Application of KLHL23 in the treatment of colorectal cancer

Abstract

The present invention proposes the application of KLHL23 in the treatment of colorectal cancer. The present invention has found that KLHL23 is overexpressed in primary CRC patient specimens and is associated with a poor prognosis, suggesting that the gene may be associated with the malignant phenotype of CRC from a clinical level. Through molecular biological means, interfering with KLHL23 can significantly inhibit the proliferation, cloning and migration of CRC, thereby showing that KLHL23 has an important function in the occurrence and development of colorectal cancer. In a mouse subcutaneous tumor model, silencing KLHL23 can significantly inhibit tumor growth. In addition, the present invention also pioneered the discovery that the key cell proliferation protein CCND1 is regulated by KLHL23 through proteomic sequencing, indicating that KLHL23 can promote the proliferation and growth of tumors, and its expression level can be used as a marker gene for the prognosis of colorectal cancer, which has important clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of disease diagnosis and treatment, and in particular to the application of KLHL23 in the treatment of colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors in the world, accounting for about 10% of all tumors in terms of incidence and mortality. Currently, there are limited diagnostic reagents and targeted treatment methods for colorectal cancer. It is very meaningful to prepare diagnostic reagents and targeted treatment reagents for colorectal cancer.

[0003] For colorectal cancer with distant metastasis, vascular endothelial growth factor inhibitors (bevacizumab and aflibercept) and kinase inhibitors (regrafenib) are often used. It is worth noting that cetuximab and panitumumab can only be used for patients with non-RAS gene mutations; about 5% of colorectal cancer patients with MSI-H have good results through immune checkpoint inhibitors, such as pembrolizumab, a PD-1 monoclonal antibody and a potential CTLA-4 monoclonal antibody. However, the treatment effect of patients with metastatic colorectal cancer is still poor, and the 5-year survival rate of patients with unresectable colorectal cancer is only 15.6%.

[0004] Therefore, it is imperative to study a new therapeutic drug for colorectal cancer. Summary of the invention

[0005] The inventors found through research that KHLH23 is significantly highly expressed in CRC patients.

[0006] KHLH23 is a ubiquitin E3 ligase that can promote the reduction of protein ubiquitination. The present invention found through proteomics experiments that silencing KHLH23 significantly reduced the expression of CCND1, which significantly stabilized CCND1 and promoted cell proliferation.

[0007] The present invention found that high expression of KHLH23 in CRC is associated with poor prognosis of cancer.

[0008] The inventors found that by inhibiting KHLH23, tumor cell proliferation, clone formation, migration and invasion can be inhibited, and tumor growth in vivo can be reduced.

[0009] On the one hand, because high expression of KHLH23 in CRC is associated with poor prognosis of cancer, reducing the expression of KHLH23 in CRC patients can be an important countermeasure. Therefore, inhibitors of KHLH23 can play an important role in the preparation of drugs for the treatment of colorectal cancer.

[0010] In some embodiments, the KHLH23 inhibitor can be a substance that inhibits the activity of the KHLH23 protein, or a substance that degrades the KHLH23 protein, or a genetic tool that reduces the level of the KHLH23 protein.

[0011] In some embodiments, the substance that inhibits the activity of KHLH23 protein is selected from compounds.

[0012] In some embodiments, the genetic tool for reducing the level of KHLH23 protein is RNA interference, microRNA, gene editing or gene knockout material.

[0013] In some embodiments, the KHLH23 inhibitor is a tumor-targeted KHLH23 inhibitor.

[0014] On the other hand, the present invention provides a use of a KHLH23 detection reagent in the preparation of a colorectal cancer diagnostic reagent / kit.

[0015] In some embodiments, the diagnostic reagent / kit is a diagnostic reagent / kit for colorectal cancer prognosis.

[0016] In some embodiments, the detection reagent is used to detect the gene expression level of KHLH23.

[0017] In some embodiments, the detection reagent detects the mRNA expression level of KHLH23.

[0018] In some embodiments, the detection reagent detects the expression level of KHLH23 protein.

[0019] In some embodiments, the detection reagent is one or more of a fluorescent quantitative PCR dye, a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody, an antibody functional fragment, and a conjugated antibody.

[0020] In some embodiments, the kit is selected from one or more of a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit, a protein precipitation kit, an immunofluorescence kit, and an electrochemiluminescence detection kit.

[0021] In some embodiments, the kit is selected from one or more of a protein precipitation kit, an immunofluorescence kit, an immunoblotting kit, and an immunohistochemistry kit.

[0022] On the other hand, the present invention provides a diagnostic reagent / kit for colorectal tumors, wherein the diagnostic reagent / kit comprises a detection reagent for KHLH23.

[0023] In some embodiments, the detection reagent is used to detect the gene expression level of KHLH23.

