阅读说明：本技术 宫颈癌干细胞特异性透膜肽和干扰RabJ基因的组合物的抑癌用途 (Cancer inhibition application of composition of cervical cancer stem cell specific membrane penetrating peptide and interference RabJ gene ) 是由 王阳 曹帅 于 2020-05-31 设计创作，主要内容包括：本发明涉及宫颈癌干细胞特异性透膜肽和干扰RabJ基因的组合物的抑癌用途,该组合物可以有效地抑制内源性RabJ基因的表达,从而抑制肿瘤细胞生长。(The invention relates to a cancer suppressor application of a composition of cervical cancer stem cell specific membrane-penetrating peptide and interference RabJ gene, which can effectively suppress the expression of endogenous RabJ gene so as to suppress the growth of tumor cells.)

1. A conjugate for interfering with the RabJ gene in cervical cancer stem cells, wherein: the amino acid sequence is shown as SEQ ID NO: 3 and the sequence of the cervical cancer stem cell membrane-penetrating peptide shown in SEQ ID NO: 1, coupling the siRNA of the RabJ gene.

2. The conjugate as claimed in claim 1, wherein the specific coupling method is as follows: 1ug of siRNA was diluted to 25ul with PBS; respectively preparing the polypeptide samples into 1mg/ml solutions, measuring the polypeptide solutions according to the polypeptide/siRNA charge ratio N/P of 10, and diluting the polypeptide solutions to 25ul by using PBS; then, the polypeptide solution is gradually dripped into the siRNA solution, the siRNA solution is evenly blown and beaten by a pipette, the vortex is carried out for 10s, and the mixture is placed at room temperature for incubation by 3Omin to ensure that the cationic polypeptide and the DNA are fully combined to form a nano compound; finally, the cells were diluted to 500ul in serum-free DMEM medium for cell transfection.

3. Use of a conjugate according to claim 1 or 2 for inhibiting cervical cancer cells.

Technical Field

The invention relates to the field of biology, in particular to a cancer inhibition application of a composition of cervical cancer stem cell specific membrane penetrating peptide and interference RabJ gene.

Background

The transactivator of the human immunodeficiency virus (HIV-1) transcription protein was the first factor to be found to be able to cross the cell membrane, and dorussi, d. It was subsequently found that a short stretch of basic amino acid regions in the TAT protein, with amino acid residues exerting a membrane-penetrating effect in the short 11-amino acid peptide TAT (Tyr-Gly-Arg-Lys-Arg-Arg-Arg-Gln-Arg-Arg) of 47-57, whereas 16 amino acid regions in the third helix homologous region in the Drosophila antennaria transcription factor protein exerting a membrane-penetrating activity, were subsequently named penetratin (Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Lys). Based on TAT and penetratin, a novel polypeptide carrier, namely a transmembrane peptide, capable of delivering various molecules is developed.

In recent years, various interdisciplinary studies have reported various types of membrane-penetrating peptides as delivery tools to deliver nucleic acids, proteins, liposomes, nanoparticles, and the like into cells. The main characteristics of the membrane-penetrating peptide are low toxicity, the delivery efficiency is dose-dependent, and the size and the type of the biomolecule or nanoparticle to be delivered are not limited. The number of amino acids of the membrane-penetrating peptide is usually less than 40, and the membrane-penetrating peptide enters cells through various routes (mainly endocytic routes), and can be combined with nucleic acid, protein, small molecule and the like in a covalent or non-covalent mode to mediate the entry of the nucleic acid, the protein, the small molecule and the like into the cells. The membrane-permeable peptide generally contains a large number of cationic lysines and arginines (a few are uncharged or negatively charged) in the sequence, has a large number of positive charges under physiological conditions, and can be combined with nucleic acid through electrostatic interaction to form a polypeptide/nucleic acid nano-complex, so that the nucleic acid is mediated to enter cells. Scientists have utilized TAT polypeptides to deliver antisense nucleic acids to inhibit the expression of P _ glycoprotein by tumor cells.

