NL2028912A

NL2028912A - Specific peptide targeting oncoprotein boris and use and encoding gene thereof

Info

Publication number: NL2028912A
Application number: NL2028912A
Authority: NL
Inventors: Tu Linglan; Li Chao; Xu Hao; Fang Mengdie; Ren Juan; Zheng Xiaoliang; Zhang Yanmei; Liu Chen
Original assignee: Hangzhou medical college
Priority date: 2021-01-07
Filing date: 2021-08-03
Publication date: 2022-07-22
Also published as: CN112679583A; CN112679583B; NL2028912B1

Abstract

The present disclosure provides a specific peptide targeting the oncoprotein BORIS and use and encoding gene thereof. The disclosure relates to the field of biomedical technology. An amino acid sequence of the specific peptide is shown as SEQ ID No.1. The peptide suppresses a tumor by inhibiting the function of BORIS. The peptide may suppress the cell growth of non-small cell lung cancer and prostate cancer, and inhibit the subcutaneous tumor progression of non-small cell lung cancer.

Description

SPECIFIC PEPTIDE TARGETING ONCOPROTEIN BORIS AND USE AND

ENCODING GENE THEREOF Field of the invention The present disclosure relates to the field of biomedical technology, and in particular to a specific peptide targeting the oncoprotein BORIS and use and encoding gene thereof. Background of the invention Brother of Regulator of Imprinted Sites (BORIS) has been frequently reported to associate with malignant carcinoma or drug resistance. Almost all types of cancer express BORIS including lung cancer, breast cancer, prostate cancer, and leukemia et a/ [1]. The pathological information of The Cancer Genome Atlas (TCGA) collected by THE HUMAN PROTEN ATLAS database showed that elevated BORIS expression correlated with short life spans to various degrees according to the types of cancer. BORIS is expressed highly only in testis but expressed very low or undetermined in other tissues in healthy people [6]. Female mice developed and reproduced normally even BORIS gene was knocked out. Though the fertility of BORIS knockout male mice was weak, the offsprings developed normally. Increased BORIS expression in carcinoma was due to the demethylation of BORIS promoter or copy number alteration of BORIS gene [3, 7]. The specific expression in carcinoma but not in adjacent normal tissue is an attractive advantage of BORIS to be applied in clinical diagnosis or therapy [1, 5]. BORIS expression was up-regulated along with the development of the resistance after ALK inhibition in neuroblastoma, suggesting that BORIS played an important role after the development of resistance in neuroblastoma. Therefore, BORIS may have a prospect in diagnosis or therapy in cases of drug resistance [8]. Cytotoxic T cell (CTL) immunotherapy targeting on BORIS resulted in significant inhibition of cervical cancer progression and lung cancer cell proliferation [9-11]. Above evidences supports that BORIS is a potential therapeutic target for cancers.

The expression of BORIS is necessary for malignant proliferation of colorectal cancer cells, lung cancer cells, neuroblastoma ef a? [5, 8, 12]. BORIS supported the genome stability of cancer cells [12]. In neuroblastoma, BORIS increased along with the switch of cellular dependence from MYCN to BORIS following the resistance of ALK inhibition [8]. BORIS located in both cytoplasm and nucleus in colorectal cancer cells [5]. The distribution of BORIS in cancer cells depends on cell types. The dual localization of BORIS in nucleus and cytoplasm differs from its paralogue CTCF (CCCTC binding factor, locates only in nucleus) [13]. The cytoplasmic BORIS suppressed apoptosis suggested that cytoplasmic BORIS play totally different roles from CTCF [5]. Though immunotherapy strategies targeting on BORIS acquired curative effects on cervical cancer or breast cancer in animal experiments [9, 11, 14-16], BORIS has not been found on cell membrane. Immunotherapy targeting on intracellular BORIS may not be the best strategy. Due to lack of understanding of BORIS structure, design of inhibitors on BORIS is infeasible.

Summary of the invention The objective of the disclosure is to provide a specific peptide targeting the oncoprotein BORI and use and encoding gene thereof. The specific peptide provided in the disclosure may inhibit the function of BORIS.

