The application discloses siRNA, shRNA and a lentivirus vector for targeting and inhibiting circZBTB44 and application thereof.

Abstract

This invention discloses a siRNA, shRNA, and lentiviral vector targeting and inhibiting circZBTB44, and their applications. This invention provides a siRNA targeting and inhibiting circZBTB44, and constructs the corresponding shRNA lentiviral expression vector and lentivirus. In vitro experiments demonstrate that the above-mentioned siRNA can inhibit the invasive and migratory abilities of colorectal cancer cells. In vivo experiments demonstrate that the above-mentioned shRNA can inhibit the binding of circZBTB44 with NF90 and PABPC1 to form a ternary complex, thereby reducing the stability of PD-L1 mRNA and downregulating PD-L1 protein expression, ultimately inhibiting the migration and invasion of colorectal cancer cells. The proposed interfering molecule targeting and inhibiting circZBTB44 and its lentiviral vector have significant clinical application value and promising prospects in the preparation of drugs for treating colorectal metastases.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical / anti-tumor drug development in the biopharmaceutical industry, specifically involving a siRNA, shRNA, lentiviral vector that targets and inhibits circZBTB44 and their applications. Background Technology

[0002] Circular RNAs (circRNAs) are a class of single-stranded, closed-loop non-coding RNAs formed through backsplicing, characterized by high stability, evolutionary conservation, and tissue-specific expression. Recent studies have revealed that circRNAs play important regulatory roles in tumorigenesis and development, with some circRNAs acting as miRNA sponges or protein scaffolds in signaling pathways. However, the specific functions and mechanisms of most circRNAs in colorectal cancer metastasis remain unclear.

[0003] The ZBTB44 (zinc finger protein 44) gene is located on human chromosome 11 and encodes a transcription factor containing a BTB domain. Existing research indicates that ZBTB44 is involved in the progression of certain tumors, but the circular RNA produced by the ZBTB44 gene—circZBTB44—has not yet been reported in colorectal cancer.

[0004] RNA interference (RNAi) is currently an effective tool for silencing specific gene expression. Small interfering RNAs (siRNAs) degrade target RNAs by forming an RNA-induced silencing complex (RISC). Designing siRNAs targeting circRNAs presents unique technical barriers: because circRNAs have a completely identical major sequence to the linear mRNA produced by the host gene, traditional siRNA design is highly prone to "off-target" issues, meaning that while knocking down circRNAs, linear mRNAs are also damaged, causing unintended side effects. Therefore, designing crosslinker siRNAs targeting the unique junction sites of circRNAs is crucial for achieving specific intervention. Currently, although there are reports of siRNA studies targeting specific circRNAs (such as circHIPK3 and circ-ITCH), this field remains unexplored in the area of ​​colorectal cancer metastasis. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a siRNA, shRNA, lentiviral vector for targeting and inhibiting circZBTB44, and their applications. This invention aims to solve the problems of a lack of targeted intervention methods for colorectal cancer metastasis and the limited efficacy of existing therapies.

[0006] The technical solution provided by this invention is as follows:

[0007] This invention provides a siRNA that targets and inhibits circZBTB44, consisting of a sense strand and an antisense strand as shown below: The sense and antisense strands are nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; Alternatively, the sense and antisense strands can be nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0008] The present invention also provides a shRNA that targets and inhibits circZBTB44, wherein the nucleotide sequences of the sense and antisense strands of the shRNA are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0009] The present invention also provides a lentiviral vector that targets and inhibits circZBTB44, which is constructed using the shRNA that targets and inhibits circZBTB44 described above.

[0010] Furthermore, the lentiviral vector is constructed by using pLKO.1-PURO as the backbone vector, linearizing it by double digestion with restriction endonucleases AgeI and EcoRI, and then linking it with the encoding double-stranded DNA of the shRNA.

[0011] The present invention also provides a lentivirus comprising the above-mentioned lentivirus vector, viral packaging helper plasmid, and transfection reagent, wherein the viral packaging helper plasmid includes pSPAX2 plasmid and pMD2.G plasmid.

[0012] The present invention also provides the use of the above-described siRNA, shRNA, or lentiviral vector in the preparation of drugs for treating colorectal cancer metastasis.

[0013] Furthermore, the drug inhibits the migration, invasion, and metastasis of colorectal cancer cells through the NF90 / circZBTB44 / PABPC1 / PD-L1 pathway.

[0014] The present invention also provides a pharmaceutical composition for treating metastatic colorectal cancer, comprising the siRNA or shRNA or lentiviral vector described above, and a pharmaceutically acceptable vector.

[0015] Furthermore, the pharmaceutical composition is administered in combination with an immunotherapy drug; the immunotherapy is PD-1 antibody therapy.

[0016] Furthermore, the pharmaceutical composition is an injectable or liquid oral formulation.

[0017] The present invention also provides a combination drug for inhibiting PD-L1 expression in the treatment of colorectal cancer metastasis, the active ingredients of which consist of a circZBTB44 inhibitor and a PABPC1 inhibitor, wherein the circZBTB44 inhibitor and the PABPC1 inhibitor are each independent drug delivery units, or the circZBTB44 inhibitor and the PABPC1 inhibitor together form a combined drug delivery unit; the circZBTB44 inhibitor includes the siRNA, shRNA, or lentiviral vector described above.

[0018] The present invention also provides a kit for targeting and inhibiting circZBTB44, containing the siRNA, shRNA, or lentiviral vector described above.

[0019] Beneficial effects

[0020] This invention utilizes a crosslinker design strategy to develop siRNA sequences that can accurately distinguish between circular RNA and linear mRNA, overcoming the off-target defects of traditional design methods. This invention clarifies the drug's action pathway (circZBTB44 / NF90 / PD-L1 axis), providing a theoretical basis for combination therapy (such as combination with PD-L1 antibodies).