Most of cell membrane-penetrating peptides have their membrane-penetrating mechanism independent of energy, and most of them adopt endocytosis mechanism when transferring large molecular weight DNA and other biological macromolecules. For example, TAT is directly transmembrane in an energy-independent manner, whereas TAT/DNA complexes transfected into HepG2 and CHO1 cell lines enter the cell via the caveolin-mediated endocytic pathway. Generally, the cation membrane-penetrating peptide is combined with glycosaminoglycan matrix with negative charge outside the cell membrane, such as heparin and the like, a small part of the cation membrane-penetrating peptide is directly transduced into cells, and the vast majority of the cation membrane-penetrating peptide is inserted into the cell membrane and enters the cells through endocytosis or large endocytosis mediated by various proteins (such as caveolin protein and clathrin). The size of the transmembrane peptide/nucleic acid complex has a great influence on a cell transmembrane mechanism, a part of the complex with smaller size can be directly transduced into cells, and the endocytosis mediated by caveolin and clathrin are gradually increased along with the increase of the size of the complex.

RNA interference (RNAi) is a process in which activation of a small interfering RNA (sirna) regulated intracellular pathway consisting of 21-23 nucleotides (nt) leads to degradation of specific targeted mrnas (reviewed in 1, 2). To cause RNAi-mediated gene silencing in human cells, double-stranded sirnas are transfected into cells. Upon entry into the cell, the siRNA duplexes undergo 5' phosphorylation, melt, and bind to the RNA-induced silencing complex (RISC). Activated RI SC (RISC) and the melted antisense strand complementary to the target mRNA and mRNA target. Single site specific cleavage of the mRNA target then occurs, the position of which is determined with reference to the 5' end of the siRNA antisense strand. Once cleavage occurs, the target mRNA degrades and the RISC cycle is used for another cleavage reaction. RNAi is widely used in a variety of laboratory applications and in future clinical therapies due to its effectiveness in silencing specific targeted genes.

Due to the wide potential application of RNAi in biology and medicine, it is important to understand the mechanism of RNAi and to develop new methods for successful delivery of siRNA to target cells. Many approaches to delivery of siRNA have been explored recently. One approach is to deliver a DNA or RNA template encoding the siRNA sequence into a cell that can be transcribed to express the siRNA. These DNA and RNA based siRNA expression methods rely on plasmid or viral vector delivery and require transfection, stable vector integration and selection to maintain expression at generations 8, 9, 10, 11, 12, 13, 14, 15, other successful methods focus on delivering siRNA directly into cells, and the fidelity of siRNA uptake by cells is critical to RNAi using this approach. Currently, the most commonly used method of siRNA delivery is Lipofectamine transfection. However, the use of this method is limited to specific cell types and this method can be toxic to cells and animals.

Human RabJ is a new small G protein family member derived from dendritic cells derived from healthy adult peripheral blood mononuclear cells, and is characterized by comprising three functional domains of Nuclear Localization Signal (NLS) at the N end (1-18 amino acids) and the middle (210-216 amino acids), Rab-like functional domain (19-209 amino acids) in the middle and J functional domain (217-273 amino acids) at the C end in the protein composition, and has higher homology with Ras family molecules. Three functional domains mediate the nuclear localization of RabJ respectively; interacting with P85 subunit and P53 subunit of extracellular signal-regulated kinase (ERKl/2) kinase (ERK kinase) MEK1/2, Protein Kinase C (PKC), phosphoinositide-3 kinase (PI 3K); and interaction with HSC70 and Raf (Ras-associated factor). The nucleic acid sequence and amino acid sequence of RabJ and their preparation are disclosed in Chinese patent application CN 01126826.3.

siRNA against RabJ has been previously available, but the designed siRNA has room for improvement in both inhibition efficiency and transfection efficiency.

Disclosure of Invention

The invention provides siRNA specific to RabJ gene.

The sense strands of the siRNA aiming at the RabJ gene are respectively shown as follows:

SiRabJ1:5’-gcagatgccattcgcagaat-3’(SEQ ID N0:1)

SiRabJ2:5’-tagcagtgctagtttcacc-3’(SEQ ID N0:2)

according to another embodiment of the present invention, 1 to 3 nucleotides may be ligated to the 3 'end of the sense strand, such that a 3' overhang consisting of the 1 to 3 nucleotides is formed at least one end of the double-stranded structure after the sense strand and the antisense strand are complementary to form the double-stranded structure. Among them, it is preferable that the 3' overhang is composed of two consecutive deoxythymine nucleotides dTdT or two consecutive uracil nucleotides UU.

According to another embodiment of the invention, the sense strand and the antisense strand each comprise at least one group of modified nucleotides therein. Wherein, the modified nucleotide group is at least one modified nucleotide group of phosphate group, ribose group or base. Preferably, the modified nucleotide group is a nucleotide group in which the 2' -hydroxyl group of the ribose group is substituted with methoxy or fluorine.