[0024] In some embodiments, the detection reagent detects the mRNA expression level of KHLH23.

[0025] In some embodiments, the detection reagent detects the expression level of KHLH23 protein.

[0026] In some embodiments, the detection reagent is one or more of a fluorescent quantitative PCR dye, a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody, an antibody functional fragment, and a conjugated antibody.

[0027] In some embodiments, the kit is selected from one or more of a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit, a protein precipitation kit, an immunofluorescence kit, and an electrochemiluminescence detection kit.

[0028] In some embodiments, the kit is selected from a protein precipitation kit, an immunofluorescence kit, an immunoblotting detection kit, and an immunohistochemistry detection kit.

[0029] In another aspect, the present invention provides a colorectal cancer diagnosis system, the diagnosis system comprising:

[0030] Detection component: The detection component is used to detect the expression level of KHLH23;

[0031] Result judgment component: The result judgment component is used to output the disease result of the tumor patient according to the result of the expression amount of KHLH23 detected by the detection component.

[0032] In some embodiments, the expression level of KHLH23 is one or more of gene expression level, mRNA expression level and / or protein expression level.

[0033] In some embodiments, the result judgment component comprises an input module, an analysis module and an output module; the input module is used to input the expression level of KHLH23; the analysis module is used to analyze the possibility of the disease risk result of the tumor patient according to the expression level of KHLH23; the output module is used to output the analysis result of the analysis module.

[0034] In some embodiments, the detection component comprises one or more of a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometric analysis kit, an immunohistochemistry detection kit, an ELISA kit, a protein precipitation kit, an immunofluorescence kit, an electrochemiluminescence detection kit, a qPCR instrument, an immunoblotting detection device, a flow cytometer, an immunohistochemistry detection device, an ELISA detection device, an electrochemiluminescence detection device, and an immunofluorescence detection device.

[0035] In some embodiments, the kit is selected from a protein precipitation kit, an immunofluorescence kit, an immunoblotting detection kit, and an immunohistochemistry detection kit.

[0036] In the present invention, the detection sample of the KHLH23 detection reagent is tissue.

[0037] In some embodiments, the test sample is a tissue.

[0038] Compared with the prior art, some of the embodiments of the present invention have the following beneficial effects:

[0039] (1) The present study found that KHLH23 was overexpressed in primary CRC patient specimens, suggesting that this gene may be associated with the occurrence and development of CRC at the clinical level.

[0040] (2) Through molecular biological methods, interfering with KHLH23 can significantly inhibit the proliferation, cloning, and migration of CRC, thus showing that KHLH23 plays an important role in the occurrence and development of colorectal cancer.

[0041] (3) In addition, the present invention also pioneered the discovery through proteomic sequencing that the key cell proliferation protein CCND1 is regulated by KLHL23, indicating that KLHL23 can promote tumor proliferation and growth. Studies have shown that KLHL23 promotes the growth and metastasis of colorectal cancer, and its expression level can be used as a marker gene for the prognosis of colorectal cancer, which has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The mRNA levels of KLHL23 in normal tissues and colorectal cancer tissues are derived from the GSE9348 colorectal cancer database.

[0043] Figure 2 The figure is a waterfall plot of the relative KLHL23 mRNA levels in colorectal cancer tissues and paired adjacent normal tissue samples from 20 CRC patients detected by fluorescent quantitative PCR.

[0044] Figure 3Figure 2 is the Kaplan-Meier survival curve based on the expression of KLHL23 in CRC tissues; ROC curve is used to define the cutoff value, and Log-rank is used to test significance. The figure lists the colorectal cancer databases from GSE39582, GSE29621, and GSE17538 respectively.

[0045] Figure 4 To silence the expression of KLHL23 in colorectal cancer cells infected with the specified shRNA, qPCR experiments were performed to verify the silencing efficiency of shRNA. Cell proliferation was detected using CCK8 reagent, and the data are presented as mean ± standard deviation. shKLHL23-1 refers to the knockdown of KLHL23 gene expression by the hairpin structure of fragment 1; shKLHL23-2 refers to the knockdown of KLHL23 gene expression by the hairpin structure of fragment 2; Scramble refers to the control group.

[0046] Figure 5 Colorectal cancer cells were infected with the indicated shRNAs to silence KLHL23 expression, and the clonogenicity of cells was determined. Data are presented as mean ± SD.

[0047] Figure 6 Colorectal cancer cells were infected with the indicated shRNA to silence KLHL23 expression, or transfected with KLHL23 plasmids to overexpress KLHL23, and cell migration was measured using Transwell chambers. Data are presented as mean ± SD.

