Shrna targeting aldob and its application in inhibiting colorectal cancer

Abstract

This invention belongs to the field of anti-tumor technology, specifically relating to a shRNA targeting ALDOB and its application in inhibiting colorectal cancer. This invention designs and screens specific shRNAs targeting the human ALDOB gene; these shRNAs target and inhibit ALDOB, significantly suppressing colorectal cancer cell viability, colony formation ability, and DNA synthesis ability, promoting apoptosis, and inhibiting glycolysis in colorectal cancer cells. They also inhibit tumor growth in subcutaneous tumor-bearing models and reduce Ki67 expression in tumor tissue, providing experimental evidence and technical reference for developing nucleic acid intervention agents targeting ALDOB for colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of anti-tumor technology, specifically relating to a shRNA that targets ALDOB and its application in inhibiting colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, characterized by high incidence, heavy disease burden, and significant patient heterogeneity. Beyond clinical diagnosis and treatment, molecular intervention studies focusing on the proliferation capacity, apoptosis regulation, and in vivo tumorigenicity of colorectal cancer cells still hold significant value for basic research and translational development.

[0003] ALDOB (aldolase B) is a member of the aldolase family. Current research suggests that the biological role of ALDOB in different tumors is significantly tissue- and disease-specific, and its expression changes and functional significance are not entirely consistent across specific tumors. The inventors previously analyzed public databases and validated their findings using cell and clinical tissue samples, discovering that ALDOB is highly expressed in colorectal cancer, suggesting a possible association with the biological behavior of colorectal cancer.

[0004] However, current research on ALDOB mainly focuses on general molecular expression phenomena or functional discussions in the context of other diseases. There is still a lack of clear and directly implementable technical solutions regarding the specific ALDOB shRNA sequence itself, the nucleic acid encoding the shRNA, the recombinant expression vector containing the nucleic acid, and its application in inhibiting the growth of colorectal cancer cells, promoting apoptosis, and inhibiting tumorigenesis in vivo.

[0005] Therefore, it is necessary to provide a specific shRNA targeting ALDOB, the nucleic acid encoding the shRNA, and its recombinant expression vector, and to verify its application value in inhibiting the growth of colorectal cancer cells, promoting apoptosis, and inhibiting tumorigenesis in vivo, so as to provide a technical basis for subsequent nucleic acid intervention research and formulation development. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a specific shRNA targeting ALDOB. This shRNA specifically targets and inhibits ALDOB, significantly reducing the viability, colony-forming ability, and DNA synthesis capacity of colorectal cancer cells, and promoting apoptosis, thereby inhibiting the growth of colorectal cancer cells. Specifically, it includes the following:

[0007] In a first aspect, the present invention provides an application of ALDOB as a target in screening anti-colorectal cancer drugs, wherein the drug inhibits the expression of ALDOB.

[0008] Preferably, the drug is an shRNA targeting ALDOB, and the target sequence of the shRNA targeting the ALDOB gene is shown in SEQ ID NO.1 or 5.

[0009] Preferably, the transcript sequence of the shRNA is as shown in SEQ ID NO.2 or 6.

[0010] Preferably, the nucleotide sequence of the sense strand of the shRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.4; or the nucleotide sequence of the sense strand of the shRNA is as shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.8.

[0011] Secondly, the present invention provides an shRNA targeting ALDOB, wherein the target sequence of the ALDOB gene is shown in SEQ ID NO.1 or 5.

[0012] Preferably, the transcript sequence of the shRNA is as shown in SEQ ID NO.2 or 6.

[0013] Preferably, the nucleotide sequence of the sense strand of the shRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.4; or the nucleotide sequence of the sense strand of the shRNA is as shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.8.

[0014] Thirdly, the present invention provides the application of the shRNA described in the second aspect above in the preparation of antitumor drugs.

[0015] Preferably, the tumor is colorectal cancer.

[0016] Fourthly, the present invention provides a nucleic acid construct containing a gene fragment encoding the shRNA described in the second aspect above.

[0017] Fifthly, the present invention provides the application of the nucleic acid constructs described in the fourth aspect above in the preparation of antitumor drugs.

[0018] Preferably, the tumor is colorectal cancer.

[0019] In a sixth aspect, the present invention provides a recombinant vector comprising the shRNA described in the second aspect above.

[0020] Preferably, the recombinant vector is a lentiviral vector.

[0021] Preferably, the lentiviral vector is LV3 (H1 / GFP & Puro).

[0022] In a seventh aspect, the present invention provides the use of the recombinant vector described in the sixth aspect above in the preparation of antitumor drugs.

[0023] Preferably, the tumor is colorectal cancer.

[0024] Eighthly, the present invention provides a lentivirus, which is prepared by viral packaging of the nucleic acid construct described in the fourth aspect above with the assistance of a lentivirus packaging plasmid and a cell line.

[0025] In a ninth aspect, the present invention provides the use of the lentivirus described in the eighth aspect above in the preparation of antitumor drugs.

[0026] Preferably, the tumor is colorectal cancer.

[0027] In a tenth aspect, the present invention provides a pharmaceutical composition for treating colorectal cancer, wherein the active ingredient of the pharmaceutical composition comprises the shRNA described in the second aspect above, or the nucleic acid construct described in the fourth aspect above, or the recombinant expression vector described in the sixth aspect above, or the lentivirus described in the eighth aspect above.