The present disclosure provides the following technical solutions to realize the above objectives: The present disclosure provides a specific peptide targeting the oncoprotein BORIS and an amino acid sequence of the peptide is shown as SEQ ID No.1.

The present disclosure provides a gene encoding the specific peptide in the above technical solution and a nucleotide sequence of the peptide is shown as SEQ ID No.2.

The present disclosure provides the use of the specific peptide in the above technical solution for the preparation of a drug for tumor therapy.

The present disclosure provides the use of the specific peptide in the above technical solution for the preparation of a drug for inhibiting BORIS function and inducing cancer cell apoptosis.

In some embodiments, the tumors may include non-small cell lung cancer and/or prostate cancer.

In some embodiments, the drugs may include the specific peptide and acceptable excipients in pharmacy.

The present disclosure provides a specific peptide targeting the oncoprotein BORIS, use and encoding gene thereof. An amino acid sequence of the specific peptide is shown as SEQ ID No.1. The specific peptide provided in this disclosure regulates the expressions of ERCC! and cMYC by inhibiting BORIS, inducing genome unstability and promoting cancer cell apoptosis for tumor therapy.

Examples of the present disclosure showed that the specific peptide inhibited non-small cell lung cancer and prostate cancer and inhibited subcutaneous tumor progression of non-small cell lung cancer. Brief description of the drawings FIG. 1 is a selection procedure of SEQ ID No.l, in which, (A) is expressions and purifications of BORIS Ni.2ss and BORIS del N;.25s antigens for the selection of SEQ ID No.1, (B) is a selection procedure of SEQ ID No.1, and (C) is a statistic of the eluted phage titers after each rounds of enrichment.

FIG. 2 is an examination of the binding affinity between SEQ ID No.l and BORIS protein, in which (A) is an examination of the binding affinity between BORIS protein and SEQ ID No.1, and (B) is an examination of the binding affinity between SEQ ID No.1 and BORIS protein reversely.

FIG. 3 is an examination of the interaction between SEQ ID No.1 and BORIS protein which was expressed in mammalian cells, in which (A) is an examination of the binding affinity between SEQ ID No.1 and BORIS protein which was expressed in mammalian cells, (B) is an examination of the interaction between SEQ ID No.1 and BORIS protein which was expressed in mammalian cells. (C) is the detection of the cell membrane penetration efficiency of SEQ ID No.1 and (D) is an examination of the specificity of SEQ ID No.1 for the detection of BORIS compared with the commercial monoclone antibody of BORIS.

FIG. 4 is an analysis of the binding site of SEQ ID No.1 on BORIS, in which (A) is an analysis of the binding site of SEQ ID No.1 on BORIS, and (B) is test of the interaction between SEQ ID No.1 and BAT3 which was reported to interact with BORIS.

FIG. 5 is an analysis of the inhibition effect of SEQ ID No.1 on cancer cells, in which (A) is an inhibition of cell proliferation of non-small cell lung cancer cell H1299 by SEQ ID No.1, (B) is an analysis of half maximal inhibitory concentration (IC50) of SEQ ID No.1 on H1299 cells, and (C) shows that SEQ ID No.1 did not affect the cell proliferation of normal cells which did not express BORIS.

FIG. 6 is SEQ ID No.l induced DNA damage and apoptosis of cancer cells, in which (A) is SEQ ID No.l induced DNA damage of cancer cells, (B) is SEQ ID No.1 induced apoptosis of cancer cells, and (C) shows that SEQ ID No.1 suppressed ZRCC'! and cMYC expressions.

FIG. 7 shows that SEQ ID No.l inhibited the progression of subcutaneous tumor of non-small cell lung cancer, in which (A) is the experiment procedure, (B) shows that SEQ ID No. 1 inhibited the progression of subcutaneous tumor of non-small cell lung cancer, and (C) is an analysis of the weight of subcutaneous tumors.

