CN111544445B

CN111544445B - Cancer inhibition application of composition of cervical cancer stem cell specific membrane penetrating peptide and interference RabJ gene

Info

Publication number: CN111544445B
Application number: CN202010481248.XA
Authority: CN
Inventors: 马洪松; 王阳; 曹帅
Original assignee: Fubin Biotechnology Tianjin Co ltd
Current assignee: Fubin Biotechnology Tianjin Co ltd
Priority date: 2020-05-31
Filing date: 2020-05-31
Publication date: 2021-03-26
Anticipated expiration: 2040-05-31
Also published as: CN111544445A

Abstract

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.

Description

Cancer inhibition application of composition of cervical cancer stem cell specific membrane penetrating peptide and interference RabJ gene

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 RISC (RISC) and the melted antisense strand complementary to the target 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 cervical cancer stem cell, comprising introducing an siRNA conjugate as described above into a cervical 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 the siRNA conjugate as described above in the preparation of a medicament for the treatment and/or prevention of cervical cancer.

Advantageous effects

The invention obtains the specific membrane-penetrating peptide of the cervical cancer stem cell by screening, can provide corresponding gene delivery capacity aiming at the cervical 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

Example 1 preparation of cervical cancer stem cells

Taking fresh tumor tissue edge without necrosis, calcification and electrocoagulation part from cervical cancer operation, aseptically taking material, and placing in culture medium (DMEM/F12, containing 10% FBS). Pruning to remove necrotic tissue and residual blood vessels, washing with PBS 3 times, cutting tissue pieces, digesting with 0.14% collagenase type I and 0.25% trypsin-EDTA 37 deg.C for 30min, discarding supernatant, collecting cell-containing precipitate, transferring to culture flask, culturing with DMEM/F12 medium containing 10% FBS, 100U/ml penicillin and 100mg/ml streptomycin at 37 deg.C and 5% CO₂Culturing for 48h in a saturated humidity incubator, washing off non-adherent cells, and continuing culturing. The inoculation is completed within 2h from the drawing of the material. Performing the liquid change for the 1 st time when the primary culture is carried out for 3-5 days, and then changing the liquid for 1 time every 3-4 days, wherein the liquid is changed according to the proportion of 1: passage 2. Each primary cervical cancer cell is independently cultured.

Cervical carcinoma SP and non-lateral population NSP cells were sorted using BD FACS flow cytometry. Selecting cells in logarithmic growth phase, digesting with 0.25% trypsin-EDTA, and adjusting cell concentration to 1 × 10⁶And/ml. Adding Hoechst33342 to the cell suspension to a final concentration of 10. mu.l, keeping out of the light, incubating at 37 ℃ for 110min, and mixing 1 time every 15 min. Centrifuging at 4 deg.C for 10min at 1000r/min, removing supernatant, washing cells with precooled PBS for 1 time, resuspending with PBS containing 2% FBS, filtering with 40um filter screen, sorting with inflow cytometer to obtain cervical cancer SP cells, i.e. cervical cancer stem cells,the proportion of SP cells was (1.43. + -. 0.50)%.

Example 2 screening of cell Membrane penetrating phage short peptides

After subculturing the cervical cancer stem cells prepared in example 1, the culture solution in a 10cm dish was discarded, adherent stem cells were washed 3 times with a DMEM medium, and cultured in a DMEM medium containing 0.1% BSA at 37 ℃ for 1 hour. Add 10. mu.l of stock solution of random dodecapeptide phage display library (titer 3X 10)¹³pfu/ml), placing in a shaker at 37 ℃, shaking gently for 15min, and continuing to incubate in a cell incubator for 1.5h, wherein the shaking is 70 r/min. Immediately after incubation for 1h at 37 ℃, the plates were placed on ice for 5min, terminating internalization of the phage. Unbound phage supernatant was discarded and cells were washed 6 times at room temperature using 5ml DMEM medium containing 0.1% BSA. The stem cells were digested with 2ml PBS and 1.5ml 0.25% trypsin mix and placed in an incubator and incubated until the cells were observed under the mirror to collapse and detach from the dish wall. Centrifuging at 4 deg.C for 2min at 1000r/min, removing supernatant, adding 3ml PBS, gently suspending 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. The screening method is repeated for 5 times, the recovery rate of the screened phage is gradually improved, the number of the phage internalized into the stem cells is improved by about 500 times, 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.