[0028] The beneficial effects of this invention are:

[0029] (1) This invention first verified the expression of ALDOB in a public database of colorectal cancer, cells and clinical tissue samples, and found that ALDOB is highly expressed in colorectal cancer, suggesting that ALDOB can be used as a candidate target for nucleic acid intervention in colorectal cancer. This invention, combined with the verification results of expression in clinical tissues of colorectal cancer, further illustrates the rationality of ALDOB as a target for nucleic acid intervention in colorectal cancer.

[0030] (2) Based on this, the present invention provides a specific shRNA targeting ALDOB, the nucleic acid encoding the shRNA and its recombinant expression vector, with a clear technical target and a clear structure, which facilitates subsequent construction, verification and application.

[0031] (3) The present invention demonstrates through in vitro functional experiments that the shRNA-targeted inhibition of ALDOB can significantly reduce the viability, clonogenic ability and DNA synthesis ability of colorectal cancer cells, promote cell apoptosis, and inhibit their glycolytic metabolism, suggesting that the technical solution can directly act on the malignant biological behavior of colorectal cancer cells.

[0032] (4) The present invention further demonstrates through a subcutaneous tumor-bearing model that the shRNA-targeted inhibition of ALDOB can inhibit tumor growth in vivo and reduce Ki67 expression in tumor tissue, indicating that the technical solution not only has an inhibitory effect in vitro, but also has tumor-suppressing potential in vivo.

[0033] (5) In summary, this invention provides experimental basis and technical reference for the study of nucleic acid intervention for colorectal cancer targeting ALDOB and the development of related preparations. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the technical route of the present invention, which selects ALDOB as an intervention target based on its high expression in colorectal cancer and uses specific shRNA to implement nucleic acid intervention.

[0035] Figure 2 Figure 1 shows the expression, prognosis, bioinformatics analysis, and experimental validation results of ALDOB in colorectal cancer. Specifically: A represents the differential expression analysis results of ALDOB in colorectal cancer tissues and normal colorectal tissues from a public database; B represents the validation results of ALDOB expression in colorectal cancer tissues and normal tissues from an independent external dataset; C represents the prognostic analysis results of ALDOB based on a public database; D represents the differential expression analysis results of ALDOB in pan-cancer based on a public database; E represents the qPCR detection results of ALDOB mRNA expression levels in normal intestinal epithelial cells and colorectal cancer cell lines; F represents the Western blot detection results of ALDOB protein expression levels in normal intestinal epithelial cells and colorectal cancer cell lines; G represents the qPCR detection results of ALDOB mRNA expression levels in paired colorectal cancer tissues and adjacent normal tissues; H represents the Western blot detection results of ALDOB protein expression levels in paired colorectal cancer tissues and adjacent normal tissues; and I represents the immunohistochemical staining results of ALDOB in representative colorectal cancer tissues and paired adjacent normal tissues.

[0036] Figure 3 Figure 1 shows the results of the construction and validation of the ALDOB silencing model. A represents the qPCR results of ALDOB mRNA expression levels in the shNC, shALDOB-673, shALDOB-384, and shALDOB-112 groups in HCT116 and Caco2 cells; B represents the Western blot results of ALDOB protein expression levels in the shNC, shALDOB-673, shALDOB-384, and shALDOB-112 groups in HCT116 and Caco2 cells.

[0037] Figure 4 The figure shows the effect of ALDOB silencing on the growth-related phenotypes of colorectal cancer cells; where A represents the cell viability test results of each group in HCT116 and Caco2 cells; B represents the colony formation test results of each group in HCT116 and Caco2 cells; and C represents the EdU test results of each group in HCT116 and Caco2 cells.

[0038] Figure 5The figure shows the effect of ALDOB silencing on the apoptosis rate of colorectal cancer cells; where A represents the results of flow cytometry detection of apoptosis in each group of HCT116 cells; and B represents the results of flow cytometry detection of apoptosis in each group of Caco2 cells.

[0039] Figure 6 The figure shows the effect of ALDOB silencing on glucose metabolism in colorectal cancer cells; where A represents the glucose consumption of HCT116 and Caco2 cells in each group; B represents the lactate production of HCT116 and Caco2 cells in each group; and C represents the ATP content of HCT116 and Caco2 cells in each group.

[0040] Figure 7 The figures show the results of subcutaneous tumor-bearing models and in vivo imaging verification of the effect of targeted inhibition of ALDOB on tumorigenesis in vivo. Among them, A is a schematic diagram of the experimental process of the subcutaneous tumor-bearing model; B is the in vivo fluorescence imaging result of tumor-bearing mice; C is the actual image of the tumor removed at the experimental endpoint; D is the statistical graph of tumor weight at the endpoint; E is the tumor volume growth curve; F is the qPCR and Western blot detection results of ALDOB expression level in tumor tissue; and G is the immunohistochemical detection results of Ki67 and ALDOB in tumor tissue. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art; the experimental materials used, unless otherwise specified, can be obtained from commercial sources or prepared under conventional conditions in the art. Unless otherwise stated, the cell culture, nucleic acid extraction, real-time quantitative PCR, protein extraction, Western blot, immunohistochemistry, cell function experiments and animal experiments used in this invention can be appropriately adjusted according to well-known technical conditions in the art.

[0042] Bioinformatics analysis: To validate the rationale for ALDOB as a nucleic acid intervention target in colorectal cancer, expression profile data from publicly available databases can be used to analyze ALDOB expression in colorectal cancer. Preferably, publicly available transcriptome data from colorectal cancer tissues and normal colorectal tissues are obtained, and the differences in ALDOB expression levels are compared. Further validation is then performed using independent external datasets. The analysis process may include standard steps such as data download, standardization, difference comparison, and result visualization. If necessary, prognostic analysis and pan-cancer differential expression analysis of ALDOB can be performed using publicly available databases to further evaluate its clinical relevance and expression characteristics in different cancer types. Through the above analysis, the expression characteristics of ALDOB in colorectal cancer can be evaluated, providing a basis for subsequent target selection.