More importantly, the invention relates to a membrane-penetrating peptide specifically aiming at cervical cancer stem cells.

More specifically, the invention provides a method for screening a membrane-permeable peptide, which comprises the steps of adding cervical cancer stem cells into a DMEM medium containing 0.1% BSA for incubation, adding a stock solution of a random dodecapeptide phage display library, placing the mixture into a shaking table at 37 ℃, carrying out incubation by gentle shaking, and continuing incubation in a cell incubator. Internalization of the phage was terminated after incubation for 1h at 37 ℃. Unbound phage supernatant was discarded and cells were washed. And digesting the stem cells by using pancreatin mixed liquor, placing the stem cells in an incubator, and keeping the temperature until the cells are observed to be shrunk and separated from the dish wall under the mirror. Centrifuging, removing supernatant, adding gently resuspended cells, centrifuging, and repeatedly washing cells for 3 times. The precipitated cells were repeatedly frozen and thawed 5 times in liquid nitrogen and 37 ℃ constant temperature water bath, and sufficiently shaken. The frozen and thawed material was added with 2ml of 1% Triton X-100 containing PBS (containing PMSF and Cocktail) and allowed to act at room temperature for 2h to lyse the cells. Centrifuging at 5000r/min at 4 deg.C for 10 min. The supernatant after centrifugation is the separation liquid for screening. And repeating the screening method for 5 times, wherein the recovery rate of the screened phage is gradually improved, the number of the phage internalized into the stem cells is improved, and the target phage is obviously enriched. Mu.l of phage clone amplification solution obtained by screening is heated for 10min in a metal bath at 96 ℃, and 1 mu.l of supernatant is taken as a DNA template. Primers were designed based on the consensus sequence of the dodecapeptide insert upstream and downstream, and PCR was performed. And (3) carrying out sequencing on the PCR product of the phage clone identified as the insert by electrophoresis. The amino acid sequence of the foreign dodecapeptide fused with the pIII protein is deduced according to the reading frame of the phage pIII gene in the coding chain. According to the sequencing result, 1 sequence has 4 repeats, the high-peak dodecapeptide sequence is TM-2 polypeptide with the permeability characteristic of stem cells, and the sequence is SEQ ID NO: 3, respectively.

In addition, the invention also provides a method for coupling the membrane-penetrating peptide and siRNA, and particularly, the siRNA is diluted by PBS; preparing a polypeptide sample into a solution, measuring the polypeptide solution according to the charge ratio N/P of the polypeptide/siRNA as 10, and diluting the polypeptide solution with PBS; then, the polypeptide solution is gradually dripped into the siRNA solution, the siRNA solution is evenly blown and beaten by a pipette, the vortex is carried out for 10s, and the mixture is placed at room temperature for incubation by 3Omin to ensure that the cationic polypeptide and the DNA are fully combined to form a nano compound; finally, the cells were diluted to 500ul in serum-free DMEM medium for cell transfection.

In another aspect, the present invention provides a method for inhibiting RabJ gene expression in a breast cancer stem cell, comprising introducing an siRNA conjugate as described above into a breast cancer stem cell, thereby allowing the siRNA to sequence-specifically induce inhibition of the RabJ gene expression.

In another aspect, the present invention provides the use of an siRNA conjugate as described above in the manufacture of a medicament for the treatment and/or prevention of breast cancer.

Advantageous effects

The invention obtains the specific transmembrane peptide of the breast cancer stem cell by screening, can provide corresponding gene delivery capacity aiming at the breast cancer stem cell specifically, simultaneously designs specific siRNA which can mediate the inhibition of RabJ gene expression in sequence specificity and can effectively inhibit the expression of endogenous RabJ gene, thereby inhibiting the growth of tumor cells.

Drawings

FIG. 1 shows the effect of TM-2 polypeptides on cell viability, with the TM-2 polypeptides represented in black and the TAT polypeptides represented in gray.

FIG. 2 shows the results of examining the influence of siRNA on the gene expression level.

FIG. 3 shows the results of the detection of the protein expression level, which represent, in order from left to right, TM-2 polypeptide + SEQ ID NO: 1siRNA, TAT polypeptide + SEQ ID NO: 1siRNA, TM-2 polypeptide + SEQ ID NO: 2siRNA, TAT polypeptide + SEQ ID NO: 2siRNA, negative control.

Detailed Description