Example 3 Effect of TM-2 Polypeptides on cell viability

(1) The cervical cancer stem cells isolated in example 1 cultured in the logarithmic growth phase were collected at 1X10⁴Inoculating the cells/well into a 96-well plate for conventional culture at the inoculation density of 100 mu l per well, setting 3 multiple wells for each cell, and culturing at 37 ℃;

(2) when the logarithmic phase is reached, the culture solution is changed into a serum-free RPMI-1640 culture solution, and the culture is continued for 1 h;

(3) respectively changing into serum-free RPMI-1640 culture solutions with TM-2 polypeptide concentration gradient of 10 μ M, 20 μ M, 30 μ M, 40 μ M and 50 μ M, and continuously culturing for 24 hr;

(4) after the incubation time is over, adding PBS (100 mu l per well) for washing for 2min multiplied by 3 times;

(5) mu.l of a normal culture medium containing serum and 20. mu.l of MTT (stock solution concentration 5mg/ml, i.e., 0.5% MTT) solution were added to each well, and the culture was continued at 37 ℃ for 4 hours, and the culture was aspirated after the termination of the culture. Adding dimethyl sulfoxide into 200 μ l/hole, oscillating for 10min until the crystal is fully dissolved, detecting light absorption value A with wavelength of 570nm on a full-wavelength microplate reader, and taking the average value of 3 multiple holes per group. OD490 was measured. Repeat 3 times to calculate the cell survival rate. Cell viability was calculated as follows: cell viability ═ 100% (experimental well OD value-control well OD value-blank well OD value)/(control OD value-blank well OD value).

TAT polypeptide (C18-CLLHHLLHHLLHHCGRKKRRQRRR-NH) which is a short peptide of 11 amino acids commonly used in the field₂) The control was treated in the same manner as described above.

The experiment adopts an MTT method to determine the influence of TM-2 polypeptides with different concentrations on the cell viability after being treated for 24 hours. MTT analysis data of stem cells treated by TM-2 polypeptide with different concentrations show that the cells treated by TM-2 polypeptide with concentration higher than 50mM still maintain more than 98% and have little influence on the vitality of the cells (figure 1). This suggests that the TM-2 polypeptide does not affect cell viability in the 50mM range, whereas the control TAT polypeptide has a cell viability of 90% at 50mM, which is not conducive to studying the actual inhibition of stem cell genes by siRNA.

Example 4 identification of transmembrane efficiency of transmembrane peptide

(1) Design of siRNA

Based on the gene sequence of IKCA1, the applicant designed 1siRNA sequence, the sense strand of which is Si-IKCA1-2: 5'-GCCTGGATGTTCTACAAACAT-3', siRNA sequence, synthesized by Shanghai, wherein the siRNA is loaded on plasmid DNA, and the siRNA refers to the siRNA loaded on the plasmid DNA.

(2) Conjugation of siRNA to TM-2 Polypeptides

Plasmid DNA was labeled with YOYO-1 fluorescent label, 2.5. mu.l YOYO-1 (10. mu.M) per. mu.g siRNA was incubated in an air bath at 37 ℃ for 30min to allow the YOYO-1 fluorescent label to be completely embedded in the DNA base pair.

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. Conjugation was performed under the same conditions as for the short peptide TAT polypeptide as a positive control.

The stem cells of example 1 were seeded 24h before siRNA/peptide complexes in NEST 15mm glass culture dishes at 3X 10⁴Individual cells, 5% CO at 37 ℃₂Culturing in an incubator. Sucking out the culture solution after 24h, adding 1ml of DMEM culture solution which is serum-free and contains YOYO-1 fluorescence labeled polypeptide/siRNA compound, incubating for 4h in an incubator at 37 ℃, sucking out the culture solution after 4h, washing for 3 times by using PBS precooled at 4 ℃, fixing for 10min by using 4% formaldehyde/PBS solution, washing for 3 times by using PBS, staining cell nucleus for 15min by using 2.0 mu g/ml DAPI/PBS solution, and washing for 3 times by using PBS (in the above process, the cell is washed by using dropping solution slowly attaching to wall to avoid cell drop as much as possible). The samples were photographed under a CarlZeiss LSM510 laser confocal microscope and observed under a 60-fold oil microscope, and the excitation wavelengths were 543nm respectively. The result shows that the membrane-penetrating peptide has higher fluorescent cell number and better membrane-penetrating efficiency than the membrane-penetrating peptide in the prior art, and the membrane-penetrating efficiency reaches more than 97.5 percent of cell number.

Example 5 specific identification of transmembrane peptides

The siRNA and the Tm-2 polypeptide are introduced into a human pancreatic cancer CFPAC-1 cell, a human myeloblast leukemia cell, an AL7P/HL-60R cell, a human immortalized epidermal cell HaCaT cell and an HEK293F cell according to the operation mode of example 4, and after identification, the cells all show very weak fluorescence activity and number, and the HEK293F cell with the strongest membrane permeability has only 5% membrane permeability efficiency, so that the transmembrane peptide provided by the invention has better specificity, and the primary analysis reason is that the transmembrane peptide has the surface specificity binding and activating activity of cervical cancer stem cells, and can specifically stimulate the cervical cancer cells to identify and endocytose the polypeptide so as to realize the delivery of external nucleic acid.