[0043] Cells, vectors, and experimental animals: This embodiment preferably uses normal human intestinal epithelial cells and human colorectal cancer cell lines for research. Preferably, the normal intestinal epithelial cells can be HIEC cells; the colorectal cancer cell lines can include HT29, SW480, HCT116, and / or Caco2 cells; the virus packaging cells can be 293T cells. In this embodiment, the vector used to construct the ALDOB silencing model is preferably an LV3 (H1 / GFP&Puro) lentiviral expression vector containing the ALDOB target sequence. The target sequence of the negative control sh-NC is 5'-TTCTCCGAACGTGTCACGT-3'. The experimental animals used for in vivo experiments are 4-5 week old female BALB / c-Nude nude mice, purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. The animals are preferably housed in an SPF-grade environment with free access to food and water, and under constant temperature and humidity conditions. All operations involving animal experiments should comply with the ethical requirements of the institution's experimental animal guidelines, with animal ethics approval number MEC120250027.

[0044] Clinical tissue samples: To further verify the expression of ALDOB in colorectal cancer, pathologically confirmed colorectal cancer tissues and their paired adjacent normal tissues were selected as clinical samples. The clinical samples consisted of 40 pairs of pathologically confirmed colorectal cancer tissues and their paired adjacent normal tissues. The collection and use of relevant tissue specimens should strictly adhere to medical ethics guidelines and obtain approval from the relevant medical ethics committee (ethics approval number 21YF5FA112). These clinical tissue samples can be used to detect the mRNA and / or protein expression levels of ALDOB, preferably using qPCR, Western blot, and immunohistochemistry to verify the expression trend of ALDOB in colorectal cancer tissues.

[0045] Unless otherwise specified, the reagents and consumables used in the embodiments of this invention can be selected from conventional commercially available products in the field. The reagents and consumables mainly include: cell culture-related reagents, such as penicillin-streptomycin mixture, RPMI 1640 medium, fetal bovine serum, serum-free cell cryopreservation solution, PBS phosphate buffer, and 0.25% trypsin digestion solution; nucleic acid detection-related reagents, such as total RNA extraction reagents, chloroform or chloroform substitutes, premixed reagents for genomic-de-sense cDNA first-strand synthesis, and premixed reagents for quantitative real-time PCR; protein detection-related reagents, such as PVDF membranes, gel preparation kits, electrophoresis buffers, transfer buffers, TBST buffer, blocking reagents, antibody dilution buffers, protein loading buffers, BCA protein concentration assay kits, RIPA lysis buffers, protease inhibitors, and ECL chemiluminescence detection reagents; cell function detection-related reagents, such as CCK-8 assay kits, crystal violet staining solution, EdU cell proliferation assay kits, and Annexin V / PI cell apoptosis assay kits; and histological detection-related reagents, such as tissue fixation solutions, paraffin embedding reagents, section staining reagents, antigen retrieval solutions, and immunohistochemical staining reagents. All of the above reagents can be selected from suitable products known in the field, depending on the experimental requirements.

[0046] Antibodies and primers: Unless otherwise specified, the antibodies and primers used in the embodiments of this invention can be conventional commercially available products in the art. Preferably, the primary antibody includes ALDOB antibody, β-actin antibody, and Ki67 antibody. Preferably, ALDOB antibody and β-actin antibody can be used for Western blot detection, and Ki67 antibody can be used for immunohistochemical detection of tumor tissue proliferation activity. Primers used for real-time quantitative PCR detection preferably include ALDOB primers and β-actin primers (internal reference gene). Preferably:

[0047] The ALDOB forward primer sequence can be: 5'-TGTCTGGTGGCATGAGTGAAG-3';

[0048] The ALDOB reverse primer sequence can be: 5'-GGCCCGTCCATAAGAGAAACTT-3';

[0049] The forward primer sequence for β-actin can be: 5'-TTGTTACAGGAAGTCCCTTGCC-3';

[0050] The reverse primer sequence for β-actin can be: 5'-ATGCTATCACCTCCCCTGTGT-3'.

[0051] Unless otherwise specified, the instruments and equipment used in the embodiments of this invention can be selected from conventional commercial equipment in the field, including but not limited to: clean bench, NanoDrop spectrophotometer, chemiluminescence imaging system, cell culture incubator, low-temperature high-speed centrifuge, low-speed centrifuge, electrophoresis apparatus, constant temperature metal bath, autoclave, real-time fluorescence quantitative PCR instrument, ultra-low temperature freezer, decolorizing shaker, inverted biological microscope, electric thermostatic water bath, pipette, paraffin microtome, constant temperature slide oven, tissue cryo-grinding instrument, flow cytometer, multi-functional microplate reader, broadband small animal in vivo imaging system, research-grade inverted microscopy imaging system, and enzyme-linked immunosorbent assay (ELISA) reader, etc.

[0052] Each in vitro experiment of this invention preferably involves at least three independent replicates; parallel replicates may be included in each replicate. Animal experiments are conducted according to a predetermined sample size, and grouping, modeling, observation, and sampling are performed under identical conditions. Experimental data can be expressed as mean ± standard deviation (mean ± SD). For comparisons between two groups, a two-tailed Student's t-test can be used; for comparisons of three or more groups, one-way ANOVA can be used. When comparing tumor volumes at multiple time points, repeated measures ANOVA or other appropriate statistical methods may be used depending on the experimental design. A p-value < 0.05 is used as the criterion for statistical significance.