[0021] This invention reveals a novel mechanism by which circZBTB44 recruits NF90 to stabilize PD-L1 mRNA via a "molecular scaffold." Experiments demonstrate that circZBTB44 can simultaneously bind to the RNA-binding proteins NF90 and PABPC1, forming a ternary complex, thereby increasing PD-L1 mRNA stability and upregulating PD-L1 protein expression on the cell surface, ultimately promoting the migration, invasion, and metastasis of colorectal cancer cells. Knockdown of circZBTB44 significantly inhibits this process. Therefore, circZBTB44 is a potential diagnostic biomarker and anti-metastatic therapeutic target for colorectal cancer.

[0022] This invention, from molecular mechanisms and cell phenotypes to animal metastasis models, fully demonstrates the potent inhibitory effect of this siRNA sequence on the invasion and metastasis of colorectal cancer.

[0023] (1) Compared with linear lncRNA, the circRNA (circZBTB44) targeted by this invention has a closed circular structure, is extremely stable in plasma and exosomes, and is more suitable as a therapeutic target; and the drug designed by this invention is based on its unique "connector", which is much more specific than drugs targeting ordinary sequences.

[0024] (2) The siRNA of the present invention can not only physically inhibit the movement of cancer cells (inhibit EMT), but also downregulate PD-L1, which has the dual advantages of "anti-metastasis + sensitization immunotherapy".

[0025] (3) Compared with existing nucleic acid drugs, which often cause toxicity due to accidental damage to homologous mRNA, the sequence rigorously screened in this invention has been verified to not affect the ZBTB44 parent gene mRNA, thus ensuring the maintenance of normal physiological function of ZBTB44 and having higher safety. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the generation mechanism of circZBTB44.

[0027] Figure 2 The circZBTB44 inverse splicing site was verified using Sanger sequencing.

[0028] Figure 3 The cyclic structure of circZBTB44 was verified by RNase R digestion experiments.

[0029] Figure 4 This involves screening and efficiency verification of knockdown sequences targeting circZBTB44.

[0030] Figure 5 The study investigated the effect of knocking down circZBTB44 on the migration and invasion abilities of colorectal cancer cells. A represents the scratch imaging in the cell scratch assay; B is a statistical graph of A; C is a staining image from the Transwell assay; and D is a statistical graph of C.

[0031] Figure 6 The study aimed to reverse NF90 overexpression-induced lung metastasis in colorectal cancer cells by knocking down circZBTB44; where A represents the density of lung surface metastases in RKO cell and HT29 cell lung metastasis models; and B represents the expression levels of Ki67 and NF90 detected by HE staining and immunohistochemical staining (IHC).

[0032] Figure 7 This study examines the effects of circZBTB44 knockdown on PD-L1 expression and its regulatory relationship with PABPC1. Specifically, A represents the statistical analysis of the number of differentially expressed genes between NC and si-circZBTB44-1 and si-circZBTB44-2; B represents polya length validation (PCR validation results using universal primers (U) and specific primers (S) in NC and si-circZBTB44 samples); C represents the detection of PD-L1 mRNA expression levels after knockdown of circZBTB44 and PABPC1; D represents the detection of the half-life of PDL1 after knockdown of circZBTB44 and PABPC1; and E represents the detection of changes in PD-L1 protein levels after knockdown of circZBTB44 and PABPC1. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0034] The experimental materials mainly used in the embodiments of this invention are as follows:

[0035] 1. Laboratory animals

[0036] The mice used in this experiment were purchased from Shanghai Southern Model Biotechnology Co., Ltd., and were raised and managed strictly in accordance with the "Guidelines for the Breeding of Experimental Animals at the Experimental Animal Center of Soochow University".

[0037] cell lines

[0038] HT29 and the human colorectal cancer cell line RKO (high metastatic potential) were both obtained from the Chinese Academy of Sciences Cell Bank and were mycoplasma negative. RKO cells were cultured in DMEM complete medium, and HT29 cells were cultured in 5A complete medium.

[0039] 3. Medicines and reagents

[0040] The pharmaceutical reagents used in this embodiment are listed in Table 1. Unless otherwise specified, all pharmaceutical reagents are standardized reagents from commercial sources, and their storage and use are in accordance with the manufacturer's standard manual.

[0041] Table 1. Main Drugs and Reagents

[0042]

[0043] The core technical solution of this invention is as follows: confirming the circular structure of circRNA; designing specific siRNA targeting the backsplicing site and screening effective sequences; elucidating the mechanism by which it regulates the stability of PD-L1 mRNA through the NF90 / PABPC1 complex; and verifying its anti-transfer effect through in vitro and in vivo models.

[0044] Example 1: Screening and identification of circular RNA molecules that bind to NF90 protein

[0045] This study first collected 44 pairs of colorectal cancer (CRC) tissues with complete clinicopathological information and their paired adjacent normal tissue samples. To comprehensively analyze the changes in protein expression profiles during the development and progression of CRC, we systematically analyzed these samples using quantitative proteomics. We compared the protein expression levels of CRC tissues and their paired adjacent normal tissues, and screened the samples according to a pre-defined significance threshold (fold change > 2.0, and statistical p < 0.05). The results showed significant differences in protein expression between CRC tissues and adjacent normal tissues, with 405 proteins upregulated and 94 proteins downregulated in CRC tissues. To further explore the potential regulatory mechanisms of RNA-binding proteins (RBPs) in CRC, we further screened 11 candidate proteins with RNA-binding functions from the upregulated proteins. Among these 11 RBPs, nuclear factor 90 (NF90) showed particularly significant differential expression folds and very high statistical significance, suggesting that it may play a key regulatory role in the occurrence or progression of CRC.