Example 6 measurement of siRNA inhibitory Effect

(1) Design of siRNA

Based on the gene sequence of RabJ, the applicant researches and designs 2siRNA sequences, wherein the sense strands of the siRNA sequences are SiRabJ1: 5'-gcagatgccattcgcagaat-3' (SEQ ID N0:1) and SiRabJ2: 5'-tagcagtgctagtttcacc-3' (SEQ ID N0:2), the 2siRNA sequences are synthesized by Shanghai, the siRNA is loaded on plasmid DNA, and the siRNA refers to the siRNA loaded on the plasmid DNA.

(2) Conjugation of siRNA to TM-2 Polypeptides

(3) Complex transfection

The stem cells of example 1, 24h before administration, were seeded at 3X 10 in NEST 15mm glass culture dishes⁴Individual cells, 5% CO at 37 ℃₂Culturing in an incubator. Sucking out the culture solution after 24h, adding 1ml of DMEM culture solution which is serum-free and contains YOYO-1 fluorescence labeled polypeptide/siRNA complex, and incubating in an incubator at 37 DEG CCulturing for 4h, sucking out the culture solution after 4h, washing with PBS for 3 times, culturing with RPMI-1640 culture solution for 36h, and harvesting cells for detection.

(4) mRNA detection by RT-PCR and PCR product quantitative analysis method

Total RNA was extracted according to TRIzol Total RNA extraction kit. 2.5ug of total RNA was added to a 50u1 reverse transcription reaction system, and reverse transcription was performed using SuperScriptTMII reverse transcription kit according to the instructions.

PCR amplification PCR reaction system was conventional (reagents from Invitrogen).

RabJ primers 1: 5'-ATG CCG AAG AGG AAG GAG CCC-3';

RabJ primer 2: 5'-GTT TCA CCA AAG AAC AAG CAG-3';

5'-GCATCGTGATGGACTCCG-3' as beta-actin primer;

beta-actin primer 2: 5'-TCGGAAGGTGGACAGCGA-3', the above sequences are all synthesized by Shanghai Bioengineering Co.

Cycle conditions were 94 ℃ denaturation for 45 seconds, 58 ℃ annealing for 1 minute, 72 ℃ extension for 1 minute, and final extension for 10 minutes. And the amplification product of beta-actin is used as an internal reference control.

And (3) quantification of PCR products, namely taking 10u1 amplification products to carry out electrophoresis on 15g/L agarose gel electrophoresis, taking PCR mark (Huamei company) as a molecular mass standard, and carrying out voltage of 100V for 20 minutes. Staining with ethidium bromide, developing with ultraviolet reflectometer, and taking pictures. Scanning analysis was used for semi-quantification of the gene of interest (the expression level of RabJ mRNA is reflected in the ratio of RabJ/beta-actin cDNA).

The results are shown in fig. 2, and the cells treated with siRNA for 36h detected that SiRabJ1 can down-regulate mRNA expression compared with the negative control group compared with SiRabJ2, but SiRabJ1 has more significant down-regulation effect. Particularly, in the presence of the transmembrane peptide of the present invention, the effect of accelerating interference is more remarkable than the inhibitory effect without the transmembrane peptide, and an unexpected technical effect is obtained.

And collecting cells of each group for 36h, preparing cell lysate, performing Western blot detection by using RabJ polyclonal antibody, and detecting the expression of the RabJ protein level. The results in FIG. 3 show that the polypeptide of SEQ ID NO: 1 after 36h, has faster and better inhibition effect than that without adding TM-2, and adopts TM-2 polypeptide mediated SEQ ID NO: 2 was also faster and better than without TM-2 after 36h, but clearly, SEQ ID NO: 1 to SEQ ID NO: 2 has a better effect.

Example 7 inhibition of tumorigenicity in tumor cells in vivo following RabJ siRNA interference with RabJ expression

Cervical cancer stem cells (1X 10) transfected (final concentration of 100nmol) with the transmembrane peptide-siRNA conjugate described in example 4⁶) The mouse was injected subcutaneously into the lower right flank of Balb/C nude mice, and then observed for five weeks, and the incidence of tumor (number of mice presenting tumor/number of nude mice injected with cells) was calculated. In addition, SEQ ID NO: 1 and SEQ ID NO: 2 cervical cancer stem cells were transfected according to the same experimental protocol as described above, as a control group.

The results show that the TM-2 polypeptide-mediated SEQ ID NO: 1, and TM-2 polypeptide-mediated SEQ ID NO: 2, the tumorigenicity of the siRNA is (30.52 +/-1.17)%, and the siRNA has the sequence shown in SEQ ID NO: 1, the tumorigenicity of the siRNA is (17.59 +/-1.01)%, and the siRNA has the sequence shown in SEQ ID NO: 2, the tumorigenicity of the siRNA is (40.88 +/-2.13)%, which fully shows that the transmembrane peptide can improve the penetration rate and the penetration efficiency of the siRNA and can obviously inhibit the tumorigenicity of cervical cancer stem cells in animal bodies.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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Claims

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, and carrying out coupling on siRNA of the RabJ gene shown in the specification, wherein the siRNA is loaded on plasmid DNA.

2. Use of the conjugate of claim 1 in the preparation of an agent for inhibiting cervical cancer cells.