[0053] Example 1: Bioinformatics analysis of ALDOB and validation of its expression in colorectal cancer

[0054] First, the expression of ALDOB in colorectal cancer was analyzed using the TCGA database. Transcriptome expression data of colorectal cancer tissues and normal colorectal tissues were obtained, and the expression levels of ALDOB were compared and analyzed; further validation was performed using the independent external dataset GSE41657. The results are as follows: Figure 2 As shown in Figures A and B, compared with normal tissues, the expression level of ALDOB was increased in colorectal cancer tissues, suggesting that ALDOB may be related to the occurrence and development of colorectal cancer and could serve as a candidate target for subsequent nucleic acid intervention studies.

[0055] Further analysis of the relationship between ALDOB and the prognosis of colorectal cancer patients was conducted using the PanCanSurvPlot database. Results are as follows: Figure 2 As shown in Figure C, high ALDOB expression is associated with poorer prognosis in patients, suggesting that in addition to its abnormally high expression in colorectal cancer, ALDOB may also have some prognostic value.

[0056] Furthermore, the expression of ALDOB in pan-cancer studies was analyzed using the TIMER database. Transcriptome expression data from various tumor tissues and corresponding normal tissues were obtained, and the expression levels of ALDOB in different cancer types were compared and analyzed. Results are as follows: Figure 2 As shown in Figure D, ALDOB exhibits differential expression characteristics in different cancer types, suggesting that ALDOB may play a role in the occurrence and development of various tumors, among which its abnormal expression in colorectal cancer has further research value.

[0057] To further verify the expression of ALDOB in colorectal cancer cells, normal intestinal epithelial cells and various colorectal cancer cell lines were selected for detection. Normal intestinal epithelial cells could be HIEC cells, and colorectal cancer cell lines could include one or more of HT29, SW480, HCT116, and Caco2. ALDOB mRNA expression levels were detected by qPCR, and ALDOB protein expression levels were detected by Western blot. Results are as follows: Figure 2 As shown in Figures E and F, compared with normal intestinal epithelial cells, the expression levels of ALDOB mRNA and protein in various colorectal cancer cell lines showed an increasing trend, suggesting that ALDOB has high expression in colorectal cancer cells.

[0058] Further validation of ALDOB expression in clinical tissues was conducted. The tissue samples preferably included pathologically confirmed colorectal cancer tissues and their paired adjacent normal tissues. Forty pairs of colorectal cancer tissues and their paired adjacent normal tissues were used for analysis. qPCR was used to detect ALDOB mRNA expression levels in the paired tissues, Western blot was used to detect ALDOB protein expression levels, and immunohistochemistry was used to observe ALDOB protein expression in representative tissue sections. Results are as follows: Figure 2 As shown in Figure G, the overall expression level of ALDOB mRNA in colorectal cancer tissues was higher than that in paired adjacent normal tissues; Figure 2 As shown in Figure H, the overall expression level of ALDOB protein in colorectal cancer tissues was higher than that in the paired adjacent normal tissues; Figure 2 As shown in Figure I, the intensity of ALDOB immunohistochemical staining in colorectal cancer tissue is higher than that in adjacent normal tissue.

[0059] The above-mentioned public database analysis, prognostic analysis, pan-cancer differential expression analysis, cell line validation, and clinical tissue validation results collectively indicate that ALDOB is highly expressed in colorectal cancer and is associated with patient prognosis, providing a basis for its use as a nucleic acid intervention target in colorectal cancer.

[0060] Example 2: Construction and Validation of the ALDOB Silent Model

[0061] Based on Example 1, this example further designs and screens specific shRNAs targeting ALDOB, and conducts subsequent functional verification experiments.

[0062] 1. Construction of shRNA sequence, shDNA template sequence and vector targeting ALDOB.

[0063] Three candidate shRNA sequences were designed targeting the human ALDOB gene, named shALDOB-673, shALDOB-384, and shALDOB-112. All candidate shRNAs were constructed in LV3(H1 / GFP&Puro) lentiviral expression vectors and verified by sequencing. The candidate sequences, corresponding shDNA template sequences, and transcripts are as follows:

[0064] (1) shALDOB-673, whose target sequence is: 5'-GGAACACTGCCAGTATGTTAC-3' (SEQ ID NO. 9); whose shDNA sense strand (S) sequence is: 5'-GATCCGGAACACTGCCAGTATGTTACTTCAAGAGAGTAACATACTGGCAGTGTTCCTTTTTTG-3' (SEQ ID NO. 11); whose shDNA antisense strand (A) sequence is: 5'-AATTCAAAAAAGGAACACTGCCAGTATGTTACTCTCTTGAAGTAACATACTGGCAGTGTTCCG-3' (SEQ ID NO. 12); whose transcription product sequence is: 5'-GGAACACTGCCAGTATGTTACTTCAAGAGAGTAACATACTGGCAGTGTTCCTT-3' (SEQ ID NO. 10). The corresponding vector name is LV3(H1 / GFP&Puro)-shALDOB-673.

[0065] (2) shALDOB-384, whose target sequence is: 5'-TCGTGGTGGGAATCAAGTTAG-3' (SEQ ID NO. 5); whose shDNA positive strand (S) sequence is: 5'-GATCCGTCGTGGTGGGAATCAAGTTAGTTCAAGAGACTAACTTGATTCCCACCACGATTTTTTG-3' (SEQ ID NO. 7); whose shDNA antisense strand (A) sequence is: 5'-AATTCAAAAAATCGTGGTGGGAATCAAGTTAGTCTCTTGAACTAACTTGATTCCCACCACGACG-3' (SEQ ID NO. 8); whose transcription product sequence is: 5'-TCGTGGTGGGAATCAAGTTAGTTCAAGAGACTAACTTGATTCCCACCACGATT-3' (SEQ ID NO. 6). The corresponding vector name is LV3(H1 / GFP&Puro)-shALDOB-384.

