ShRNA (short hairpin Ribonucleic Acid) interference library for inhibiting expression of CT45A3, vector and application of shRNA interference library in anti-colon cancer medicine

Abstract

The invention discloses a shRNA (short hairpin Ribonucleic Acid) interference library for inhibiting expression of CT45A3, a vector and application of the shRNA interference library in anti-colon cancer drugs, and belongs to the technical field of gene drugs. The shRNA interference library comprises one or more of the sequences as shown in SEQ ID NO.1-3. The invention also discloses a method for preparing the shRNA interference library. The shRNA interference library or the expression vector thereof can efficiently and specifically down-regulate the expression level of CT45A3 in colorectal cancer cells, so that abnormal activation of a downstream cancer promoting signal channel mediated by the shRNA interference library or the expression vector is blocked, and the development of malignant phenotypes is remarkably inhibited. The shRNA interference library has potential clinical application value in targeted therapy of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of gene drug technology, specifically relating to a shRNA interference library and vector for inhibiting CT45A3 expression and its application in anti-colon cancer drugs. Background Technology

[0002] The development of colorectal cancer is a complex process involving multiple factors and steps, influenced by genetic background, environmental factors, and lifestyle. Family history, especially hereditary colorectal cancer syndromes, is a significant risk factor. In addition, obesity, a long-term high-fat, high-calorie diet, excessive consumption of red meat and processed meat products, type 2 diabetes, insulin resistance, chronic inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease), insufficient dietary fiber intake, smoking, and excessive alcohol consumption have also been proven to be closely related to the development of colorectal cancer.

[0003] Currently, for patients with early-stage colorectal cancer, surgical resection is the primary treatment, often combined with preoperative or postoperative adjuvant chemotherapy, which usually yields a good prognosis. However, for patients with metastatic colorectal cancer, since the optimal surgical window has passed, treatment strategies mainly include chemotherapy, targeted therapy, and immunotherapy. But even with these systemic treatment regimens, patients still face a very high risk of recurrence, and the overall prognosis is generally poor.

[0004] The aforementioned bottlenecks in clinical treatment have made the discovery of novel therapeutic targets at the molecular level a key research focus for overcoming current challenges. Colorectal cancer is often accompanied by epigenetic regulatory disorders, particularly alterations in gene methylation, leading to the abnormal activation of genes not expressed in normal somatic cells. Cancer / testis antigen (CT antigen) is a class of proteins expressed only in the testes in normal tissues but abnormally expressed in various tumors. Their encoding genes are typically highly methylated in normal somatic cells, with transcription strictly repressed; however, in tumors such as colorectal cancer, due to abnormal methylation, these genes are re-expressed and participate in the malignant transformation and proliferation of tumors. Therefore, CT antigen has significant clinical application potential and can serve as a diagnostic biomarker and a candidate molecule for targeted therapy.

[0005] Currently, over a hundred CT antigen genes have been identified in the human genome, but research on their specific functions and regulatory mechanisms in tumors remains relatively limited, and CT antigens that can serve as effective intervention targets are even scarcer. Therefore, discovering novel CT antigen targets that play a key driving role in colorectal cancer and developing corresponding efficient and specific intervention strategies has become an urgent need to promote the optimization of clinical treatment for colorectal cancer. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide an shRNA interference library and vector for inhibiting CT45A3 expression, and its application in anti-colon cancer drugs.

[0007] In a first aspect, the present invention provides an shRNA interference library for inhibiting CT45A3 expression, the shRNA interference library comprising one or more of the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0008] In some embodiments, the shRNA interference library contains the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0009] In some embodiments, the molar ratio of the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in the shRNA interference library is 1:1:1.

[0010] In some embodiments, the expression vector contains the shRNA interference library described above.

[0011] In a second aspect, the present invention provides the use of the above-described shRNA interference library or the above-described expression vector in the preparation of a drug that inhibits CT45A3 expression.

[0012] In some embodiments, the drug is a drug that inhibits the expression of CT45A3 in colon cancer cells.