[0046] Given that NF90 is an RNA-binding protein, its function is often achieved by recognizing and specifically binding to specific RNA molecules (such as circular RNA, circRNA, and circRNA). To identify circRNAs that specifically bind to NF90 in CRC cells, we employed RNA immunoprecipitation (RIP) to enrich NF90-binding RNA molecules using anti-NF90 antibodies, followed by comprehensive screening using high-throughput sequencing (RIP-seq). Specifically, we first lysed CRC cell lines and performed RIP experiments using NF90 antibodies. The enriched RNA fragments were then used to construct circRNA-specific libraries, followed by high-throughput sequencing. Through bioinformatics analysis, we identified a series of circRNA molecules significantly enriched by NF90. To ensure the reliability of the candidate circRNAs, we rigorously filtered the sequencing results according to a pre-defined screening threshold (fold change > 2, P < 0.05), and validated this result using independent RIP experiments.

[0047] After the above screening and verification, we found one candidate circRNA (hsa_circ_0002484, hereinafter referred to as circZBTB44, a circular RNA formed by backsplicing of exon 2 of the human ZBTB44 gene). Figure 1The sequence exhibited the strongest NF90 enrichment signal and the highest confidence level. To definitively confirm the molecular structure of circZBTB44, we further employed Sanger sequencing technology. The sequencing results are as follows: Figure 2 As shown, this clearly confirms that the molecule possesses a typical closed-loop circRNA structure, where its 3' and 5' ends are covalently closed circularly linked via reverse splicing. Next, to verify the stability of this circular structure under physiological conditions, we performed an RNase R digestion experiment. RNase R is an exonuclease that specifically degrades linear RNA while retaining circular RNA. By incubating total RNA with RNase R for different times (e.g., 0, 15, and 30 minutes), the remaining amounts of linear RNA and circular RNA products were measured. The experimental results are as follows: Figure 3 As shown, with prolonged digestion time, the linear RNA product was rapidly degraded to almost undetectable levels, while the circular signal of hsa_circ_0002484 remained stable, almost unaffected by RNase R treatment. This result further functionally demonstrates that circZBTB44 possesses highly stable circular structural characteristics.

[0048] Example 2: Design and screening of siRNAs specifically targeting circZBTB44

[0049] This embodiment aims to design and screen siRNA sequences that can efficiently and specifically knock down circZBTB44 without affecting the expression of linear ZBTB44 mRNA.

[0050] 1. siRNA design

[0051] Three specific siRNA candidate sequences (si-circZBTB44) spanning the backsplicing linker region of circZBTB44 (hsa_circ_0002484) were designed and synthesized. These were named si-circZBTB44-1, si-circZBTB44-2, and si-circZBTB44-3, respectively. A universally scrambled sequence with no significant homology to the human genome was used as a negative control sequence (si-NC), provided by Gemma Biosciences. All three candidate siRNA sequences and the negative control sequence were synthesized by Gemma Biosciences and purified by HPLC before use.

[0052] 2. Cell transfection

[0053] Using the human colorectal cancer cell line RKO as the research subject, a 50 nM transient transfection was performed, with a universal negative control (si-NC) set up. The specific steps are as follows:

[0054] Dissolve the lyophilized siRNA powder in RNase-free water to a storage concentration of 20 μM, aliquot and store at -80°C. 24 hours before transfection, trypsinize logarithmic growth phase CRC cells (RKO), centrifuge and resuspend, seed at an appropriate density into 6-well culture plates, add complete culture medium containing 10% fetal bovine serum (FBS), and incubate overnight at 37°C in a 5% CO2 cell culture incubator. The cell confluence in the wells should reach 60%~80% at the time of transfection.

[0055] Preparation of the transfection complex: Under aseptic conditions, two 1.5 mL EP tubes with RNase removed were used: 150 μL of serum-free Opti-MEM medium was added to tube A, followed by an appropriate amount of siRNA stock solution. 150 μL of Opti-MEM medium was added to tube B, followed by an appropriate amount (7.5 μL per well for a 6-well plate) of Lipofectamine™ RNAi MAX reagent. The liquid in tube B was added drop by drop to tube A, and the mixture was slowly aspirated and mixed. The mixture was then incubated at room temperature for 10-15 minutes to allow the siRNA-liposome transfection complex to fully form.

[0056] After incubation, the complex was added dropwise to each well of a 6-well plate, with a final volume of 2 mL per well and a final siRNA concentration of 50 nM. The plates were incubated for 6 hours after transfection, then the supernatant was discarded, replaced with fresh complete culture medium, and incubated for another 24 hours. Each group was divided into three replicates, and the experiment was independently repeated three times.

[0057] 3. Total RNA extraction and reverse transcription

[0058] Cells were collected from each group 24 hours after transfection. The culture medium was discarded, and 1 mL of TRIzol reagent was added to each well. Cells were lysed by repeated pipetting and transferred to RNase-free 1.5 mL EP tubes. Total RNA was extracted using a Kangwei Century kit (CW0597S).

[0059] Take 2 μL of RNA sample and determine the RNA concentration and OD260 / 280 ratio using a NanoDrop 2000 micro-volume spectrophotometer. All samples had an OD260 / 280 ratio between 1.8 and 2.2, indicating that the RNA purity met the requirements. Perform reverse transcription according to the instructions of the reverse transcription kit (Promega, A5001). Store the obtained cDNA at -20℃ for later use.

[0060] 4. RT-qPCR detection

[0061] Real-time quantitative PCR was performed using the SYBR Green assay. 2 -ΔΔCtThe relative expression level of circZBTB44 in each group of cells was calculated.

[0062] 5. Experimental Results

[0063] Test results as follows Figure 4 As shown in Table 2, this invention quantitatively compared the knockdown effects of the three sequences mentioned above, and screened out two effective sequences that could significantly knock down circZBTB44 (inhibition rate > 90%) without affecting linear ZBTB44 mRNA expression.