[0066] (3) shALDOB-112, whose target sequence is: 5'-GAAGAAGGAGCTCTCAGAAAT-3' (SEQ ID NO.1); whose shDNA positive strand (S) sequence is: 5'-GATCCGAAGAAGGAGCTCTCAGAAATTTCAAGAGAATTTCTGAGAGCTCCTTCTTCTTTTTTG-3' (SEQ ID NO.3); whose shDNA antisense strand (A) sequence is: 5'-AATTCAAAAAAGAAGAAGGAGCTCTCAGAAATTCTCTTGAAATTTCTGAGAGCTCCTTCTTCG-3' (SEQ ID NO.4); whose transcription product sequence is: 5'-GAAGAAGGAGCTCTCAGAAATTTCAAGAGAATTTCTGAGAGCTCCTTCTTCTT-3' (SEQ ID NO.2). The corresponding vector name is LV3(H1 / GFP&Puro)-shALDOB-112.

[0067] All three shRNA lentiviral expression vectors were verified to be correct by sequencing and can be used for the construction of ALDOB silencing cell models. After initial screening, shALDOB-112 and shALDOB-384 showed higher interference efficiency and were named shALDOB-1 and shALDOB-2, respectively, for subsequent in vitro functional experiments. Among them, shALDOB-112 (i.e., shALDOB-1) was the optimal intervention sequence and was used as a representative shRNA for verification in subsequent in vivo experiments.

[0068] 2. Lentiviral packaging, infection, and screening of stable cell lines

[0069] The aforementioned candidate shRNAs targeting ALDOB were constructed into LV3 series lentiviral vectors, with a negative control shNC included. The lentiviruses were packaged in 293T cells using a three-plasmid system, comprising a vector plasmid carrying the shRNA expression cassette, a psPAX2 packaging plasmid, and a pMD2.G envelope plasmid. Viral supernatants were collected at 48 h and 72 h post-transfection and used directly or further concentrated as needed for experiments.

[0070] When infecting HCT116 and Caco2 cells, polybrene can be added to improve infection efficiency, with a preferred final concentration of 5 μg / mL. The corresponding lentivirus should be added at an MOI of 10. Replace with fresh complete culture medium 12–24 h post-infection, and continue culturing for another 24–48 h before detecting target gene expression or performing drug screening.

[0071] For stable cell line screening, puromycin was used, with the working concentration determined to be 1 μg / mL through a minimum lethal concentration (MLD) pre-experiment. Screening was continued for at least 2 weeks to obtain cell lines with stable ALDOB knockdown. Subsequently, stable cell lines with high interference efficiency were screened based on qPCR and Western blot results for functional and in vivo experiments.

[0072] 3. qPCR detection method

[0073] In this invention, qPCR detection is preferably used to evaluate the inhibitory effect of candidate shRNA on ALDOB mRNA expression level, and to detect ALDOB expression in clinical tissues and cells.

[0074] For cell samples, adherent cells were seeded in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and cultured at 37°C in a 5% CO2 incubator. When the cells were in good growth condition and the confluence reached 70%–90%, the medium was discarded, and the cells were slowly washed twice with pre-cooled PBS. Total RNA extraction reagent lysis buffer was added to each well, and the cells were incubated at room temperature for 5 min to allow for complete cell lysis.

[0075] For tissue samples, an appropriate amount of tissue can be placed in a pre-cooled homogenization tube, and after adding total RNA extraction reagent lysis buffer, it can be thoroughly homogenized using a tissue homogenizer or tissue cryo-grinder to ensure that the tissue sample is fully lysed.

[0076] Subsequently, the lysis buffer was transferred to an RNase-free centrifuge tube, and chloroform was added according to the instructions of the total RNA extraction reagent. The mixture was vigorously vortexed and allowed to stand at room temperature for 10 min, followed by centrifugation at 12000×g for 10 min at 4°C to separate the sample into layers. The upper aqueous phase was transferred to a new RNase-free centrifuge tube, and an equal volume of isopropanol or precipitation reagent was added according to the reagent instructions. The mixture was then incubated at room temperature and centrifuged at 12000×g at 4°C to precipitate the total RNA. The supernatant was discarded, and the RNA precipitate was washed twice with 75% ethanol. After air drying, an appropriate amount of RNase-free ddH2O was added to dissolve the RNA. RNA concentration and purity were determined using NanoDrop.

[0077] Take an equal volume of total RNA, remove genomic DNA if necessary, and then reverse transcribe according to the reverse transcription kit instructions to obtain cDNA. Store the reverse transcription product at -20℃ or -80℃ for later use. Use the obtained cDNA as a template and perform detection using a SYBR real-time PCR kit. Ideally, each sample should be set up with 3 technical replicates and at least 3 independent biological replicates. The qPCR reaction system and amplification program are preferably set according to the kit instructions; in one embodiment, the total reaction volume is 10 μL, including 1.0 μL of cDNA template, 5.0 μL of 2×SYBR Mix, 0.3 μL of upstream primer, 0.3 μL of downstream primer, 0.2 μL of ROX correction solution, and RNase-free ddH2O to bring the total volume to 10 μL. The PCR amplification program can be 95℃ pre-denaturation, followed by 40 cycles: 95℃ denaturation, 60℃ annealing / extension. After amplification, perform melting curve analysis to evaluate amplification specificity. β-actin is used as an internal control gene, and 2^ -ΔΔCt The relative expression level of the target gene is calculated using this method.

