Colon cancer stemness inhibiting polypeptide, expression vector and application thereof

Abstract

This invention discloses a colorectal cancer stem cell sex-inhibiting polypeptide, its expression vector, and its applications, belonging to the field of bioengineering technology. This invention is the first to discover the crucial role of ZCCHC14 protein in maintaining the characteristics of colorectal cancer stem cells, and based on this, develops an inhibitory polypeptide targeting ZCCHC14. Experiments have shown that this polypeptide can significantly downregulate the expression of colorectal cancer stem cell markers, upregulate the expression of differentiation markers, inhibit the spheroidization ability of stem cells, and significantly reduce EdU incorporation rate, effectively weakening the characteristics of colorectal cancer stem cells, promoting cancer cell differentiation, and inhibiting proliferation. It can be applied to the preparation of anti-colorectal cancer drugs, especially drugs that inhibit the proliferation of colorectal cancer cells, and has significant clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a colorectal cancer stem cell sex-inhibiting polypeptide, its expression vector, and its applications. Background Technology

[0002] Cancer stem cells are a subpopulation of cells in tumor tissue that possess stem cell characteristics, including self-renewal capacity and multi-lineage differentiation potential. Studies have shown that cancer stem cells are a significant factor contributing to chemotherapy resistance, distant metastasis, and disease recurrence in cancer. On one hand, they highly express ABC family drug efflux pumps, actively pumping chemotherapeutic drugs out of the cells, preventing them from reaching effective cytotoxic concentrations. On the other hand, they are typically in a relatively quiescent cell cycle state (G0 phase), while most chemotherapeutic drugs primarily target actively dividing cells, thus being ineffective against quiescent cancer stem cells. These two mechanisms work together to allow cancer stem cells to survive chemotherapy, becoming a root cause of tumor recurrence.

[0003] Colorectal cancer is a common malignant tumor of the digestive system. Current research indicates that the occurrence of colorectal cancer is closely related to mutations in key genes within intestinal stem cells. Intestinal stem cells are fundamental to maintaining intestinal epithelial renewal, and under normal circumstances, they maintain the stability of the stem cell pool through symmetrical and asymmetrical division. When tumor suppressor genes such as APC, TP53, and SMAD4 mutate, the self-renewal signals of intestinal stem cells are continuously activated, differentiation signals are blocked, and apoptosis mechanisms fail, leading to uncontrolled stem cell proliferation. These mutated stem cells further acquire enhanced self-renewal capacity and tumorigenic potential, transforming into tumor stem cells. Studies have shown that loss-of-function mutations in APC, abnormal inactivation of the TP53 gene, and activating mutations in the KRAS gene occur frequently in colorectal cancer tissues.

[0004] Therefore, finding new molecular targets that can inhibit the characteristics of colon cancer stem cells and developing corresponding intervention strategies are of great clinical significance for improving the treatment effect of CRC. Summary of the Invention

[0005] The purpose of this section is to provide a colorectal cancer stem cell sex-inhibiting polypeptide, its expression vector, and its applications.

[0006] Based on previous systematic research, the inventors discovered that a gene, ZCCHC14, whose function is not yet fully understood, plays a crucial role in maintaining and promoting the characteristics of colon cancer stem cells. The ZCCHC14 protein contains a typical CCHC-type zinc finger domain and possesses RNA-binding capabilities. Based on this, this application successfully developed an inhibitory polypeptide targeting the ZCCHC14 protein. This polypeptide can significantly weaken the stem cell characteristics of colon cancer cells, promote the transformation of cancer cells into a differentiated state, and inhibit their proliferative capacity, thereby effectively delaying tumor progression.

[0007] In a first aspect, the present invention provides a colorectal cancer stem cell sex-inhibiting polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a nucleotide sequence encoding the above-mentioned colorectal cancer stem cell sex-inhibiting polypeptide.

[0009] In some embodiments of the present invention, the nucleotide sequence is as shown in SEQ ID NO:2.

[0010] In a third aspect, the present invention provides a recombinant expression vector comprising the above-described nucleotide sequence.

[0011] In some embodiments of the present invention, the recombinant expression vector uses pLKO-CMV as the backbone vector.

[0012] In a fourth aspect, the present invention provides a method for constructing the above-mentioned recombinant expression vector, comprising the following steps:

[0013] The nucleotide sequence shown in SEQ ID NO:2 was synthesized from the entire genome.