FIG. 8 is SEQ ID No.l induced DNA damage of non-small cell lung cancer subcutaneous tumor, in which (A) shows that SEQ ID No.l induced DNA damage of subcutaneous tumor and (B) shows that SEQ ID No.1 did not induce liver and kidney toxicity. Detailed description of the embodiments The present disclosure provides a specific peptide targeting the oncoprotein BORIS. The amino acid sequence of peptide is shown as SEQ ID No.1 and is as follows: VHWDFRQWWOPSGGRKKRRORRRG.

The present disclosure also provides a gene encoding the specific peptide in the above technical solution. The nucleotide sequence of the gene is shown as SEQ ID No.2 and is as follows:

GTGCATTGGGATTTTCGGCAGTGGTGGCAGCCTTCTGGTGGCCGTAAAAAGC GCCGACAACGGAGAAGGGGA.

The present disclosure further provides the use of the specific peptide in the above technical solution in the preparation of a drug for inhibiting BORIS and inducing cancer cell apoptosis. The drug may preferably include the specific peptide and acceptable excipients in clinic. The specific peptide was the only active component in drugs in the present disclosure. The dosage form and the preparation method of the drugs were not particularly limited in the present disclosure. Those skilled in the art may routinely choose medically acceptable dosage forms and preparation methods according to the specific peptide.

The present disclosure further provides the use of the specific peptide in the above technical solution in preparation of drugs for tumor therapy. The tumors may preferably include 5 non-small cell lung cancer and/or prostate cancer in the present disclosure. The drugs may preferably include the specific peptide and acceptable excipients in clinic in the present disclosure. The specific peptide is the only active component in drugs in the present disclosure. The dosage form and the preparation method of the drug are not particularly limited in the present disclosure. Those skilled in the art may routinely choose medically acceptable dosage forms and preparation methods according to the specific peptide. In the present disclosure, the specific peptide regulates the expressions of ERCC'! and cMYC by inhibiting the oncoprotein BORIS, and induces cancer cell apoptosis for tumor therapy.

In the present disclosure, the peptide concentrations used for cell treatment are 5-100 pM.

In animal experiments, the peptide is dissolved in PBS for treatment by dosage of 16~25mg/kg.

In order to further illustrate the present disclosure , the following describes the disclosure in detail with examples below, but they should not be understood as limiting the protection scope of the present disclosure.

Example 1 Selection and characterization of BORIS binding peptide (SEQ ID No.3 VHWDFRQWWQPS) In the present example, the N-terminal 1-258 AA of BORIS specific section was used to screen the specific peptide targeting BORIS. Experimental method: BORIS N-terminal section between 1-258AA (BORIS-N:258) was constructed into the plasmid of Pfnók (Promega Corporation) and tagged with tag peptide (HQHQHQ) at N-terminal for expression and screen of BORIS specific protein section that target the peptide and subsequent protein purification. Moreover BORIS N-terminal section deletion (BORIS-del N1.258) was also constructed in Pfnók plasmid for the expression and purification of BORIS unspecific section, aiming to remove peptides which did not bind BORIS. Both the above plasmids containing BORIS del Ni-2ss and BORIS-N:.258 were expressed in Single step Competent cells protein expression system (KRX) and were purified by Ni-NTA column chromatography(FIG. 1A). BORIS-del Ni.2ss was firstly used for pre-clear of phages that does not bind to the antigen of BORIS-N:-258 in the Ph.D. ™-12

Phage Display Peptide Library (purchased from New England Biolabs). Then BORIS-N:-258 was used in further enrichment of BORIS-N:-258 binding phage clones (FIG. 1B). After two rounds of enrichment until the eluted phage titers did not change (FIG. 1C), sixty phage clones were selected randomly for sequencing of the displayed peptides. The peptide of VHWDFRQWWOQPS (the precursor of SEQ ID No.1) showed the highest frequency in the sequenced clones.

