A PTEN gene downstream nucleotide sequence and polypeptide encoded by the same

Abstract

The present invention discloses a PTEN gene downstream nucleotide sequence and a polypeptide encoded by it. The nucleotide sequence is shown in the sequence table SEQ ID NO: 1, and the amino acid sequence of the polypeptide is shown in the sequence table SEQ ID NO: 2. The polypeptide can be endogenously expressed in glioma cells and normal brain glial cells. The expression level of 48aa-dORF in various glioma samples and normal samples was detected, and it was found that the expression level of 48aa-dORF in glioma tissue samples was significantly reduced compared with normal brain tissue. PTTN-48aa can significantly inhibit the growth and proliferation of glioma cells, and can provide a new polypeptide drug for the clinical treatment of glioma.

Description

Technical Field

[0001] The invention belongs to the field of biological gene technology, and specifically relates to a PTEN gene downstream nucleotide sequence and a polypeptide encoded by the same. Background Art

[0002] The PTEN gene is a tumor suppressor gene with dual specific phosphatase activity discovered after p53. The gene has 9 exons and 8 introns. The ATG and CGG repeat sequences at the 5′ end of the cDNA form the structural basis for methylation. Its lipid phosphatase activity depends on the structure homologous to the protein serine / threonine enzyme and protein tyrosine phosphatase catalytic center. The PTEN gene mainly exerts its tumor suppressor function through apoptosis, cell cycle arrest, cell migration and other links. The key to regulation lies in the balance of PI3K / AKT and MAPK signaling pathways. The PTEN gene encoding product can specifically induce CDKs inhibitors p21, p27 and p51 to participate in the regulation of the cell cycle or lead to changes in the apoptosis process by antagonizing the AKT signaling pathway. The PI3K / AKT signal transduction pathway is closely related to cell survival. The PTEN protein mainly affects the cell cycle process through this pathway, leading to abnormal cell proliferation, inhibiting cell apoptosis, and affecting cell migration and invasion.

[0003] Several studies have shown that the expression of PTEN gene in endometrial cancer is lower than that in normal endometrium and is correlated with malignant evaluation indicators such as differentiation degree of endometrial adenocarcinoma cells, pathological grade, clinical stage, and lymph node metastasis. Xue Shuai et al. showed through Meta-analysis that the expression of PTEN protein in thyroid cancer is lower than that in normal thyroid or benign thyroid lesions. This low expression mainly occurs in the progressive stage of tumor rather than in the early stage. PTEN gene mutation or inactivation can lead to an increased risk of benign and malignant thyroid tumors. The expression levels of PTEN gene mRNA and protein show a gradual decrease in the three stages of atypical hyperplasia of breast ductal epithelium, intraductal carcinoma, and invasive ductal carcinoma. As a tumor suppressor gene with dual specific phosphatase activity, the research on PTEN gene is currently focused on the loss of expression in different tumors. The quantitative detection of the reduced expression of PTEN gene in tumors, the mechanism of loss of expression in different tumor tissues, and the study of multiple links such as the effect on tumor cell growth and lymphatic vessel growth will provide more molecular biological theoretical basis for the differential diagnosis and treatment of tumors and propose more clinical diagnostic methods. Further understanding of the PTEN gene-related signal transduction pathways and targets such as tumor cell apoptosis and cell cycle regulation will also provide more new ideas and basis for the diagnosis and treatment of tumors at the genetic level.

[0004] In the traditional concept, "translation" is limited to open reading frames encoding proteins only. With the development and progress of high-throughput deep sequencing and protein spectrum analysis technology, this concept is being changed by new research results. Researchers have found that in some areas of mammalian cells that were previously thought to be unable to encode proteins, there are many small open reading frames that can encode polypeptides of less than 100 amino acids and have important biological functions. These regions include the 5′UTR non-coding region and the 3′UTR non-coding region of the encoding gene mRNA. Patent 201710429319.X discloses a 45aa-uORF nucleotide sequence of the upstream open reading frame of the PTEN gene and the application of the polypeptide encoded by it. The molecular weight of the micropeptide is particularly small, and it can easily enter glioma cells and penetrate into tumor tissues. It can also pass through the blood-brain barrier well to treat gliomas. At present, no active peptides have been found in the downstream open reading frame of the PTEN gene. Summary of the invention

[0005] The purpose of the present invention is to provide a PTEN gene downstream nucleotide sequence and the polypeptide encoded by it.

[0006] A PTEN gene downstream nucleotide sequence, the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing.

[0007] The polypeptide encoded by the PTEN gene downstream nucleotide sequence has an amino acid sequence as shown in SEQ ID NO: 2 in the sequence listing.

[0008] Primers for amplifying the downstream nucleotide sequence of the PTEN gene.

[0009] An expression vector containing the downstream nucleotide sequence of the PTEN gene.

[0010] A host cell containing an expression vector for the downstream nucleotide sequence of the PTEN gene.