[0013] In a third aspect, the present invention provides the use of the above-described shRNA interference library or the above-described expression vector in the preparation of anti-colon cancer drugs.

[0014] In some embodiments, the antitumor drug is used to inhibit the expression of oncogenes MYC and CyclinD1 in colon cancer cells.

[0015] In some embodiments, the antitumor drug is used to inhibit the proliferation of colon cancer cells.

[0016] Compared with existing technologies, this invention utilizes bioengineering techniques to construct multiple DNA sequences corresponding to CT45A3 shRNAs into the pLKO.1 eukaryotic expression vector. After successful vector construction was confirmed by PCR identification and sequence analysis, this eukaryotic shRNA library was transfected into colorectal cancer cells. Immunoblotting demonstrated its intervention in CT45A3 expression. Studies on the malignant phenotype of colorectal cancer revealed that it can inhibit the expression of oncogenes MYC and Cyclin D1 in colorectal cancer, thereby inhibiting the proliferation of colorectal cancer cells. This drug shows significant development potential for the clinical treatment of colorectal cancer. Attached Figure Description

[0017] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments taken in conjunction with the accompanying drawings, in which:

[0018] Figure 1 Electrophoresis image of PCR identification of shRNA expression vector targeting CT45A3 (M: DNA marker; negative control: sterile water; lanes 1-3: corresponding clones of SEQ ID NO.1-3).

[0019] Figure 2 Statistical plot of silencing efficiency of CT45A3 protein expression by a single shRNA and shCT45A3 library (**, P < 0.01; ***, P < 0.001).

[0020] Figure 3 The figure shows the results of detecting the inhibitory effect of the shCT45A3 library on CT45A3 protein expression in colon cancer cells (*, P < 0.001).

[0021] Figure 4 Figure 1 shows the results of detecting the downregulation of MYC and Cyclin D1 mRNA expression in colon cancer cells by the shCT45A3 library (*, P < 0.001).

[0022] Figure 5 The image shows the results of detecting the inhibitory effect of the shCT45A3 library on the in vitro proliferation of colon cancer cells (***, P < 0.001). Detailed Implementation

[0023] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Some embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of the invention will be thorough and complete.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] Example 1

[0026] Through high-throughput screening combined with phenotypic validation, the research group discovered that CT45A3 is highly expressed in some colorectal cancer tissues, and that intervention in its expression can significantly inhibit the abnormal activation of proto-oncology pathways and tumor cell proliferation. Based on this, the research group designed and constructed several independent shRNA encoding genes targeting CT45A3, the nucleotide sequences of which are shown in SEQ ID NO.1-3.

[0027] shCT45A3-1: 5'-AGAAACTGAAACGTATGATTT-3' (SEQ ID NO. 1).

[0028] shCT45A3-2: 5'-TAGATCCTGAAACTGTGTTTA-3' (SEQ ID NO. 2).

[0029] shCT45A3-3: 5'-ACTGCAGTCAGGAAGCGATTT-3' (SEQ ID NO. 3).

[0030] The forward and reverse primers for the sequences shown in SEQ ID NO.1-3 are shown in Table 1.

[0031] Table 1. Forward and reverse primers for sequences shown in SEQ ID NO. 1-3

[0032]

[0033] Example 2

[0034] (1) Primer annealing: Forward primers and their corresponding reverse primers targeting the sequence shown in SEQ ID NO.1 were synthesized. Equal volumes of forward and reverse primers were mixed to a final concentration of 5 μM, and 10% of 10×Tango buffer was added. The volume was then brought to 100 μL with ddH2O. The mixture was placed in a boiling water bath for 5 minutes, followed by natural cooling for 3 hours to complete primer annealing and obtain the double-stranded DNA insert corresponding to SEQ ID NO.1.