The amino acid sequence of BORIS-N:-258 (SEQ ID No.6) is shown as follows :

MAATEISVLSEQFTKIKELELMPEKGLKEEEKDGVCREKDHRSPSELEAERTSGA FODSVLEEEVELVLAPSEESEKYILTLQTVHFTSEAVELQDMSLLSIQQQEGVQVVVQQP GPGLLWLEEGPRQSLQQCVAISIQQELYSPQEMEVLQFHALEENVMVASEDSKLAVSLA ETTGLIKLEEEQEKNQLLAERTKEQLFFVETMSGDERSDEIVLTVSNSNVEEQEDQPTAG QADAEKAKSTKNQRKTKGAKGTFH.

The peptide was synthesized by chemical synthesis and measured its physical interaction affinity with BORIS-Ni.2ss by Bio-Layer Interferometry(BLI technology) as following : BORIS-N:.258 protein was conjugated with biotin and loaded on SSA sensor. The synthesized peptide VHWDFRQWWQPS was diluted with the PBS buffer with 0.02 % Tween 20 to be 29.5 UM tol594 uM series concentrations and used to detect the interaction affinity with BORIS-Niass by ForteBio OctetRed96 detecting system. The peptide (VHWDFRQWWGQPS) was shown to have strong binding affinity (Kd) of 86.38 uM with BORIS-N;258 (FIG. 2A).

A reverse binding experiment was conducted by labeling the peptide with biotin, loading it on SA sensor and detecting the affinity of the peptide and the antigen by series concentrations of BORIS-N:-258 (0.25 to 0.625 uM of protein which diluted by PBS with 0.02 % Tween 20) in ForteBio OctetRed96 system. The Kd value of the infinity between peptide and the antigen was shown to be 5.30 nM (FIG. 2B).

BORIS binding peptide SEQ ID No.1 specifically bound to BORIS in mammalian cells BORIS-N:.258 antigen used for selecting peptides was purified from bacteria. To confirm the interaction between SEQ ID No.3 and BORIS-N:.258 which was expressed in mammalian cells, BORIS-N:-258 was expressed and purified from HEK293 cells, which do not express BORIS. Then the binding affinity between SEQ ID No.3 (VHWDFRQWWQPS) and mammalian BORIS-N:-258 was verified by BLI technology. The Kd value was 6.37 nM which was comparable with the affinity between SEQ ID No.3 and bacteria expressed BORIS-Ni.2ss antigen (FIG. 3A). The results indicated that the obtained peptide interacted with BORIS which was expressed in mammalian cells. Then SEQ ID No.3 was synthesized to fuse with HIV-TAT peptide (the amino acid sequence of HIV-TAT is shown as SEQ ID No.4: GGRKKRRQRRRG; the nucleotide sequence of HIV-TAT is shown as SEQ ID No.5: GGTGGCCGTAAAAAGCGCCGACAACGGAGAAGGGGA) to penetrate cell membrane and form SEQ ID No.l which was used to inhibit BORIS in cancer cells and induce cancer cell apoptosis. In the present example, SEQ ID No.1 was labeled by biotin for tracing (referred as SEQ ID No.1-biotin in the following) and used for detecting the interaction with BORIS in cells. IO Myc tagged BORIS was overexpressed in HEK293 cells. The cell lysis was collected and supplied with 25 uM SEQ ID No.1-biotin for detecting the interaction between SEQ ID No. 1-biotin and BORIS by co-immunoprecipitation. SEQ ID No.l-biotin was pulled down by streptavidin conjugated magnetic beads. The detection of BORIS by Myc antibody showed that SEQ ID No. 1-biotin bound to full length of BORIS and BORIS-N:-258 but not BORIS-del Ni-2ss (FIG. 3B). The results indicated that SEQ ID No.1 specifically bound to BORIS in cells and bound to the 1-258 amino acid section of BORIS. Because BORIS located in cancer cells but not on cell membrane, the present example examined whether SEQ ID No.l penetrated cell membrane. SEQ ID No.1-biotin or VHWDFRQWWQPS-biotin (for comparison) were supplied in individual cell cultures for 4 hours of incubation and then the cell membrane penetration efficiencies of the peptides were detected by immunoflueresence with Fluorescein Isothiocyanate (FITC) coupled anti-biotin secondary antibody. The present example showed that SEQ ID No.1-biotin but not VHWDFRQWWOPS-biotin penetrated cells (FIG. 3C), i.e. SEQ ID Nol but not VHWDFRQWWQPS penetrated cells. In addition, to verify the specificity of SEQ ID No.1 to BORIS, BORIS was knocked down by small interfering RNA (siRNA) in HCT116 cells in the present example. BORIS expression levels were examined by BORIS antibody (monoclone antibody catalog #: sc-377085 which was purchased from Santa Cruz Biotechnology) or SEQ ID No. 1-biotin. BORIS knockdown efficiency could be detected by either BORIS antibody or SEQ ID No.1-biotin, indicating that SEQ ID No.1 specifically bound to BORIS in cells (FIG. 3D). The above evidences suggested that SEQ ID No.1 penetrated cell membrane and specifically bound to BORIS in cells.