[0014] The synthesized nucleotide sequence was double-digested with the pLKO-CMV eukaryotic expression vector using restriction endonucleases.

[0015] Recombinant expression plasmids are constructed by ligating enzyme-digested nucleotide fragments with linearized vectors.

[0016] In some embodiments of the present invention, the restriction endonucleases are EcoRI and KpnI.

[0017] In a fifth aspect, the present invention provides the application of the above-mentioned polypeptide, nucleotide sequence, and recombinant expression vector in the preparation of a drug for treating colon cancer.

[0018] In some embodiments of the present invention, the drug for treating rectal cancer is a drug that inhibits the proliferation of colon cancer cells.

[0019] In a sixth aspect, the present invention provides a method for screening candidate drugs that inhibit colorectal cancer stem cells, the method comprising: using ZCCHC14 protein as a target, screening candidate substances that can inhibit the activity or expression of ZCCHC14 protein.

[0020] Compared with existing technologies, this invention is the first to discover the crucial role of ZCCHC14 protein in maintaining the characteristics of colorectal cancer stem cells, and based on this, a ZCCHC14-targeting inhibitory peptide (SEQ ID NO:1) was developed. Experiments demonstrated that this peptide significantly downregulated the expression of colorectal cancer stem cell markers (LGR5, ASCL2), upregulated the expression of differentiation markers (KRT20, CEACAM1), inhibited stem cell spheroidization ability, and significantly reduced EdU incorporation rate (***P < 0.001), effectively weakening the characteristics of colorectal cancer stem cells, promoting cancer cell differentiation, and inhibiting proliferation. This invention provides a novel molecular target and candidate drug for colorectal cancer treatment, and has significant clinical application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0022] Figure 1 Electrophoresis image for PCR identification of a clone of a sex-inhibiting peptide for colon cancer stem cells;

[0023] Figure 2 The image shows the results of Western blot detection of sex-inhibiting peptides in colon cancer stem cells.

[0024] Figure 3 A statistical graph showing the effect of sex-inhibiting peptides on the expression of stem cell markers in colon cancer cells;

[0025] Figure 4 Statistical chart of the results of detecting the ability of colorectal cancer stem cell sex-inhibiting peptides to inhibit the spheroidization of colorectal cancer stem cells;

[0026] Figure 5 A statistical graph showing the EdU detection results of the inhibitory peptide on DLD1 cell proliferation in colorectal cancer stem cells. Detailed Implementation

[0027] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] This invention is based on the fact that ZCCHC14 protein can significantly promote colon cancer stem cell activity, and designs a colon cancer stem cell activity inhibitory peptide that can potentially target and intervene in the process of colon cancer.

[0031] The amino acid sequence of the colorectal cancer stem cell sex-inhibiting polypeptide is designated as SEQ ID NO:1, as shown below:

[0032] 1 MVEKRCPLQR DGVYRWFSEL

[0033] 21 PSPQRVEFLC GLLDLCIPLE

[0034] 41 LRFLGSCLED LARKDYHSLR

[0035] 61 DSEIKANNPA DLGSLTNLTD

[0036] 81 EVVRSKLLVS LALLGSEQRE

[0037] 101 AAGVLYRTLT HIDSIIHNYG

[0038] 121 LQLNEGRTGD EFLLLFTMAS

[0039] 141 NHPAFSFHQK QVLRQELTQI

[0040] 161 QSSLNGGGGH GGKGAPGPGG

[0041] 181 ALPTCPACHK ITPRTEAPVS

[0042] 201 SVSNSLENAL HTSAHSTEES

[0043] 221 LPKRPLGKHS KVSVEKIDLK

[0044] The cDNA sequence encoding the sex-suppressing polypeptide of colon cancer stem cells is designated as SEQ ID NO:2, as shown below:

[0045] 1 ATGGTTGAGA AGCGTTGTCC TCTGCAACGT GATGGTGTTT ACCGTTGGTT CAGCGAGCTG

[0046] 61 CCAAGTCCAC AACGTGTTGA GTTCCTTTGT GGTCTTCTTG ATCTTTGCAT TCCACTTGAG

[0047] 121 CTTCGTTTCC TTGGTAGCTG TCTTGAGGAT CTTGCTCGTA AGGATTACCA CAGCCTTCGT

[0048] 181 GATAGCGAGA TTAAGGCTAA TAATCCTGCT GATCTTGGTA GCCTTACTAA TCTTACTGAT

[0049] 241 GAGGTTGTTC GTAGCAAGCT TCTTGTTAGC CTTGCTCTTC TTGGTAGCGA GCAGCGTGAG

[0050] 301 GCTGCTGGTG TTCTTTACCG TACTCTTACT CACATTGATA GCATTATTCA CAATTACGGT

[0051] 361 CTTCAACTTA ATGAGGGTCG TACTGGTGAT GAGTTCCTTC TTCTTTTCAC TATGGCTAGC

[0052] 421 AATCACCCTG CTTTCAGCTT CCACCAAAAG CAAGTTCTTC GTCAAGAGCT TACTCAAATT

[0053] 481 CAAAGCAGCC TTAATGGTGG TGGTGGTCAC GGTGGTAAGG GTGCTCCAGG TCCAGGTGGT

[0054] 541 GCTCTTCCAA CTTGTCCTGC TTGTCACAAG ATTACTCCTC GTACTGAGGC TCCTGTTAGC

[0055] 601 AGCGTTAGCA ATAGCCTTGA GAATGCTCTT CACACTAGCG CTCACAGCAC TGAGGAGAGC

[0056] 661 CTTCCAAAGC GTCCTCTTGG TAAGCACAGC AAGGTTAGCG TTGAGAAGAT TGATCTTAAA

[0057] Example 1: Construction and liposome delivery of a colon cancer stem cell sex-inhibiting peptide expression vector

[0058] 1. Construction of mammalian cell expression vector for sex-inhibiting peptides in colon cancer stem cells

[0059] The cDNA sequence encoding the colorectal cancer stem cell sex inhibitory peptide (nucleotide sequence shown in SEQ ID NO:2) was synthesized using a whole-genome synthesizer (manufactured by Shanghai Sangon Biotech). EcoRI and KpnI were inserted upstream and downstream of this sequence, respectively. 4 μg of the obtained sample nucleic acid was added to 34 μl of sterile purified water, followed by 4 μl of 10× restriction enzyme buffer, and 1 μl each of EcoRI and KpnI restriction endonucleases. Simultaneously, 1 μg of the pLKO-CMV vector was diluted in 34 μl of sterile purified water, and 4 μl of 10× restriction enzyme buffer, and 1 μl each of EcoRI and KpnI restriction endonucleases were added. Both samples were incubated at 37°C for 2 h, and the DNA fragments were recovered using a PCR nucleic acid recovery kit. The enzyme-digested cDNA encoding the colorectal cancer stem cell sex inhibitory peptide and the pLKO-CMV vector were mixed at a 7:1 ratio, and 2 μl of 10× T4 ligase buffer and 1 μl of T4 ligase were added. Sterile purified water was added to a total volume of 20 μl, and the mixture was incubated at room temperature for 1 h. 2 μl of the ligated recombinant expression vector sample was transformed into 100 μl of DH5α competent Escherichia coli and plated onto Amp agar plates. The plates were incubated overnight at 37°C. Single colonies were then inoculated into LB broth and cultured for 8 h. The recombinant expression vector and the empty control vector were identified by colony PCR. Electrophoresis results are shown below. Figure 1 As shown, compared with the control empty vector, the recombinant expression vector contained a characteristic PCR fragment consistent with the molecule, indicating a positive PCR identification. The identified recombinant vector clones were then verified for sequence correctness using Sanger sequencing, demonstrating the successful construction of the recombinant expression vector for the colorectal cancer stem cell sex repressor peptide.

[0060] 2. Liposome delivery of a colon cancer stem cell sex-inhibiting peptide expression vector into human colon cancer cells.

[0061] Take DLD1 cells in good growth condition, at a density of approximately 2 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 6-well plates and cultured overnight at 37°C and 5% CO2 in DMEM medium containing 10% fetal bovine serum, allowing the cells to adhere to the plate and reach approximately 70% confluence. One hour before transfection, the medium was replaced with Opti-MEM medium free of antibiotics and serum. Following the Lipofectamine 2000 transfection reagent instructions, appropriate amounts of expression vector (experimental group) and empty vector control (control group) were diluted separately in 125 μL of Opti-MEM. In another tube, 5 μL of Lipofectamine 2000 reagent was diluted with 125 μL of Opti-MEM. After standing at room temperature for 5 minutes, the two solutions were mixed, gently mixed, and incubated at room temperature for 20 minutes to form a DNA-liposome complex. Subsequently, 250 μL of the complex was added dropwise to each well, and the plate was gently shaken to distribute it evenly. The plates were then incubated at 37°C for 6 hours, after which the medium was replaced with complete medium.