[0011] A kit for tumor diagnosis comprises a probe for detecting a nucleotide sequence downstream of a PTEN gene, a primer for amplifying a nucleotide sequence downstream of a PTEN gene, or an antibody against a polypeptide encoded by a nucleotide sequence downstream of a PTEN gene.

[0012] The tumor is endometrial cancer, thyroid cancer or glioma.

[0013] A pharmaceutical composition for treating tumors, comprising the PTEN gene downstream nucleotide sequence of claim 1 and the polypeptide encoded by the PTEN gene downstream nucleotide sequence of claim 2.

[0014] The tumor is endometrial cancer, thyroid cancer or glioma.

[0015] Beneficial effects of the present invention: The present invention discovered that there is a small open reading frame 48aa-dORF at the 3′ end of PTEN, which can encode a short peptide PTTN-48aa of 48 amino acids. Antibodies to PTTN-48aa were prepared, and Western blot showed that 48aa-dORF can indeed be endogenously expressed in glioma cells and normal brain glial cells. Simultaneous detection of the expression levels of 48aa-dORF in a variety of glioma samples and normal samples revealed that the expression level of 48aa-dORF in glioma tissue samples was significantly reduced compared with that in normal brain tissues.

[0016] PTTN-48aa can significantly inhibit the growth and proliferation of glioma cells and can provide a new polypeptide drug for the clinical treatment of glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Western analysis was performed using a specific antibody for the polypeptide encoded by 48aa-dORF to detect the endogenous expression of 48aa-dORF in cells.

[0018] Figure 2 The number of clones formed by U251 cells and cells overexpressing 48aa-dORF by lentivirus was counted for the plate clone formation experiment.

[0019] Figure 3 The number of clones formed by U251 cells and cells overexpressing 48aa-dORF by lentivirus was counted for the soft agar colony formation experiment.

[0020] Figure 4 The growth volume of U251 subcutaneous transplanted tumor in nude mice in the 48aa-dORF encoding polypeptide group and the random peptide control group. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0022] Example 1

[0023] By analyzing the mRNA sequence of the PTEN gene, a 147nt open reading frame (ORF) was found in the 3′UTR non-coding region, which can encode a short peptide of 48 amino acids. The inventor named it: 48aa-dORF, and its nucleotide sequence is shown in the sequence table SEQ ID NO: 1. The short peptide encoded by it is named: PTTN-48aa, and the amino acid sequence of the short peptide is shown in the sequence table SEQ ID NO: 2. The molecular weight of the protein predicted by the protein molecular weight prediction software http: / / www.bio-soft.net / sms / prot_mw.html is 5.54KD.

[0024] According to the amino acid sequence of 48aa-dORF, a polyclonal antibody that specifically recognizes the polypeptide and can be detected by western blot is designed. The specific method is as follows: by chemically synthesizing peptides, a segment of 48aa-dORF, IIMGFCTVIIFPLECEGLN amino acid sequence, is synthesized as an immunogen, injected and immunized New Zealand white rabbits, and then the target antibody is purified for detecting the 48aa-dORF encoded polypeptide in cells. CRISPR / Cas9 specifically knocks out a segment in the middle of 48aa-dORF on the genome, silences the expression of 48aa-dORF polypeptide at the genome level, and obtains a 293 cell line with 48aa-dORF knockout. The endogenous expression and changes of 48aa-dORF polypeptide are detected by western using a 48aa-dORF-specific antibody.

[0025] Establishment of CRISPR / Cas9 knockout cell line: According to the sequence of 48aa-dORF, the sequence of gRNA was designed to target different regions of 48aa-dORF, specifically delete a section of 48aa-dORF, and obtain a cell line with genome knockout of 48aa-dORF.

[0026] The specific gRNA sequence information is as follows:

[0027] 48aa-cas9 / gRNA-1: 5′-GUCAUAAAUAAUGACAUUA-3′;

[0028] 48aa-cas9 / gRNA-2: 5′-CAAGGCUUCUCAAAAACAUUGA-3′.

[0029] The pX330-Puro-PTEN-SgRNA plasmid was transiently transfected into HEK293T cells, and its efficiency was detected by sequencing. The results showed that 48aa-SgRNA2 was a high-efficiency SgRNA. The 48aa-SgRNA2 plasmid was selected for subsequent experiments and transfected into HEK293 cells. The positive clones were selected after screening with puromycin. After DNA sequencing, the clones with frameshift mutations in the PTEN-48aa sequence in the cloned daughter cells met the requirements, and a cell line with genome knockout of 48aa-dORF was obtained.

[0030] Cell culture: HEK-293T cells were cultured in DMEM supplemented with 10% fetal bovine serum and 10 U / mL penicillin-streptomycin in a 5% CO2, 37°C constant temperature humidified cell culture incubator. The cells were passaged every three days.