BORIS binding peptide SEQ ID No.1 specifically bound to BORIS on the amino acid region of S2AA-172AA

To determine the binding site of SEQ ID No.l-biotin on BORIS, five truncations of BORIS-Nia2ss were constructed. The deletion regions of the truncations were as follows: A1:2-51AA, A2:52-102AA, A3:103-171AA, A4: 172-217AA and AS: 215-258AA (FIG. 4A). The expression plasmids of A1-A5 truncations were transfected in HEK293 cells respectively, and then cell lysis containing the above Al-A5 BORIS protein were harvested. Cell lysis were supplied with 25 uM SEQ ID No.l-biotin for 12 hours at 4 °C and then incubated with streptavidin conjugated magnetic beads (streptavidin conjugated magnetic beads was used to pull down biotin tagged SEQ ID No.1) for 2 hours at room temperature for detecting the interactions between A1-A5 and SEQ ID No.1-biotin. The results indicated that SEQ ID No. I-biotin bound to JO Al. A4, A5 but not to A2 or A3. A2 lacked 52-102AA and A3 lacked 103-171AA. Therefore, the reason why SEQ ID No.1 did not bound to A2 or A3 was that SEQ ID No.1 bound to BORIS at 52AA-172AA (FIG. 4A). To validate the reliability of the results of the present example, whether interaction between BAT3 and SEQ ID No.l was mediated by BORIS 52AA-172AA was tested with the reported interaction between BAT3 and BORIS at 1 AA-50AA as a positive reference[17]. It was indicated that SEQ ID No. 1-biotin did not interact with BAT3 when BORIS lacked 1-172AA, i.e., in cases of BORIS A1/2/3 (FIG. 4B). The above results indicated that SEQ ID No. 1-biotin interacted with BORIS at 52AA-172AA and with BAT3 indirectly with the help of BORIS. Example 2 SEQ ID No.1 induced cancer cell apoptosis and suppressed cancer cell proliferation As SEQ ID No.1 binds to BORIS and BORIS is important for carcinoma, the effect of SEQ ID No.l on carcinoma cells in cell proliferation and apoptosis was examined. A gradient treatments of SEQ ID No.1 between 25-100 pM on H1299 non-small lung cancer cells were performed and measured by MTT assay and cell counting. SEQ ID No.1 suppressed H1299 cells proliferation by 25-100 uM treatment compared with the negative control peptide SEQ ID No.7 for 3 days treatment. SEQ ID No.7 was constructed by twelve tandem repeated histidines fused with HIV-TAT peptide, the its amino acid sequence was HHHHHHHHHHHHGGRKKRRQRRRG (SEQ ID No.7) and the nucleotide sequence was

CATCATCACCATCACCATCATCATCACCATCACCATGGTGGCCGTAAAAAGCGCCGA CAACGGAGAAGGGGA (SEQ ID No.8). The inhibition efficiencies of different concentrations of SEQ ID No.1 on H1299 lung cancer cells were as follows: 25 pM SEQ ID No.1 inhibited 27.8%

cell amount and 16.4% cell viability, SO uM SEQ ID No.1 inhibited 54.2% cell amount and 66% cell viability, and 100 pM SEQ ID No.linhibited 83.1% cell amount and 93.5% cell viability (FIG. 5A). The half maximal inhibitory concentration (IC50) of SEQ ID No.l treatment was