[0062] Thirty-six hours after transfection, cells were collected and lysed directly with 200 μl of SDS-PAGE loading buffer. After boiling for 5 minutes, 10 μl of the sample was loaded onto a 10% SDS-PAGE gel for electrophoresis to separate protein samples. Western blotting was used to detect the expression of colorectal stem cell sex inhibitory peptides in DLD1 cells. The results are as follows: Figure 2 As shown, a specific band was detected at approximately 20 kDa, indicating that the polypeptide was successfully expressed.

[0063] Example 2: Target peptide induces downregulation of colon cancer stem cell marker expression and upregulation of differentiation markers.

[0064] Collect cells from the control and experimental groups 36 h after transfection. Discard the culture medium, gently wash cells twice with pre-cooled PBS, add 1 mL of TRIzol reagent to each well, and incubate at room temperature for 5 minutes to allow for complete cell lysis. Use a pipette to repeatedly pipette the lysate to break up cells and shear DNA, reducing sample viscosity. Transfer the lysate to RNase-free centrifuge tubes. Following the standard TRIzol assay, add chloroform for phase separation, precipitate RNA with isopropanol, wash the precipitate with 75% ethanol, and finally dissolve the RNA in an appropriate amount of RNase-free water. Determine RNA concentration and purity using Nanodrop (A260 / A280 ratio should be between 1.8 and 2.0), and assess RNA integrity by agarose gel electrophoresis.

[0065] Subsequently, reverse transcription was performed using the RevertAid first-strand cDNA synthesis kit. 1 μg of total RNA was added to a mixture of oligo(dT)18 primers and dNTPs, and incubated at 70°C for 5 minutes, then immediately placed on ice. Next, 5× reaction buffer, RiboLock RNase inhibitor, and RevertAid M-MuLV reverse transcriptase were added, bringing the total volume to 20 μL. The reaction was incubated at 42°C for 60 minutes, followed by heating at 70°C for 5 minutes to terminate the reaction. The resulting cDNA product was stored at −20°C for later use.

[0066] Finally, SYBR Green real-time quantitative PCR was used to detect the expression of target genes. Specific primers were designed targeting stem cell markers LGR5 and ASCL2, and differentiation markers KRT20 and CEACAM1, using ACTB (β-actin) as an internal reference gene.

[0067] The primer sequences for the target gene and the internal reference gene are shown in Table 1:

[0068] Table 1 Primer sequences for target gene and internal reference gene

[0069]

[0070] The 20 μL reaction system contained 10 μL SYBR Green premix, 0.4 μL upstream and downstream primers (final concentration 0.2 μM), 2 μL cDNA template, and 7.2 μL nuclease-free water. The reaction program was: 95℃ pre-denaturation for 5 minutes; followed by 40 cycles of 95℃ denaturation for 15 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds; finally, melting curve analysis was performed to verify amplification specificity. Three technical replicates were set for each sample. The relative expression level of the target gene was calculated using the 2−ΔΔCt method. By comparing the expression differences of stem cell markers and differentiation markers between the experimental and control groups, the results showed that compared with the control group, the expression of stem cell markers was significantly downregulated and the expression of differentiation markers was significantly upregulated in the experimental group (***, P < 0.001). Figure 3 As shown in the figure. This indicates that the colorectal cancer stem cell sex-inhibiting peptide can significantly reduce the expression of colorectal cancer stem cell markers and promote the expression of tumor differentiation markers, proving that this peptide can inhibit colorectal cancer stem cell sex at the gene level.

[0071] Example 3: Colon cancer stem cell inhibition peptides inhibit the ability of colon cancer stem cells to form spheroids.

[0072] DLD1 colon cancer cells in the logarithmic growth phase were collected by trypsin digestion and centrifugation, and washed twice with PBS to completely remove serum. The cells were then resuspended in a pre-prepared stem cell spheroidization medium based on DMEM / F12, supplemented with 1×B27, 1×N2, 20 ng / mL recombinant human basic fibroblast growth factor, and 20 ng / mL recombinant human epidermal growth factor to selectively support stem cell proliferation and spheroidization.

