saos-2 cells;

FIG. 13 is a graph of 5-AZA versus U2 OS and Saos-2 induced cell cycle arrest.

FIG. 14 is a schematic diagram showing fluorescence emission of a recombinant plasmid expressing TAZ gene and a recombinant plasmid expressing YAP gene fused to each other in SW 1353 cells.

Detailed Description

The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.

The following are specific examples of the present invention, and raw materials, equipments and the like used in the following examples can be obtained by purchasing them unless otherwise specified.

YAP gene was given by the central laboratory tumor group of the first hospital affiliated to the university of river-south, the CDS region sequence of human TAZ (NM-000116.5) was searched according to NCBI website, and the CDS region sequence was obtained by PCR amplification of human cDNA sequence; the pCMV-HA-VC155-Linker-MCS and the pCMV-Myc-VN155-Linker-MCS plasmids are presented by the professor Huchang lantern of the medical chemistry and molecular pharmacology department of the university of Puzhu, USA.

The chondrosarcoma tissue chip is commercially available.

The present invention provides a method for synthesizing a heterodimer of YAP and TAZ