[0064] Table 2. siRNA sequences targeting the circZBTB44 backsplicing adapter region

[0065]

[0066] Example 3: Construction of the lentiviral expression vector hsa_circ_0002484

[0067] 1. DNA Oligonucleotide Sequence Synthesis and Preparation

[0068] Based on the si-circZBTB44-1 and control interference target sequence (si-NC) obtained above, single-stranded DNA oligonucleotide 1 sequence and control single-stranded DNA oligonucleotide sequence (synthesized by Sangon Biotech Co., Ltd.) were designed and synthesized. The single-stranded DNA oligonucleotide 1 sequence and control single-stranded DNA oligonucleotide sequence (shNC) were purified by PAGE.

[0069] DNA oligonucleotide 1 sequence:

[0070] The single-stranded oligonucleotide 1-1 sequence (i.e., the positive strand of shRNA1) is as follows: 5'-CCGGGTCTTCCTCTATAGGACATTTCTCGAGATGTCCTATAGAGGAAGACTTTTTTTG-3' (SEQ ID NO: 5). The single-stranded oligonucleotide 1-2 sequence (i.e., the shRNA1 antisense strand) is as follows: 5'-AATTCAAAAAGTCTTCCTCTATAGGACATTTCTCGAGATGTCCTATAGAGGAAGACTT-3' (SEQ ID NO: 6).

[0071] Reference single-stranded DNA oligonucleotide sequence:

[0072] A generic disordered sequence with no significant homology to the human genome was used as a negative control sequence, which was provided by Sangon Biotech Co., Ltd.

[0073] The single-stranded sequences obtained above and the control single-stranded DNA oligonucleotide sequence (shNC) were annealed to obtain double-stranded DNA oligonucleotide 1 with sticky ends (i.e., shRNA1 sequence) and control double-stranded DNA oligonucleotide sequence (i.e., shNC sequence). The annealing system is shown in Table 3, and the annealing program is shown in Table 4.

[0074] Table 3 Composition of the annealing system

[0075]

[0076] Table 4 Annealing Procedure

[0077]

[0078] 2. Preparation of linearized carriers

[0079] The pLKO.1-PURO vector was selected as the original vector, and the plasmid was purchased from Addgene. The original vector was double-digested with restriction endonucleases AgeI and EcoRI. Each reagent was added sequentially according to the order in Table 5 to prepare a 50 μL digestion system. The mixture was then gently aspirated and incubated in a 37°C water bath for 1–2 h to obtain the vector digestion products. The digestion products were subjected to agarose gel electrophoresis. The target band was recovered and linearized, and then incubated at 37°C for 1 h. The target fragment was then excised and recovered. The electrophoresis results confirmed the successful construction of the linearized vector.

[0080] Table 5. Vector Enzyme Digestion System

[0081]

[0082] 3. Connection of the target fragment to the vector

[0083] The prepared shRNA1 sequence was ligated with the vector digestion product, as shown in Table 6. Ligation was carried out at 37°C for 1-2 hours, or at 16°C overnight. This achieved the ligation of the linearized vector with shRNA1 (or shNC sequence) double-stranded DNA, yielding the ligation product.

[0084] Table 6 Connection Reaction System

[0085]

[0086] 4. Transformation and PCR identification and sequencing of positive clones

[0087] After removing *E. coli* Stabl 3 competent cells (purchased from Quanshijin) from the -80°C freezer, immediately place them on ice to thaw. The competent cell aliquoting process should be handled gently to minimize cell damage. Once thawed, aliquot the cells into 50 μL tubes. Add ligation product to each tube at 1 / 10 of the competent cell volume and incubate on ice for 30 min. Heat shock at 42°C for 90 s, then immediately place the tubes on ice for 2 min. In a clean bench, add 500 μL of antibiotic-free LB medium and gently invert 3–5 times. Then incubate at 37°C with shaking at 220 rpm for 45–60 min. Spread the bacterial culture evenly onto appropriately sized antibiotic plates and incubate at 37°C for 12–16 h. Single colonies were selected and identified by PCR using universal primer U6 (sequence 5'-TATATCTTGTGGAAAGGAC-3' (SEQ ID NO: 7)) and reverse primer 1-R (sequence 5'-TCCCCCCTTTTCTTTTAAAA-3' (SEQ ID NO: 8)). Positive clones were selected for sequencing (sequencing data provided by Sangon Biotech). After sequencing alignment, the positive clones were identified as successfully constructed lentiviral vectors.

[0088] 5. Plasmid extraction

[0089] Positive clones verified by sequencing underwent plasmid extraction, following the instructions of the extraction kit (purchased from Tiangen, product number DP117). The extracted plasmids, after passing QC verification, were used for cell transfection.

[0090] 6. Lentiviral packaging and titer determination

[0091] 6.1 Lentiviral Packaging

[0092] 24 h prior to transfection, 293T cells in logarithmic growth phase were digested with trypsin and seeded in DMEM complete medium containing 10% fetal bovine serum, and cultured at 37°C and 5% CO2. Transfection was performed when cell confluence reached 70%–80%. The transfection complex was prepared in two steps: first, 500 μL of Opti-MEM serum-depleted medium and 32 μL of Lipofectamine were added to a sterile centrifuge tube. TMGently mix the 3000 transfection reagent; in another tube, add 500 μL of Opti-MEM medium, followed by 10 μg of the target plasmid (specifically, shRNA1 / shNC interference vector, or overexpression vector and corresponding empty vector), 6.67 μg of psPAX2 plasmid, 3.33 μg of pMD2.G plasmid, and 40 μL of P3000 reagent, and gently mix by pipetting. Combine the two tubes and gently mix, avoiding vortexing or centrifugation throughout the process. Incubate at room temperature for 10–15 min to form the transfection complex. Finally, evenly add the complex to a pre-changed cell culture dish, gently agitate, and incubate in an incubator. No further medium changes are needed in the short term.