[0035] (2) Vector digestion: Take 4 μg of pLKO.1 plasmid, add 1 μL each of AgeI and EcoRI restriction enzymes to a 40 μL digestion system, and digest at 37°C for 2 hours. Separate the digestion products by 1% agarose gel electrophoresis (100 V, 30 min), cut the target band under UV imaging, and purify and recover using a DNA gel recovery kit.

[0036] (3) Ligation reaction: The recovered enzyme-digested pLKO.1 plasmid (1 ng) was mixed with 2 μL of annealed double-stranded DNA insert, 1×T4 DNA ligase buffer and 1 μL of T4 DNA ligase were added, and the mixture was ligated in a water bath at 16℃ for 8 hours to obtain the ligation product.

[0037] (4) Transformation and PCR verification of positive clones:

[0038] Mix 2 μL of the ligation product with 50 μL of competent cells, incubate on ice for 30 minutes, then heat shock in a 42°C water bath for 90 seconds, followed by cooling on ice for 4 minutes. Add 400 μL of LB medium and incubate at 37°C on a shaker for 45 minutes. Spread 100 μL of the bacterial culture evenly onto LB solid medium containing ampicillin and incubate overnight at 37°C. The next day, pick a single colony and inoculate it into LB liquid medium containing ampicillin, then incubate overnight at 37°C on a shaker for amplification.

[0039] One μL of bacterial culture was used as a template for PCR identification. The reaction mixture consisted of 10 μL of 2×PCR premix, 1 μL of bacterial template, 1 μL of 5 μM forward and reverse primer mixture, and ddH2O to a final volume of 20 μL. The PCR amplification program was 25 cycles: denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 30 seconds. The reaction products were analyzed by 2% agarose gel electrophoresis (120 V, 30 minutes).

[0040] The shRNA expression vectors for the sequences shown in SEQ ID NO.2 and SEQ ID NO.3 were constructed and identified using the same methods described above.

[0041] Electrophoresis images of PCR identification of shRNA expression vectors targeting the sequences shown in SEQ ID NO. 1-3 for CT45A3 are shown below. Figure 1 As shown, M is the DNA marker; the negative control is sterile water; lanes 1-3 are the clones corresponding to SEQ ID NO.1-3.

[0042] Figure 1 The results showed that each clone group amplified positive bands of the expected size, indicating that the shRNA vector targeting CT45A3 was successfully constructed.

[0043] Example 3

[0044] The three constructed shRNA vectors targeting CT45A3 (three target sites) were mixed in a molar ratio of 1:1:1 to create the shCT45A3 library targeting CT45A3.

[0045] To compare the silencing efficiency of the shCT45A3 library with that of a single target, each single shRNA vector and the shCT45A3 library were transfected into HUTU80 colon cancer cells. The specific procedures are as follows:

[0046] HUTU80 cells were seeded into six-well plates. When the cell confluence reached 70%–90%, the medium was replaced with 2 mL of Opti-DMEM medium without serum and antibiotics, in preparation for transfection.

[0047] The experiment consisted of 5 groups, each with 3 replicates, and each group underwent independent transfection.

[0048] Transfection with the empty pLKO.1 vector;

[0049] Transfect CT45A3 shRNA-1;

[0050] Transfect CT45A3 shRNA-2;

[0051] Transfect CT45A3 shRNA-3;

[0052] Transfect the shCT45A3 library.

[0053] For each of the above groups, transfection was performed using the following methods:

[0054] Take two 1.5 mL centrifuge tubes: add 100 μL of Opti-DMEM and 10 μL of Lipofectamine 2000 to tube A, and add 3 μg of the corresponding shRNA plasmid and 100 μL of Opti-DMEM to tube B. After incubating at room temperature for 15 min, gently mix the two solutions and add them dropwise to the cell culture medium. 36 h after transfection, wash the cells once with ice-cold PBS, add 1 mL of TRIzol reagent to each well, and extract total RNA using the chloroform-ethanol method. After determining the RNA concentration and purity, calculate and pipette the volume of solution required to contain 1 μg of total RNA based on the measured concentration, and extract using PrimeScript. TM The RT reverse transcription kit (Takara, catalog number RR037A) was used to synthesize cDNA via reverse transcription according to the instructions.