[0073] Cell suspensions were evenly seeded at a density of 5000 cells / well in 6-well plates with an ultra-low adsorption surface, with 2 mL of complete culture medium added to each well. The experiment was divided into two groups: the experimental group was seeded with DLD1 cells pre-stabilized and expressing a colorectal cancer stem cell sex inhibitory peptide, while the control group was seeded with DLD1 cells transfected with an empty vector. Each group had three independent replicate wells. After seeding, the culture plates were placed in an incubator at 37°C, 5% CO2, and saturated humidity for static incubation. During this period, half of the freshly prepared stem cell spheroidizing medium was replaced every 3 days to maintain growth factor activity and remove metabolic waste.

[0074] After 10 days of culture, the formation of cell spheroids in each group was observed and images were acquired under an inverted microscope. ImageJ software was used to count the formed spheroids. The selection criteria were cell spheroids with a diameter greater than 50 μm, regular morphology, and clear boundaries. The number of cell spheroids in each well was counted, and the mean ± standard deviation of three replicate wells was calculated. The results are as follows: Figure 4 As shown in the figure. The inhibitory effect of this peptide on the self-renewal and spheroidization ability of colon cancer stem cells was evaluated by comparing the number of cell spheroids formed in the experimental group and the control group. The results showed that the spheroidization ability of colon cancer stem cells was significantly downregulated after transduction of the colon cancer stem cell inhibitory peptide.

[0075] Example 4: Colon cancer stem cell inhibition peptides significantly inhibit the proliferation of human colon cancer cells.

[0076] Controlled DLD1 cells and DLD1 cells stably expressing a colorectal cancer stem cell sex repressor peptide were seeded into 24-well plates coated with cell spreaders and cultured for 48 hours. Then, 10 µM 5-ethynyl-2'-deoxyuridine (EdU) was added to the culture medium, and incubation continued for 2 hours. The culture medium was then aspirated, and cells were fixed with 10% neutral formaldehyde at room temperature for 15 minutes, followed by three washes with PBS. Next, 1% Triton X-100 solution was added for permeabilization at room temperature for 20 minutes to enhance antibody penetration. After permeabilization, cells were blocked with PBS containing 1% BSA for 1 hour to reduce non-specific binding. Then, EdU primary antibody diluted 1:1000 was added, and the cells were incubated overnight at 4°C. The next day, the primary antibody was recovered, cells were washed three times with PBS, and Alexa Fluor 488-labeled goat anti-mouse secondary antibody was added. The cells were incubated in the dark for 2 hours, followed by three more washes with PBS. Finally, cell nuclei were stained with 10 µg / mL DAPI for 5 minutes. After washing, the slides were mounted with anti-fluorescence quenching mounting medium and observed and images were acquired under a Leica DM5000 fluorescence microscope. The proportion of EdU-positive cells to the total number of cells was counted to assess the effect of the peptide on cell proliferation. The results showed that the EdU positivity rate of the experimental group was significantly lower than that of the control group (***P < 0.001). Figure 5 As shown, this indicates that the polypeptide has the effect of inhibiting the proliferation of colon cancer cells.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A colorectal cancer stem cell sex-inhibiting polypeptide, wherein the amino acid sequence of the colorectal cancer stem cell sex-inhibiting polypeptide is shown in SEQ ID NO:

1.

2. The nucleotide sequence encoding the colorectal cancer stem cell sex-inhibiting polypeptide of claim 1.

3. The nucleotide sequence according to claim 2, wherein, The nucleotide sequence is shown in SEQ ID NO:

2.

4. A recombinant expression vector comprising the nucleotide sequence of claim 2 or 3.

5. The recombinant expression vector of claim 4, wherein, The recombinant expression vector uses pLKO-CMV as its backbone vector.

6. A method of constructing the recombinant expression vector of claim 4, wherein, Includes the following steps: The nucleotide sequence shown in SEQ ID NO:2 was synthesized from the entire genome. The synthesized nucleotide sequence was double-digested with the pLKO-CMV eukaryotic expression vector using restriction endonucleases. Recombinant expression plasmids are constructed by ligating enzyme-digested nucleotide fragments with linearized vectors.

7. The method of claim 7, wherein, The restriction endonucleases are EcoRI and KpnI.

8. The use of the polypeptide of claim 1, the nucleotide sequence of claim 2 or 3, and the recombinant expression vector of claim 4 or 5 in the preparation of a medicament for treating colon cancer.

9. Use according to claim 8, characterized in that, The drug used to treat rectal cancer is a drug that inhibits the proliferation of colon cancer cells.

10. A method of screening a candidate drug for inhibiting a colon cancer stem cell, characterized by, Using ZCCHC14 protein as a target, we screened for candidate substances that could inhibit the activity or expression of ZCCHC14 protein.

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