63.12 uM on H1299 cells (FIG. 5B). SEQ ID No.l suppressed the proliferation of H1299 lung cancer and LNCaP prostate cancer cells, but not normal HEK293 cells which does not express BORIS (FIG. 5C). VHWDFRQWWOQPS did not inhibit cell proliferation because it did not penetrate cell membrane (FIG. 5C). SEQ ID No.l induced DNA damage in TUNEL assay and induced caspase 3/7 activity (FIG. 6A-B). BORIS prevented cancer cells from apoptosis and kept the genome stable, and SEQ ID No.1 treatment attenuated the protection of cancer cell genome by IO BORIS. The presented results of example 2 supported that SEQ ID No.1 treatment inhibited expressions of KRCCI and cMYC in H1299 cells, which are DNA excision repair gene and proto-oncogene (FIG. 6C). It was indicated that SEQ ID No.1 bound to 52AA-172AA of BORIS and inhibited the expressions of ERCC/and cMYC to induce cancer genome unstability and apoptosis. Example 3 SEQ ID No.l inhibited the progression of subcutaneous tumor of non-small lung cancer. To test the anticancer effects of SEQ ID No.1 in vivo, H1299 cells were used to construct xenograft model in nodltsz-scidil2rg (nsg) mice (FIG. 7A). 1+10%njection of H1299 cells were used to construct subcutaneous tumors in 12 mice for one week and then the 12 mice were divided into two groups. Each group of mice was intraperitoneally injected with 16 mg/kg SEQ ID No.1 (treatment group) or His-TAT (negative control) every other day for 3 weeks (FIG. 7B). The tumor volumes were measured every other day. Tumor weights were measured at the end of the experiment after euthanasia and surgery. The treatment of SEQ ID No.l inhibited tumor progression compared with SEQ ID No.7 treatment (FIG. 7B and 7C). Tumors were sliced and examined by TUNEL assay. Three weeks of SEQ ID No.1 treatment induced significant apoptosis of cancer cells in subcutaneous tumor (FIG. 8A). Meanwhile, SEQ ID No.l treatment did not induce liver and kidney toxicity (FIG. 8B). The above description is only the preferred embodiments of the disclosure and does not limit the present disclosure in any form. For those of ordinary skill in the art, without departing from the principle of the present disclosure, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present disclosure. Reference:

[1] Martin-Kleiner, I. BORIS in human cancers -- a review. European journal of cancer. 2012, 48, 929-35.

[2] Hoivik, E.A., Kusonmano, K., Halle, MK, Berg, A., Wik, E., Werner, H.M. et al. Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene. Oncotarget. 2014, 5, 1052-61.

[B] Renaud, S., Pugacheva, E.M., Delgado, M.D., Braunschweig, R., Abdullaev, Z., Loukinov, D., et al. Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors. Nucleic acids research. 2007, 35, 7372-88.

[4] Sleutels, F., Soochit, W., Bartkuhn, M., Heath, H., Dienstbach, S., Bergmaier, P., et al. The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner. Epigenetics & chromatin. 2012, 5,8.

[5] Zhang, Y., Fang, M., Song, Y., Ren, J., Fang, J., Wang, X. Brother of Regulator of Imprinted Sites (BORIS) suppresses apoptosis in colorectal cancer. Scientific reports. 2017, 7, 40786.

[6] Loukinov, D.I, Pugacheva, E., Vatolin, S., Pack, S.D., Moon, H., Chernukhin, I, et al. BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proceedings of the National Academy of Sciences of the United States of America. 2002, 99, 6806-11.

[7] Woloszynska-Read, A, James, S.R., Link, P.A, Yu, J, Odunsi, K., Karpf, AR. DNA methylation-dependent regulation of BORIS/CTCFL expression in ovarian cancer. Cancer immunity. 2007, 7, 21.

i

[8] Debruyne, D.N,, Dries, R., Sengupta, S., Seruggia, D., Gao, Y., Sharma, B, et al. BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells. Nature. 2019, 572, 676-80.