[0055] Using cDNA obtained from each group as templates, the mRNA expression level of the CT45A3 gene was detected by real-time quantitative PCR using PowerTrack SYBR Green (ThermoFisher, catalog number A46109). With β-actin as an internal reference gene, the relative expression level of the CT45A3 gene in each group was calculated using the 2ΔΔCt method. The silencing efficiency of each individual shRNA and the shCT45A3 library was analyzed. Results Figure 2 As shown.

[0056] The RT-PCR primer sequences for CT45A3 and the internal reference gene β-actin (ACTB) are shown in Table 2:

[0057] Table 2. RT-PCR primer sequences for CT45A3 and the internal reference gene β-actin (ACTB)

[0058]

[0059] Figure 2 The results showed that the silencing efficiency of the shCT45A3 library was significantly higher than that of any single shRNA (P < 0.01; *, P < 0.001). This indicates that the shCT45A3 library has lower off-target effects and higher targeting efficiency compared to single shRNAs, meaning it has a significant advantage in targeting.

[0060] Example 4

[0061] Western blot was used to detect CT45A3 protein expression levels. The specific procedures were as follows:

[0062] HUTU80 colon cancer cells were seeded into six-well plates. When the cell confluence reached 70%–90%, the medium was replaced with 2 mL of serum- and antibiotic-free Opti-DMEM. Transfection complexes were prepared in two 1.5 mL centrifuge tubes: tube A contained 100 μL of Opti-DMEM and 10 μL of Lipofectamine 2000; tube B contained 3 μg of the shCT45A3 library plasmid targeting CT45A3 and 100 μL of Opti-DMEM. After incubation at room temperature for 15 minutes, the two solutions were gently mixed and added dropwise to the cell culture medium. Six hours after transfection, the medium was replaced with fresh complete medium, and cells were harvested after 48 hours of culture.

[0063] Cells were washed three times with PBS, and an appropriate amount of immunoprecipitation lysis buffer (RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors) was added. Lysis was performed on ice for 30 minutes, with repeated pipetting and grinding during this period. The supernatant was collected by centrifugation at 4°C. A portion of the protein supernatant was taken and 5× loading buffer was added at a volume ratio of 4:1. The protein was denatured by heating at 100°C for 7 minutes. The expression level of CT45A3 protein was detected by Western blotting: SDS-PAGE electrophoresis (80 V), wet transfer (300 mA), blocking with 5% skim milk powder for 2 hours, incubation with primary antibody at 4°C overnight, washing, incubation with HRP-labeled secondary antibody, washing again, and then imaging using a Bio-Rad chemiluminescence imaging system. Statistical analysis was performed, and the results are shown below. Figure 3 As shown.

[0064] Western Blot results ( Figure 3 The results showed that the CT45A3 protein band was significantly weakened in the transfected shCT45A3 library group, indicating that the constructed shCT45A3 library can effectively inhibit the expression of CT45A3 in colon cancer cells.

[0065] Example 5

[0066] HUTU80 colon cancer cells were seeded in six-well plates. When the cells adhered and reached 70%–90% confluence, the medium was replaced with 2 mL of serum- and antibiotic-free Opti-DMEM. An shRNA library transfection group and a negative control group were set up. Following the method described in Example 4, the shCT45A3 library plasmid targeting CT45A3 and the packaging plasmid (control) were co-transfected into HUTU80 cells. Six hours after transfection, the medium was replaced with fresh complete medium, and cells were collected after 48 hours of culture. Cells were washed three times with PBS, and total RNA was extracted using TRIzol reagent. After determining the concentration and purity, 1 μg of total RNA was reverse transcribed to synthesize cDNA. Using cDNA as a template, real-time quantitative PCR was performed using the SYBR Green assay. The target genes were the oncogenes MYC and Cyclin D1 (CCND1), and the internal reference gene was β-actin (ACTB). The reaction primers are shown in Table 3.