[0093] Viral supernatant was collected twice post-transfection, at 48 h and 36 h (fresh complete medium was used after the 48 h collection). At 48 h, the medium from a 10 mm culture dish was poured into a 50 mL centrifuge tube, ensuring the dish wall did not touch the centrifuge tube opening to prevent bacterial contamination. Then, 10 mL of fresh complete medium containing 10% fetal bovine serum (FBS) was added, and the tube was incubated stably at 37°C with 5% CO2. At 36 h, the medium from a 100 mm culture dish was directly poured into a 50 mL centrifuge tube, again ensuring the dish wall did not touch the centrifuge tube opening to prevent bacterial contamination.

[0094] 6.2 Lentiviral Concentration

[0095] Filter the harvested lentivirus supernatant through a 0.45 μm filter to remove cell debris. Add the virus solution and lentivirus precipitation solution (Quanshijin) to a centrifuge tube at a volume ratio of 4:1, gently invert to mix, and incubate at 2–8°C for 45 min. Then centrifuge at 7,000 × g for 45 min at 2–8°C to remove the supernatant as completely as possible, retaining the white precipitate. Resuspend the precipitate in 1 / 10 to 1 / 100 volume of PBS before concentration to obtain the concentrated virus solution. Aliquot the solution according to the amount needed for each experiment and store at -80°C, avoiding repeated freeze-thaw cycles.

[0096] 6.3 Titer Determination and Quality Control

[0097] The titer of the concentrated lentivirus was determined using a fluorescence method, requiring a viral titer ≥ 1 × 10⁻⁶. 8 TU / mL, aliquoted and stored at -80°C; in vitro verification of the toxicity of the vector to drug-resistant colorectal cancer cells, requiring cell viability ≥90% after transfection to ensure the safety of the vector.

[0098] 7. Lentiviral infection of cells and screening of stable transfected strains

[0099] 7.1 Cell Preparation

[0100] Seed healthy target cells into 6-well plates, ensuring the cell confluence is between 30% and 50% by the time of viral infection the following day. Incubate overnight at 37°C in a 5% CO2 incubator.

[0101] 7.2 Viral infection (1 / 2 volume infection method)

[0102] Before infection, remove the virus from the refrigerator and thaw it slowly on ice. Aspirate the original culture medium and add 1 / 2 volume of fresh culture medium. Based on the determined MOI value, add an appropriate volume of virus for infection (virus volume per well (μL) = (MOI × cell number) / virus titer). 8 μg / mL of polyglobulin can be added simultaneously. After 4 hours of lentiviral infection, replenish to the complete culture volume.

[0103] 7.3 Screening and validation of puromycin

[0104] 24 hours after viral infection, the culture medium was replaced with complete medium for continued culturing. For lentivirus infection, 48-72 hours later, when the infection efficiency reached approximately 80% and cell confluence was 60-70%, puromycin was applied at the determined concentration (the specific concentration depends on cell condition; it was added after cell stability). Approximately 48 hours after drug administration, the mortality of empty cells in the control group was observed. If the cell mortality rate in the empty cell group exceeded 90%, puromycin was removed and replaced with fresh culture medium. Periodic drug screening can be performed based on the growth rate of the empty cells in later stages.

[0105] The surviving cells were collected, RNA was extracted and reverse transcribed into cDNA, and finally verified using qRT-PCR. The results showed that the lentiviral interference vector constructed in this invention has good targeting specificity and silencing efficiency.

[0106] Example 4: In vitro functional validation of circZBTB44 in colorectal cancer cells

[0107] In this embodiment, siRNA (si-NC, si-circZBTB44-1, si-circZBTB44-2) was transfected into RKO cells according to the transfection system of Example 2.

[0108] 1. Experimental Methods

[0109] 1.1 Transwell invasion experiment

[0110] 24 hours after transfection, cells from each group were collected, resuspended in serum-free DMEM medium, and the density was adjusted to 2 × 10⁶ cells / day. 5 Cells / mL. The upper chamber of the Transwell was pre-coated with Matrigel, and 200 μL of cell suspension (containing 4 × 10⁶ cells / mL) was added to the upper chamber.4 (1 cell), add 600 μL of DMEM complete medium containing 20% ​​FBS (as a chemotactic agent) to the lower chamber. After culturing for 24 hours, remove the chamber, wipe off the cells from the upper chamber surface, fix with methanol for 15 minutes, and stain with 0.1% crystal violet for 15 minutes. Randomly select 5 fields of view under a microscope to count the number of invasive cells.

[0111] 1.2 Scratch Healing Experiment

[0112] 24 hours after transfection, the cells in each group were injected with 1×10⁻⁶ cells / mL. 6 Re-seed cells at a density of cells / well in 6-well plates and cultured until fully confluent. Using a 200 μL sterile pipette tip, vertically scratch the center of the bottom of each well. Gently wash twice with PBS to remove suspended cells, then add serum-free DMEM medium. Immediately after 48 hours, photograph the scratch area under an inverted microscope. Photograph again after 48 hours of culture. Calculate the scratch area using ImageJ software, generate bar charts using Graphpad Prism, and perform differential analysis.

[0113] 2. Experimental Results

[0114] The results of the scratch healing experiment showed that ( Figure 5 (A and B): Compared with the si-NC group, the si-circZBTB44-1 and si-circZBTB44-2 groups showed significantly lower scratch healing rates at 48 hours, with an average healing rate that was about 5% lower than that of the control group, indicating that knockdown of circZBTB44 significantly inhibited the migration ability of colorectal cancer cells.

[0115] Transwell experimental results show that ( Figure 5 Compared with the si-NC group, the number of invasive cells in the si-circZBTB44-1 and si-circZBTB44-2 groups was reduced, indicating that knockdown of circZBTB44 significantly inhibited the invasive ability of colorectal cancer cells.

[0116] Example 5: In vivo verification of anti-metastatic efficacy

[0117] This embodiment uses a nude mouse tail vein injection lung metastasis model to verify the inhibitory effect of circZBTB44 knockdown on NF90-mediated colorectal cancer lung metastasis.