[0078] The results are as follows Figure 3As shown in Figure A, compared with the shNC group, shALDOB-673, shALDOB-384, and shALDOB-112 all significantly downregulated ALDOB mRNA expression levels in HCT116 and Caco2 cells. Among them, shALDOB-384 and shALDOB-112 showed more significant overall interference effects, while shALDOB-673, although also having an inhibitory effect, had a relatively lower overall interference efficiency.

[0079] 4. Western blot detection method

[0080] In this invention, Western blot detection is used to evaluate the inhibitory effect of candidate shRNAs on ALDOB protein expression levels, and to detect changes in ALDOB protein expression levels in cell samples, tissue samples, and subcutaneous tumor-bearing tissues.

[0081] Cells were collected after reaching a suitable state under standard culture conditions; for adherent cells, collection could be carried out when cell confluence reached 70%–90%. After discarding the culture medium, the cells were washed twice with pre-chilled PBS, and RIPA lysis buffer was added, with protease inhibitors added before use. Lysis was performed on ice for 30 min, with gentle pipetting or shaking every 10 min to mix. After lysis, the cells were centrifuged at 4°C and 12000×g for 10 min, and the supernatant was collected as the total protein extract. For tissue samples, an appropriate amount of tissue was minced and placed in a pre-chilled homogenization tube, with an appropriate amount of RIPA lysis buffer added, and protease inhibitors and / or phosphatase inhibitors added before use. After thorough homogenization using a tissue homogenizer or cryogenic homogenizer, the cells were placed on ice for 30 min of lysis, followed by centrifugation at 4°C and 12000×g for 10 min, and the supernatant was collected as the total protein extract. Subsequent protein quantification, electrophoresis, membrane transfer, and immunoassay steps can be performed according to the cell sample method described above.

[0082] Protein concentrations in each sample were determined using a BCA protein assay kit and adjusted to the same concentration with lysis buffer. Equal volumes of protein samples were added to 5× protein loading buffer, mixed, and heated for denaturation. 25 μg of protein was loaded into each well. Electrophoresis was performed using a 10% SDS-PAGE gel, with a stacking gel voltage of 80 V and a separating gel voltage of 120 V. After electrophoresis, the protein was transferred to a PVDF membrane, which was activated with methanol before use. The membrane was transferred using a wet transfer method at 4°C and a constant current of 200 mA for 2 h.

[0083] After transfer, the PVDF membrane was blocked in blocking solution at room temperature for 1 h. Primary antibody was then added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 10 min each time; then, the corresponding HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 h. After incubation, the membrane was washed three more times with TBST for 10 min each time. ECL chemiluminescent solution was dropped onto the membrane surface, and exposure and image acquisition were performed using a chemiluminescence imaging system. ImageJ software was used for semi-quantitative analysis of the band gray values, and β-actin was used as an internal control for normalization.

[0084] The results are as follows Figure 3 As shown in Figure B, compared with the shNC group, all three candidate shRNAs could downregulate ALDOB protein expression levels to varying degrees. Combining the results of mRNA and protein level detection in both cell types, shALDOB-384 and shALDOB-112 both exhibited relatively stable and significant interference effects.

[0085] Therefore, shALDOB-112 and shALDOB-384 were selected for subsequent studies and named shALDOB-1 and shALDOB-2, respectively. These results demonstrate that the selected shRNA sequences can effectively suppress ALDOB expression, successfully constructing an ALDOB silencing model.

[0086] Example 3: Effects of ALDOB on growth-related phenotypes of colorectal cancer cells

[0087] Cell viability was assessed using the CCK-8 assay: Cells from different treatment groups were seeded at predetermined densities in 96-well plates. CCK-8 working solution was added to each well at set time points, and after incubation for an appropriate time, the absorbance at 450 nm was measured to evaluate changes in cell viability.

[0088] The long-term proliferative capacity of cells was evaluated using a colony formation assay: Cells from different treatment groups were seeded at low density in culture plates and cultured routinely until visible colonies were formed. The culture medium was then discarded, and the cells were washed, fixed, and stained. The colony formation status was recorded and calculated.

[0089] EdU incorporation assay was used to evaluate the cellular DNA synthesis capacity: cells from different treatment groups were seeded on culture plates or cell slides, incubated with EdU working solution, fixed, permeabilized, and stained, and the nuclei were counterstained. EdU-positive cells were observed and recorded under a fluorescence microscope.

[0090] The cell groups successfully constructed in Example 2 were used for subsequent assays. In the cell viability assay, each group of cells was seeded in a 96-well plate with 1000 cells per well and at least 5 replicates per group. Cell viability changes were detected using the CCK-8 assay on days 0, 1, 2, 3, and 4 post-seeding, and absorbance at 450 nm was measured. Based on the screening results of Example 2, two stable knockdown cell lines, shALDOB-1 and shALDOB-2, with high interference efficiency were selected for subsequent functional experiments. A negative interference control group, shNC, was also included. Subsequently, each group of cells was seeded in a 6-well plate with 500 cells per well and cultured for 10 days. After fixation and crystal violet staining, the number of colonies formed was counted. The EdU reagent kit was used to detect the DNA synthesis capacity of each group of cells, with the proportion of EdU-positive cells reflecting the cellular DNA synthesis activity.