[0067] Table 3 Primer sequences for genes MYC, CCND1, and ACTB.

[0068]

[0069] The reaction system was prepared according to the kit instructions and amplified using a real-time quantitative PCR instrument. The program was set as follows: 95℃ pre-denaturation for 2 minutes; 95℃ denaturation for 15 seconds; 60℃ annealing and extension for 30 seconds, for a total of 40 cycles. After the reaction, the melting curve was analyzed, and the relative expression level of the target gene was calculated using the 2ΔΔCt method. The results are shown below. Figure 4As shown.

[0070] qRT-PCR results showed that, compared with the negative control group, the mRNA expression levels of MYC and Cyclin D1 in HUTU80 cells transfected with the shCT45A3 library targeting CT45A3 were significantly decreased, indicating that silencing CT45A3 expression can effectively inhibit the expression of oncogenes MYC and Cyclin D1 in colon cancer cells at the transcriptional level. Figure 4 ).

[0071] Example 6

[0072] HUTU80 colon cancer cells were transfected with the shCT45A3 library plasmid targeting CT45A3 and the negative control plasmid, respectively, according to the method described in Example 4. After culturing for 48 hours, the cells were digested and collected, counted, and the cell density was adjusted to 5 × 10⁹ cells / year. 4 Cells / mL. 100 μL of cell suspension was seeded into 96-well plates, 5000 cells per well, with 5 replicates per group. Cells were incubated at 37℃ in a 5% CO2 incubator for 5 days. On days 1, 2, 3, 4, and 5 post-seeding, 10 μL of CCK-8 reagent was added to each well, and incubation continued for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Cell growth curves were plotted with incubation time on the x-axis and absorbance on the y-axis. The results are shown below. Figure 5 As shown in the figure. The results showed that, compared with the negative control group, the proliferation rate of HUTU80 cells transfected with the shRNA library targeting CT45A3 was significantly slowed down, and the absorbance values ​​at each time point were significantly lower than those of the control group, indicating that silencing CT45A3 expression can effectively inhibit the in vitro proliferation ability of colon cancer cells. Figure 5 ).

[0073] The specific KDM8 interference library according to embodiments of the present invention has at least one of the following advantages:

[0074] 1. Specifically inhibits the upregulation of KDM8 expression induced by a high-fat diet, promoting its expression back to normal levels;

[0075] 2. It specifically antagonizes the induction of hepatocyte damage by upregulation of KDM8 expression and inhibits the level of ALT, a liver injury-related marker;

[0076] 3. By inhibiting KDM8 expression, it alleviates the metabolic stress on the liver, reduces lipid accumulation in hepatocytes, and inhibits the progression of hepatic steatosis and damage.

Claims

1. A library of shRNA interference that inhibits CT45A3 expression, characterized in that, The shRNA interference library contains one or more of the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.

3.

2. The shRNA interference library as described in claim 1, characterized in that, The shRNA interference library contains the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.

3.

3. The shRNA interference library according to claim 2, characterized in that, In the shRNA interference library, the molar ratio of the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is 1:1:

1.

4. An expression carrier, characterized in that, The expression vector contains the shRNA interference library as described in any one of claims 1-3.

5. The use of the shRNA interference library as described in any one of claims 1-3 or the expression vector as described in claim 4 in the preparation of a drug that inhibits CT45A3 expression.

6. The application as described in claim 5, characterized in that, The drug is a drug that inhibits the expression of CT45A3 in colon cancer cells.

7. The use of the shRNA interference library as described in any one of claims 1-3 or the expression vector as described in claim 4 in the preparation of anti-colon cancer drugs.

8. The application according to claim 7, characterized in that, The antitumor drug is used to inhibit the expression of oncogenes MYC and Cyclin D1 in colon cancer cells.

9. The application according to claim 7, characterized in that, The antitumor drug is used to inhibit the proliferation of colon cancer cells.

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