[9] Asano, T., Hirohashi, Y., Torigoe, T., Mariya, T., Horibe, R., Kuroda, T., et al. Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is involved in cervical cancer stemness and can be a target of immunotherapy. Oncotarget. 2016, 7, 11223-37.

[10] Horibe, R., Hirohashi, Y., Asano, T., Mariya, T., Suzuki, T., Takaya, A, et al. Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is a novel target of lung cancer stem-like cell immunotherapy. PloS one. 2017, 12, 0171460.

[11] Loukinov, D. Targeting CTCFL/BORIS for the immunotherapy of cancer. Cancer immunology, immunotherapy : CI. 2018, 67, 1955-65.

[12] Zhang, Y., Song, Y., Li, C., Ren, J., Fang, M., Fang, J., et al. Brother of regulator of imprinted sites inhibits cisplatin-induced DNA damage in non-small cell lung cancer. Oncology letters. 2020, 20, 251.

[13] Rosa-Garrido, M., Ceballos, L., Alonso-Lecue, P., Abraira, C., Delgado, M.D., Gandarillas, A. A cell cycle role for the epigenetic factor CTCF-L/BORIS. PloS one. 2012, 7, 39371.

[14] Dougherty, C.J, Ichim, T.E., Liu, L., Reznik, G., Min, W.P, Ghochikyan, A, et al. Selective apoptosis of breast cancer cells by siRNA targeting of BORIS. Biochemical and biophysical research communications. 2008, 370, 109-12.

[15] Mkrtichyan, M., Ghochikyan, A., Loukinov, D., Davtyan, H., Ichim, T.E., Cribbs, D.H, et al. DNA, but not protein vaccine based on mutated BORIS antigen significantly inhibits tumor growth and prolongs the survival of mice. Gene therapy. 2008, 15, 61-4.

[16] Mkrtichyan, M., Ghochikyan, A., Davtyan, H., Movsesyan, N., Loukinov, D., Lobanenkov, V., et al. Cancer-testis antigen, BORIS based vaccine delivered by dendritic cells is extremely effective against a very aggressive and highly metastatic mouse mammary carcinoma. Cellular immunology. 2011, 270, 188-97.

[17] Nguyen, P., Bar-Sela, G., Sun, L., Bisht, K.S., Cui, H,, Kohn, E., et al. BAT3 and SETIA form a complex with CTCFL/BORIS to modulate H3K4 histone dimethylation and gene expression. Molecular and cellular biology. 2008, 28, 6720-9.

Clauses

1. A specific peptide targeting the oncoprotein BORIS, wherein the amino acid sequence of the specific peptide is shown as SEQ ID No.1.

2. A gene encoding the specific peptide according to clause 1, wherein a nucleotide sequence of the gene is shown as SEQ ID No.2.

3. Use of the specific peptide according to clause | for the preparation of a drug for inhibiting BORIS function and inducing cancer cell apoptosis.

4. Use of the specific peptide according to clause 1 for the preparation of a drug for tumor therapy.

5. The use according to clause 4, wherein the tumor comprises non-small cell lung cancer and/or prostate cancer.

6. The use according to clause 3 or 4, wherein the drug comprises the specific peptide and acceptable excipients in pharmacy.

SEQLTXT

SEQUENCE LISTING <110> Hangzhou Medical College <120> SPECIFIC PEPTIDE TARGETING ONCOPROTEIN BORIS AND USE AND ENCODING

GENE THEREOF <130> GWP202107306 <150> 202110016541.3 <151> 2021-01-07 <160> 8 <170> PatentIn version 3.5 <21e> 1 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a peptide targeting the oncoprotein BORIS <400> 1 Val His Trp Asp Phe Arg Gln Trp Trp Gln Pro Ser Gly Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Gly <2105 2 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of a peptide targeting the oncoprotein BORIS <400> 2 gtgcattggg attttcggca gtggtggcag ccttctggtg gccgtaaaaa gcgccgacaa 60 Cggagaaggeg ga 72 <2105 3 <211> 12 <212> PRT <213> Artificial Sequence Pagina 1