[0031] Western blot: Total cell protein was extracted using RAPA, and the extracted protein was quantified using the BCA protein quantification method; 5% SDS PAGE concentrating gel and 15% SDS-PAGE separation gel were prepared, and 100ug of total protein was loaded; protein electrophoresis was performed at 80V for 20min and 150V for 1h; transfer to the membrane was performed at 100V for 2h; 5% skim milk was used for blocking for 1h; 48aa-dORF rabbit anti-antibody (1:500), β-actin antibody (1:3000); incubation was performed at 4 degrees overnight; the next day, rabbit secondary antibody (1:10000) was used for incubation at room temperature for 1h, TBST was used for washing 5 times for 5min each time, and then luminescence, development, and fixation were performed.

[0032] Western blot showed that the polypeptide encoded by 48aa-dORF existed in the cells. The expression level of the polypeptide in normal glial cells was significantly higher than that in glioma cells, suggesting that the polypeptide may have the function of suppressing tumors ( Figure 1 ).

[0033] Construction of a stable cell line overexpressing 48aa-dORF: A vector overexpressing 48aa-dORF-GFP (vector backbone pCDH-CMV-MCS-EF1-Puro) was synthesized and transfected into HEK-293T cells with the lentiviral backbone vectors PSPAX2 and PMD2G at a ratio of 4:3:1 to package lentivirus.

[0034] The culture medium supernatant containing lentivirus was collected at 48h and 72h, respectively, and then used to infect U251 cells. After three days of puromycin (1mg / mL) selection, puromycin was withdrawn, and the cells were then expanded normally.

[0035] Cell transfection: 500,000 293T cells were inoculated in a 6-well culture plate, and transfection was performed after the cells adhered for 24 hours. Before transfection, 100uL serum-free culture medium DMEM and plasmid were prepared into a mixed solution. 100uL serum-free culture medium DMEM and 5uL (2ug plasmid / 5uL lipo2000) liposomes were evenly mixed to make a liposome mixture. The two mixed solutions were mixed in equal proportions and placed at room temperature for 20 minutes. The operation was performed according to the transfection reagent operating instructions. The final volume of the wells in the 6-well plate was 1mL. After 6 hours of transfection, the medium was replaced with 1mL of normal culture medium (10% fetal bovine serum plus 90% DMEM medium plus 1% penicillin-streptomycin). The cell culture conditions were 37°C and 5% CO2.

[0036] CCK-8 detection of cell proliferation: U251-GFP control cells or U251-48aa-dORF-GFP cells were seeded in a 96-well plate at a number of 2000 per well, with 5 replicates for each sample. After adhesion, 10 μL of CCK-8 reagent was added to each well every 24 h, and the cells were cultured at 37°C for 3 h. The absorbance (OD) at 450λm was measured. The absorbance value was proportional to the cell number.

[0037] Clone formation assay (CFA): 2000 U251-GFP control cells or U251-48aa-dORF-GFP cells were seeded in six-well plates at 5%

[0038] The cells were cultured continuously in a cell culture incubator with a constant temperature and humidity of 37°C and CO2 for two weeks. After staining with crystal violet, the cells were photographed and the number of clones formed was counted. The experiment was repeated three times.

[0039] Soft agar colony formation test (SA): first add 2 mL of 0.6% low melting point agarose to a six-well plate, and then add 2 mL of 0.35% low melting point agarose containing 20,000 U251-GFP control cells or U251-48aa-dORF-GFP cells after solidification. Mix gently and then culture in a cell culture incubator with 5% CO2 and a constant temperature and humidity of 37°C for two weeks. After that, take pictures under an optical microscope and count the number of colonies formed. Repeat the experiment three times.

[0040] The results show Figure 2-3 As shown, in U251 glioma cells, overexpression of the polypeptide encoded by 48aa-dORF can significantly inhibit the growth and proliferation of glioma cells U251.

[0041] The anti-cancer function of 48aa-dORF polypeptide was verified at the animal level: 2 million U251 cells were inoculated on the left and right sides of nude mice at each point, and the formation of tumors was observed after 30 days. Then the tumors were evenly divided into control group and experimental group according to their size (five nude mice and ten tumors in each group). The control group was injected with control polypeptide (the 48aa-dORF encoding polypeptide sequence was randomly shuffled to synthesize the control polypeptide) every other day, and the experimental group was injected with 48aa-dORF polypeptide every other day. Each tumor was injected with 25ug of artificial synthetic polypeptide. The long and short diameters of the tumor were measured before administration. After 6 doses of administration, the nude mice were killed and the tumors were taken for photographing and weighing.

[0042] Tumor volume = 1 / 2*longest diameter*square of shortest diameter

[0043] The experimental results are as follows Figure 4 As shown, compared with the control polypeptide, the chemically synthesized 48aa-dORF encoded polypeptide can significantly inhibit the growth and formation of U251 glioma transplanted tumors, indicating that the 48aa-dORF encoded polypeptide shows good anti-tumor activity at the animal level.

[0044] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A polypeptide encoded by a nucleotide sequence downstream of a PTEN gene, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 2 in the sequence listing.

2. A pharmaceutical composition for treating glioma, characterized in that: The pharmaceutical composition comprises the polypeptide encoded by the nucleotide sequence downstream of the PTEN gene according to claim 1.

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