[0091] The results are as follows Figure 4 As shown in Figure A, in HCT116 and Caco2 cells, cell viability generally decreased after ALDOB silencing, with shALDOB-1 and shALDOB-2 showing relatively stable inhibitory effects. Figure 4 As shown in Figure B, the colony formation assay demonstrated that both shALDOB-1 and shALDOB-2 reduced the number of cell colonies formed. Figure 4 As shown in Figure C, the EdU assay further demonstrated that both shALDOB-1 and shALDOB-2 could reduce the EdU positivity rate. These results suggest that targeted inhibition of ALDOB can significantly suppress growth-related phenotypes in colorectal cancer cells.

[0092] Example 4: Effect of ALDOB on apoptosis of colorectal cancer cells

[0093] Annexin V / PI double staining combined with flow cytometry was used to evaluate cell apoptosis: After collecting cells from different treatment groups, Annexin V and PI staining was performed according to the kit instructions, and the proportion of apoptotic cells was detected by flow cytometry.

[0094] Based on the screening results of Example 2, two stable knockdown cell lines, shALDOB-1 and shALDOB-2, with high interference efficiency were selected for subsequent experiments. A negative interference control group, shNC, was also set up. The apoptosis rate of each group was detected by Annexin V-YSFluor™ 647 / PI double staining flow cytometry.

[0095] The results are as follows Figure 5 As shown in Figure A, both shALDOB-1 and shALDOB-2 can increase the apoptosis rate in HCT116 cells. Figure 5As shown in Figure B, both shALDOB-1 and shALDOB-2 can increase the apoptosis rate in Caco2 cells. These results indicate that targeted inhibition of ALDOB can promote apoptosis in colorectal cancer cells.

[0096] Example 5: Effects of ALDOB on glucose metabolism in colorectal cancer cells

[0097] Cellular glucose consumption was detected using a glucose assay kit: Cells from different treatment groups were seeded in culture plates at predetermined densities. After culture for a set time, cell culture supernatant was collected, and the changes in glucose content in the supernatant were detected. The level of cellular glucose metabolism was evaluated based on the glucose consumption in the culture system.

[0098] Lactate production in cells was detected using a lactate detection kit: Cells from different treatment groups were seeded at predetermined densities and cultured for a set time. The cell culture supernatant was then collected, and the lactate content was detected to evaluate changes in cellular glycolysis metabolism.

[0099] The ATP content of cells was detected using an ATP assay kit: Cells from different treatment groups were seeded in culture plates at predetermined densities and cultured for a set time. Cell samples were then collected, and the ATP content was detected according to the kit instructions. Normalization analysis was performed in conjunction with protein concentration to evaluate changes in cellular energy metabolism levels.

[0100] The cell groups successfully constructed in Example 2 were used for subsequent detection. Based on the screening results of Example 2, two stable knockdown cell lines with high interference efficiency, shALDOB-1 and shALDOB-2, were selected for subsequent functional experiments, while a negative interference control group, shNC, was set up. HCT116 and Caco2 cells were selected for detection, and the cells in each group were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured. When the cell confluence reached about 90%, the cell culture supernatant and cell samples were collected, and changes in glucose consumption, lactate production and ATP content were detected.

[0101] The results are as follows Figure 6 As shown in Figure A, in HCT116 and Caco2 cells, silencing ALDOB reduced cellular glucose consumption, with shALDOB-1 and shALDOB-2 showing more significant inhibitory effects. Figure 6 As shown in Figure B, lactate detection results indicate that both shALDOB-1 and shALDOB-2 can reduce cellular lactate production. Figure 6As shown in Figure C, the ATP assay results further indicate that both shALDOB-1 and shALDOB-2 can reduce cellular ATP levels. These results suggest that targeted inhibition of ALDOB can significantly reduce glucose consumption, lactate production, and ATP levels in colorectal cancer cells, thereby inhibiting glycolytic metabolism in these cells.

[0102] Example 6: Validating the effect of ALDOB silencing on in vivo tumor formation using a subcutaneous tumor-bearing model.

[0103] In in vivo experiments, a subcutaneous tumor-bearing model was used to evaluate the effect of ALDOB silencing on tumorigenicity. Cells from different treatment groups were prepared into single-cell suspensions and subcutaneously inoculated into experimental animals. After model formation, tumor size was observed and measured periodically. In vivo imaging of the animals was performed at predetermined time points, and tumor tissue was collected at the experimental endpoint for subsequent analysis. In vivo fluorescence signals, endpoint tumor weight, tumor volume changes, and the expression of ALDOB and Ki67 in tumor tissue were detected to evaluate the effect of targeted inhibition of ALDOB on tumor growth and tissue proliferation activity.

[0104] BALB / c-Nude nude mice were used to establish a subcutaneous tumor-bearing model using the HCT116 stable cell line. The HCT116 stable cell line also carries a fluorescent label that can be used for in vivo optical imaging detection. Considering that previous screening results showed that shALDOB-1 has high interference efficiency, the shALDOB-1 (designated sh-ALDOB in this experiment) stable knockdown cell line was selected as a representative intervention sequence for validation in animal experiments. The experimental groups included the sh-NC group and the sh-ALDOB group, with 6 mice in each group. The animal ethics approval number was MEC120250027.