[0118] 1. Laboratory animals and their housing conditions

[0119] Male BALB / c nude mice, 4–6 weeks old and weighing 18–22 g, were purchased from Shanghai Southern Model Biotechnology Co., Ltd. All experimental animals were housed in individually ventilated cages (IVCs) in an SPF-grade animal facility. The room temperature was strictly controlled at 22℃±2℃, and the relative humidity was maintained at 50%±10%, with a 12-hour alternating light-dark cycle. The nude mice had ample access to sterilized feed and drinking water, and all procedures involving the animals strictly adhered to the review standards and operating procedures of the Laboratory Animal Ethics and Welfare Committee.

[0120] 2. Experimental grouping and cell preparation

[0121] The experiment was divided into three groups: control group (NC), overexpression group (NF90 OE), and complementation group (NF90 OE + sh-circZBTB44). The control group used stable negative control (shNC) RKO and HT29 cells. The overexpression group used RKO and HT29 cells with stable NF90 gene overexpression (overexpression primers: upstream primer: 5'-GAATTCGCCACCATGCGTCCAATGCGAATTTTTG-3' (SEQ ID NO: 9), downstream primer: 5'-CGTGTACATTATCTGTACTGGTAGTTCATG-3' (SEQ ID NO: 10)). The complementation group used RKO and HT29 cells with stable overexpression of NF90 and knockdown of circZBTB44. Each group consisted of 10 nude mice.

[0122] The cell suspension preparation steps are as follows: Take stable transfected cells in the logarithmic growth phase from each group mentioned above, discard the culture supernatant, and wash once with PBS. Add 1 mL of 0.25% trypsin digestion solution, digest at 37℃ for 2-3 minutes until the cells become rounded and detach. Add 2 mL of complete culture medium containing 10% FBS to stop digestion, pipette evenly, transfer to a 15 mL centrifuge tube, centrifuge at 200×g for 5 minutes, and discard the supernatant. Resuspend the cell pellet in an appropriate amount of pre-chilled sterile PBS, wash once, centrifuge again at 200×g for 5 minutes, and discard the supernatant. Resuspend the cells in an appropriate amount of pre-chilled sterile PBS, perform trypan blue staining, and count viable cells under a microscope to ensure that the viable cell ratio is >95%. Adjust the cell concentration to 3×10⁻⁶ cells / mL with pre-chilled sterile PBS. 7 Cells / mL. The prepared cell suspension was kept on ice throughout the process and inoculated within 1 hour.

[0123] 3. Tail vein injection

[0124] In a sterile operating room, the tail of the nude mouse was wiped and disinfected with 75% medical alcohol. Using an insulin syringe (29G), 100 μL of cell suspension (containing 3 × 10⁻⁶ cells) was drawn. 6(1 cell), place the nude mouse in a mouse restraint device to expose the tail, locate the tail vein, and slowly inject the cell suspension along the course of the tail vein. During the injection, observe whether the blood vessel is patent. After the injection, gently press the injection site with a sterile cotton ball for a few seconds to prevent bleeding and leakage. Continue to feed the mice for 8 weeks after injection, and observe and record the general condition of the mice weekly, including activity level, food intake, weight change, and respiratory status.

[0125] 4. Sample Collection

[0126] At the end of the 8th week post-injection, the weight of nude mice in each group was measured and recorded. The mice were then euthanized by cervical dislocation. Lung tissue was completely removed, washed twice in pre-cooled PBS to remove surface blood, and the surface moisture was blotted dry with filter paper. The overall morphology of the lung tissue and surface metastatic nodules were photographed under a stereomicroscope. The lung tissue was fixed in 4% neutral formalin for 24 hours for subsequent HE staining and immunohistochemical analysis.

[0127] 5. Detection indicators and results

[0128] 5.1 Observation of lung metastatic nodules

[0129] After fixation, lung tissue was embedded in paraffin, sectioned (4 μm thick), and stained with hematoxylin and eosin (HE). The HE staining procedure included: dewaxing with xylene (I 10 min, II 10 min), gradient ethanol hydration (anhydrous ethanol I 5 min, anhydrous ethanol II 5 min, 95% ethanol 3 min, 85% ethanol 3 min, 75% ethanol 3 min), rinsing with distilled water for 2 min; hematoxylin staining for 5 min, rinsing with running water for 5 min; differentiation with 1% hydrochloric acid ethanol for 3 seconds, rinsing with running water for 10 min; eosin staining for 2 min, rinsing with distilled water; gradient ethanol dehydration (75%, 85%, 95%, anhydrous ethanol), clearing with xylene, and mounting with neutral resin. The number of micrometastases was observed and counted under a microscope.

[0130] The results show that ( Figure 6 In the RKO cell model, the density of lung metastatic lesions was significantly increased in the NF90 OE overexpression group compared to the control group, while the number of lung metastatic lesions was significantly reduced in the complementation group (NF90 OE + sh-circZBTB44) compared to the NF90 OE group. Similarly, in the HT29 cell model, the number of visible metastatic nodules and micrometastases in the lungs of nude mice in the NF90 overexpression group was significantly higher than that in the control group. Furthermore, NF90 overexpression combined with circZBTB44 knockdown significantly reduced the number of lung metastatic nodules compared to the NF90 overexpression alone group. These results indicate that NF90 promotes distant metastasis of colorectal cancer cells, while circZBTB44 knockdown can effectively reverse the pro-metastatic effect induced by NF90 overexpression.