SEQLTXT

<220>

<223> Amino acid sequence of a precursor of a peptide targeting the oncoprotein BORIS

<400> 3

Val His Trp Asp Phe Arg Gln Trp Trp Gln Pro Ser

1 5 10

<2105 4

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of HIV-TAT

<400> 4

Gly Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly

1 5 10

<216> 5

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of HIV-TAT

<400> 5 ggtggccgta aaaagcgccg acaacggaga agggga 36

<210> 6

<211> 258

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of BORIS-N1-258

<400> 6

Met Ala Ala Thr Glu Ile Ser Val Leu Ser Glu Gln Phe Thr Lys Ile

1 5 10 15

Lys Glu Leu Glu Leu Met Pro Glu Lys Gly Leu Lys Glu Glu Glu Lys Pagina 2

SEQLTXT

Asp Gly Val Cys Arg Glu Lys Asp His Arg Ser Pro Ser Glu Leu Glu 40 45 Ala Glu Arg Thr Ser Gly Ala Phe Gln Asp Ser Val Leu Glu Glu Glu 50 55 60 Val Glu Leu Val Leu Ala Pro Ser Glu Glu Ser Glu Lys Tyr Ile Leu 65 70 75 80 Thr Leu Gln Thr Val His Phe Thr Ser Glu Ala Val Glu Leu Gln Asp 85 90 95 Met Ser Leu Leu Ser Ile Gln Gln Gln Glu Gly Val Gln Val Val Val 100 105 110 Gln Gln Pro Gly Pro Gly Leu Leu Trp Leu Glu Glu Gly Pro Arg Gln 115 120 125 Ser Leu Gln Gln Cys Val Ala Ile Ser Ile Gln Gln Glu Leu Tyr Ser 130 135 140 Pro Gln Glu Met Glu Val Leu Gln Phe His Ala Leu Glu Glu Asn Val 145 150 155 160 Met Val Ala Ser Glu Asp Ser Lys Leu Ala Val Ser Leu Ala Glu Thr 165 170 175 Thr Gly Leu Ile Lys Leu Glu Glu Glu Gln Glu Lys Asn Gln Leu Leu 180 185 190 Ala Glu Arg Thr Lys Glu Gln Leu Phe Phe Val Glu Thr Met Ser Gly 195 200 205 Asp Glu Arg Ser Asp Glu Ile Val Leu Thr Val Ser Asn Ser Asn Val 210 215 220 Glu Glu Gln Glu Asp Gln Pro Thr Ala Gly Gln Ala Asp Ala Glu Lys Pagina 3

SEQLTXT 225 230 235 240 Ala Lys Ser Thr Lys Asn Gln Arg Lys Thr Lys Gly Ala Lys Gly Thr 245 250 255 Phe His <210> 7 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of a negative control peptide <400> 7 His His His His His His His His His His His His Gly Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Gly

<2105 8 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of a negative control peptide <400> 8 catcatcacc atcaccatca tcatcaccat caccatggtg gccgtaaaaa gcgccgacaa 60 Cggagaaggeg ga 72 Pagina 4

Claims

Conclusions II. A specific peptide targeted to the oncoprotein BORIS, wherein the amino acid sequence of the specific peptide is as in SEQ ID NO. 1.

A gene encoding the specific peptide of claim 1, wherein a nucleotide sequence of the gene is as in SEQ ID NO. 2.

A use of the specific peptide of claim 1 for the manufacture of a medicament for inhibiting BORIS function and inducing apoptosis of cancer cells.

A use of the specific peptide of claim 1 for the preparation of a medicament for tumor therapy.

The use of claim 4, wherein the tumor comprises non-small cell lung cancer and/or prostate cancer.

The use according to claim 3 or 4, wherein the medicament comprises the specific peptide and pharmaceutical excipients.