[0105] HCT116 cells with different treatments were suspended in PBS and each cell was seeded with 3 × 10⁶ cells. 6 A tumor-bearing model was established by subcutaneous inoculation of cells on day 0. The specific experimental procedure is as follows: Figure 7 As shown in Figure A. Tumor formation was observed from day 10, with the long and short diameters of the tumor measured every 5 days. The tumor volume was calculated using the formula V = 1 / 2 × long diameter × short diameter², and monitoring continued until day 40. To dynamically monitor tumor growth in vivo, in vivo fluorescence imaging was performed at day 25 using the DPM broadband small animal in vivo optical imaging system. Before imaging, mice were anesthetized with isoflurane inhalation at an induction concentration of 3% and a maintenance concentration of 1.5%. After stable anesthesia, the mice were placed on the imaging platform for image acquisition. During the acquisition process, exposure time, field of view, and related parameters were uniformly set, and acquisition conditions were kept consistent across groups. At the end of the experiment, the animals were euthanized, the tumors were dissected and weighed, and a portion of the tumor tissue was collected for qPCR, Western blot, and immunohistochemical detection.

[0106] Immunohistochemistry was used to detect the expression of ALDOB and Ki67 in tissue samples: Tissue samples were fixed with 4% paraformaldehyde, and after routine dehydration, clearing, paraffin embedding, and paraffin embedding, sections were prepared with a preferred section thickness of 4 μm. After dewaxing with xylene, the sections were rehydrated in a series of ethanol solutions and then rinsed with distilled water. Antigen retrieval was then performed, preferably using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0–9.0) under heated conditions. After natural cooling, the sections were washed with PBS. The sections were then incubated with 3% hydrogen peroxide at room temperature for approximately 10 min to block endogenous peroxidase activity. A suitable blocking solution, such as 5%–10% goat serum, was then added, and the sections were blocked at room temperature for approximately 20 min. After discarding the blocking solution, primary antibody was added, and the sections were incubated overnight at 4°C. The next day, after returning to room temperature, the sections were washed three times with PBS for approximately 5 min each time, and then the corresponding secondary antibody was added, followed by incubation at room temperature for approximately 20 min. After washing, DAB chromogenic solution was added for staining. The staining was observed under a microscope, and the reaction was terminated as appropriate. Subsequently, the cell nuclei were counterstained with hematoxylin, rinsed with tap water, and then blued again. The cells were then dehydrated with a series of ethanol solutions, cleared with xylene, and mounted with neutral resin. Finally, the cells were observed and images were acquired under a microscope. The expression level of the target protein was analyzed based on the staining intensity and the proportion of positive cells.

[0107] The results are as follows Figure 7 As shown in Figure B, the in vivo fluorescence signal of tumor-bearing mice in the sh-ALDOB group was weakened compared to the sh-NC group, suggesting that ALDOB silencing can inhibit tumor growth in vivo. Figure 7 As shown in Figure C, the total volume of tumors dissected in the sh-ALDOB group was smaller than that in the sh-NC group. Figure 7 As shown in Figure D, the sh-ALDOB group showed a decrease in tumor weight as the endpoint. Figure 7 As shown in Figure E, the tumor volume growth rate was slower in the sh-ALDOB group. Figure 7 As shown in Figure F, ALDOB expression is decreased in tumor tissue. Figure 7 As shown in Figure G, the immunohistochemical staining of Ki67 and ALDOB was weakened, suggesting that ALDOB silencing reduced the proliferative activity of tumor tissue.

[0108] The above results indicate that targeted inhibition of ALDOB can significantly reduce the in vivo tumorigenicity of HCT116 cells, and the difference is statistically significant (P<0.05).

[0109] This invention provides a specific shRNA targeting ALDOB, the nucleic acid encoding the shRNA, and its recombinant expression vector. In vitro experiments show that targeting and inhibiting ALDOB can suppress the growth of colorectal cancer cells, promote apoptosis, and inhibit glycolytic metabolism. In vivo experiments show that targeting and inhibiting ALDOB can suppress tumor growth in a subcutaneous tumor-bearing model and reduce Ki67 expression. Therefore, the shRNA, nucleic acid, and recombinant expression vector provided by this invention have clear and reproducible technical effects, and can be used for the research and development of nucleic acid intervention agents related to colorectal cancer, demonstrating good industrial applicability.

Claims

1. The application of ALDOB as a target in screening drugs for colorectal cancer, characterized in that, The drug inhibits the expression of ALDOB.

2. A shRNA targeting ALDOB, characterized in that, The target sequence of the shRNA targeting the ALDOB gene is shown in SEQ ID NO. 1 or 5.

3. The shRNA as described in claim 2, characterized in that, The transcript sequence of the shRNA is shown in SEQ ID NO.2 or 6.

4. The shRNA as described in claim 2, characterized in that, The nucleotide sequence of the sense strand of the shRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.4; or the nucleotide sequence of the sense strand of the shRNA is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

8.

5. A nucleic acid construct, characterized in that, The nucleic acid construct contains a gene fragment encoding any one of the shRNAs described in claims 2-4.

6. A recombinant expression vector comprising the shRNA as described in any one of claims 2-4.

7. The recombinant expression vector as described in claim 6, characterized in that, The recombinant vector is a lentiviral vector.

8. A lentivirus, characterized in that, The lentivirus is prepared by viral packaging of the nucleic acid construct described in claim 5 with the assistance of lentivirus packaging plasmids and cell lines.

9. The use of the shRNA as described in any one of claims 2-4, the nucleic acid construct as described in claim 5, the recombinant expression vector as described in any one of claims 6-7, or the lentivirus as described in claim 8 in the preparation of anti-colorectal cancer drugs.

10. A pharmaceutical composition for treating colorectal cancer, characterized in that, The active ingredient of the pharmaceutical composition includes the shRNA of any one of claims 2-4, the nucleic acid construct of claim 5, the recombinant expression vector of any one of claims 6-7, or the lentivirus of claim 8.

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