[0131] 5.2 Immunohistochemical staining

[0132] The immunohistochemical staining procedure is as follows: After dewaxing the sections to water, place them in citrate buffer (pH 6.0) and microwave until boiling. Maintain 95℃ for 15 minutes for antigen retrieval, then allow to cool naturally to room temperature. Incubate with 3% H2O2 for 10 minutes to inactivate endogenous peroxidase, and block with 5% goat serum for 30 minutes. Add primary antibody (Anti-Ki-67 or Anti-NF90, 1:200 dilution) and incubate overnight at 4℃. Wash three times with PBS, add HRP-labeled secondary antibody, and incubate at 37℃ for 30 minutes. Wash three times with PBS, develop with DAB for 2-5 minutes, controlling the degree of staining under a microscope. Counterstain with hematoxylin for 1 minute, rinse with running water for 10 minutes, dehydrate, clear, mount, and examine under a microscope. Ki-67 staining indicates positive cells with brownish-red granules in the nucleus, and NF90 staining indicates a positive signal with brownish-red granules in the cytoplasm / nucleus.

[0133] Ki-67 staining results showed that ( Figure 6 The number and density of brownish-red positive cells in the tumor tissue of the NF90 OE group were significantly higher than those in the control group and the NF90 OE+sh-circZBTB44 group. Immunohistochemical staining of NF90 showed that the number and density of brownish-red NF90 granules in the lung metastases of the NF90 OE and NF90 OE+sh-circZBTB44 groups were increased compared with the negative control group. At the same time, in HT29 cells, the number of metastatic nodules and micrometastases in the lungs of nude mice in the NF90 overexpression group were significantly higher than those in the control group, suggesting that NF90 promotes distant metastasis of cancer cells. However, after NF90 overexpression and circZBTB44 knockdown, the number of lung metastatic nodules was significantly reduced compared with the NF90 overexpression alone group.

[0134] Example 6: Validation of the mechanism by which circZBTB44 regulates PD-L1 mRNA stability

[0135] This embodiment aims to elucidate the molecular mechanism by which circZBTB44 regulates colorectal cancer metastasis and to verify the function of circZBTB44 as a protein scaffold mediating the binding of the NF90 / PABPC1 complex, thereby maintaining the stability of PD-L1 mRNA.

[0136] 1. Screening downstream target genes that significantly shorten the Poly(A) tail length after knockdown of circZBTB44 using TAIL Iso-seq technology.

[0137] To investigate the potential function of circZBTB44 in regulating the length of the poly(A) tail in mRNA, this study knocked down circZBTB44 in colorectal cancer (CRC) cells using two independent interfering sequences (si-circZBTB44-1 and si-circZBTB44-2). Subsequently, TAIL Iso-seq high-throughput sequencing was used to systematically analyze transcript expression and poly(A) tail length in cell samples from the si-circZBTB44-1, si-circZBTB44-2, and control groups. Differential analysis combined with multiple tests using p-values ​​and FDR were employed to screen for genes with significant alterations.

[0138] The results showed that the genes affected by the two interfering sequences partially overlapped. The intersection of genes that both knocked down the genes and showed a significant shortening of the Poly(A) tail was obtained, yielding a total of 9 overlapping genes (e.g., ...). Figure 7 (As shown in Table A and Table 8). This result indicates that the poly(A) tail length variations of these 9 genes depend on normal expression of circZBTB44, suggesting that they may be potential downstream targets in the circZBTB44 regulatory network.

[0139] Table 7. Nine overlapping genes that significantly shortened the Poly(A) tail length after knockdown of circZBTB44.

[0140]

[0141] 2. Detection of PD-L1 mRNA Poly(A) tail length.

[0142] RKO cells from the control group and the circZBTB44 knockdown group were collected, and total RNA was extracted from the cells using the TRIzol method. The length of the Poly(A) tail of PD-L1 mRNA was detected by 3' RACE combined with agarose gel electrophoresis: cDNA was synthesized by reverse transcription using oligonucleotide (dT) primers, and PCR amplification was performed using PD-L1 gene-specific forward primers and oligonucleotide (dT) reverse primers. The PCR products were subjected to 2.5% agarose gel electrophoresis (120V constant voltage electrophoresis for 40 minutes), and the position of the electrophoretic bands was observed under UV light to determine the changes in the Poly(A) tail length.

[0143] Agarose gel electrophoresis results showed ( Figure 7In the control group (B), knocking down circZBTB44 significantly lowered the Poly(A) tail band of PD-L1 mRNA compared to the control group, indicating a significant shortening of the Poly(A) tail length. These results fully demonstrate that circZBTB44 functions as a protein scaffold, mediating the binding of the NF90 / PABPC1 complex and specifically protecting and maintaining the length of the Poly(A) tail of the downstream target gene PD-L1 mRNA.

[0144] 3. Detection of PD-L1 mRNA expression level

[0145] Given that the length of the Poly(A) tail is a core factor determining mRNA stability and degradation rate, this study further examined the expression level of PD-L1 mRNA to confirm whether shortening the Poly(A) tail ultimately leads to the degradation of the target gene. RKO cells were divided into four groups: si-NC group (negative control), si-circZBTB44 group (circZBTB44 knockdown), si-PABPC1-1 group and si-PABPC1-2 group (see Table 8) (PABPC1 knockdown), and si-circZBTB44-1+si-PABPC1-2 group (simultaneous PABPC1 and circZBTB44 knockdown). Each siRNA (final concentration 50 nM) was transfected into RKO cells according to the transfection method described in Example 2. Forty-eight hours after transfection, total RNA was extracted from the cells, reverse transcribed into cDNA, and the PD-L1 mRNA expression level was detected by qRT-PCR using the SYBR Green assay, with GAPDH as an internal control. -ΔΔCt The relative expression level is calculated using this method.

[0146] Table 8. siRNA sequences targeting PABPC1

[0147]

[0148] The results show that ( Figure 7 In the study of PD-L1 mRNA, knocking down circZBTB44 and PABPC1 both led to a decrease in PD-L1 mRNA levels, while knocking down both PABPC1 and circZBTB44 resulted in a more significant decrease in PD-L1 mRNA levels.

[0149] 4. mRNA stability experiment

[0150] To distinguish whether this downregulation was due to reduced transcription or accelerated mRNA degradation, this study treated cells with the transcription inhibitor actinomycin D. RKO cells were divided into four groups: control group (si-NC), circZBTB44 knockdown group (si-circZBTB44-1), PABPC1 knockdown group (si-PABPC1-2), and si-circZBTB44-1+si-PABPC1-2 group (simultaneous knockdown of PABPC1 and circZBTB44), and transfected according to the method in Example 2. Forty-eight hours after transfection, the transcription inhibitor actinomycin D was added to each group of cells at a final concentration of 5 μg / mL. Cells were collected at 0, 1, 2, 3, and 4 hours after the addition of actinomycin D, and total RNA was extracted from the cells at each time point. After reverse transcription, the remaining amount of PD-L1 mRNA was detected by qRT-PCR. The relative remaining amount at each time point was calculated with the PD-L1 mRNA level at 0 hours as 100%, and mRNA degradation curves were plotted and the half-life calculated.

[0151] mRNA stability experiment results showed that ( Figure 7 In the control group (NC), PD-L1 mRNA degradation was slower, while knockdown of circZBTB44 and PABPC1, as well as simultaneous knockdown of both, accelerated mRNA degradation. These results confirm that the main mechanism of action of the NF90 / circZBTB44 / PABPC1 complex is to maintain the stability of downstream PD-L1 mRNA at the post-transcriptional level, rather than affecting its transcriptional production.

[0152] 5. Detection of PD-L1 protein expression

[0153] Previous studies have confirmed that knocking down circZBTB44 or PABPC1 leads to PD-L1 mRNA degradation by shortening the Poly(A) tail. To further confirm whether this transcriptomic instability ultimately translates into a decrease in protein expression levels, this study used Western blotting to detect PD-L1 expression in RKO cells from different treatment groups. RKO cells were divided into three groups: si-NC, si-circZBTB44-1, and si-PABPC1-2, and transfected according to the method in Example 2. Forty-eight hours after transfection, the cell supernatant was discarded, and the cells were washed twice with PBS. 100 μL of RIPA lysis buffer (containing protease inhibitors) was added to each well, and the cells were lysed on ice for 15 minutes. After centrifugation at 12,000 rpm and 4°C for 15 minutes, the supernatant was collected as total protein. Protein concentration was determined using a BCA protein quantification kit, and the loading volume of each sample was adjusted to an equal mass (20 μg / well). Prepare a 10% SDS-polyacrylamide gel and perform electrophoresis at a constant voltage of 80V for 30 minutes (adjust to 120V for 60 minutes after the proteins enter the separating gel). Transfer the separated proteins to a PVDF membrane (transfer at a constant current of 250 mA for 90 minutes). Block with 5% skim milk powder at room temperature for 1 hour, then add Anti-PD-L1 antibody (1:1000 dilution) or Anti-GAPDH antibody (1:5000 dilution) and incubate overnight at 4°C. After washing three times with TBST, add HRP-labeled secondary antibody (1:5000 dilution) and incubate at room temperature for 1 hour. Wash three times with TBST, add ECL chemiluminescent substrate, and expose and develop using a chemiluminescence imaging system.

[0154] The results show that ( Figure 7 In the middle E), knocking down circZBTB44 and PABPC1 both reduced PD-L1 protein expression.

[0155] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A siRNA that targets and inhibits circZBTB44, characterized in that, It consists of the justice chain and antithesis chain as shown below: The sense and antisense strands are nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2; Alternatively, the sense and antisense strands can be nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO:

4.

2. A shRNA that targets and inhibits circZBTB44, characterized in that, The nucleotide sequences of the sense and antisense strands of the shRNA are shown in SEQ ID NO: 5 and SEQ ID NO:

6.

3. A lentiviral vector that targets and inhibits circZBTB44, characterized in that, The shRNA that targets and inhibits circZBTB44 as described in claim 2 was constructed.

4. The lentiviral vector targeting and inhibiting circZBTB44 according to claim 3, characterized in that, The lentiviral vector is constructed by using pLKO.1-PURO as the backbone vector, linearizing it by double digestion with restriction endonucleases AgeI and EcoRI, and then linking it with the double-stranded DNA encoding the shRNA.

5. The use of the siRNA of claim 1, the shRNA of claim 2, or the lentiviral vector of any one of claims 3-4 in the preparation of a medicament for treating colorectal cancer metastasis.

6. The application according to claim 5, characterized in that, The drug inhibits the migration, invasion, and metastasis of colorectal cancer cells through the NF90 / circZBTB44 / PABPC1 / PD-L1 pathway.

7. A pharmaceutical composition for treating metastatic colon cancer, characterized in that, Includes the siRNA of claim 1, the shRNA of claim 2, or the lentiviral vector of any one of claims 3-4, as well as pharmaceutically acceptable vectors.

8. The pharmaceutical composition for treating metastatic colon cancer according to claim 7, characterized in that, The pharmaceutical composition is administered in combination with an immunotherapy drug; the immunotherapy is PD-1 antibody therapy.

9. A combination drug for inhibiting PD-L1 expression in the treatment of colorectal cancer metastasis, characterized in that, Its active ingredients consist of a circZBTB44 inhibitor and a PABPC1 inhibitor, wherein the circZBTB44 inhibitor and the PABPC1 inhibitor are each independent drug delivery units, or the circZBTB44 inhibitor and the PABPC1 inhibitor together form a combined drug delivery unit; the circZBTB44 inhibitor includes the siRNA of claim 1, the shRNA of claim 2, or the lentiviral vector of any one of claims 3 to 4.

10. A kit for targeting and inhibiting circZBTB44, characterized in that, It contains the siRNA of claim 1, the shRNA of claim 2, or the lentiviral vector of any one of claims 3 to 4.

Images