[0048] Figure 7 In mice, knocking down KHLH23 can inhibit the growth of mouse tumor tissue and reduce tumor weight. shKLHL23-1 refers to the knockdown of KLHL23 gene expression by fragment 1; shKLHL23-2 refers to the knockdown of KLHL23 gene expression by fragment 2; Scramble refers to the control group.

[0049] Figure 8 Figure 2 shows the situation of silencing KLHL23 with the specified shRNA in colorectal cancer cells. Figure a shows the number of differentially expressed genes between the shKLHL23-1 group and the control group. Figure b shows the detection of proteins between the shKLHL23-1 group and the control group by proteomics and differential protein analysis. It was found that silencing of KLHL23 cell cycle pathway-related proteins by shKLHL23-1 significantly reduced DETAILED DESCRIPTION

[0050] In order to make the technical personnel in the field understand the present invention more clearly and intuitively, the present invention will be further described below in conjunction with the accompanying drawings. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0051] Note: Examples 1-8 are specific experimental schemes.

[0052] Scramble: refers to the control, i.e., scrambled RNA: 5-GCTTCGCGCCGTAGTCTTA-3 (SEQ ID NO.5).

[0053] Example 1 Patients and tissue samples

[0054] Fresh paired frozen samples of primary colorectal cancer and adjacent normal colon tissues were obtained from the Department of Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University.

[0055] Example 2 Cell culture, reagents and transfection

[0056] All cells were obtained from ATCC and cultured at 37 °C and 5% CO2. Among them, DLD-1 cells were maintained in RPMI 1640 medium (RPMI) containing 10% (v / v) fetal bovine serum (FBS). 293T, HCT116 cells were cultured in DMEM medium (containing 10% FBS). All transient transfections of overexpression plasmids into cell lines followed the instructions of Lipofectamine 2000 transfection reagent (Thermo Fisher, #11668019).

[0057] Example 3 shRNA knockdown of KLHL23

[0058] In this example, four hairpin shRNAs targeting human KLHL23 transcripts were designed and screened, and two independent sequences were found that could reduce mRNA levels by >70%. The shRNAs were in the pLKO.1 vector (#1 and #2).

[0059] shKLHL23-1: Knock down the KLHL23 gene using shRNA of fragment 1, the nucleotide sequence of shRNA of fragment 1 is: 5-CCTCTATAATCTACTGAGCTA-3 (SEQ ID NO. 1);

[0060] shKLHL23-2: Knock down the KLHL23 gene using shRNA of fragment 2, the nucleotide sequence of shRNA of fragment 2 is: 5-ATAAGATCCGCTCCCTAATAT-3 (SEQ ID NO. 2);

[0061] shKLHL23-3: Knock down the KLHL23 gene using shRNA of fragment 3, the nucleotide sequence of shRNA of fragment 3 is: 5-ATGCTCAATGCCAGGTATTAC-3 (SEQ ID NO. 3);

[0062] shKLHL23-4: Knock down the KLHL23 gene using shRNA of fragment 4, the nucleotide sequence of shRNA of fragment 4 is: 5-CCTGTGTCTTACATGATGTTA-3 (SEQ ID NO.4).

[0063] The steps for preparing lentiviral particles are as follows: 55 cm 2 1×10 in a dish 7 HEK293T cells were co-transfected with 10 μg of pLKO.1 shRNA construct, 5 μg of psPAX2, and 5 μg of pMD2.G. Supernatants containing viral particles were collected 48 and 72 h after transfection and filtered through a Millex-GP filter (0.45 μm pore size, Millipore). To infect cancer cells with lentivirus, cells were infected twice with medium containing 2 mL of lentivirus, 200 μL of FBS, and 5 mg / mL polybrene (Sigma) at 37°C for 24 and 48 h. To increase the knockdown efficiency, infected cells were subjected to puromycin selection for several days.

[0064] Example 4 Subcutaneous colorectal cancer model in mice

[0065] This study was approved by the Animal Ethics and Welfare Committee of Sun Yat-sen University. Scramble, shKLHL23-1, and shKLHL23-2 lentiviruses were packaged and infected with HCT116 cells. Virus puromycing was performed to screen positive HCT116 cells for subcutaneous xenografting in mice, and in vivo tumor growth was measured. HCT116 cells (1×10 6 The tumors were inoculated subcutaneously into the hind flanks of 4-week-old female BALB / c-nu / nu mice. The length and width of the tumors were measured every three days and the expression of the formula (length × width) was used. 2 ) / 2 to calculate the volume.

[0066] Example 5 Cell proliferation detection

[0067] Colorectal cancer cells were infected with lentivirus containing Scramble, shKLHL23-1 and shKLHL23-2, and the cells were seeded in 6-well plates. The cell growth was monitored by a real-time cell imager, and photos were taken every two hours to analyze the cell growth rate.

[0068] Example 6 Clone Formation

[0069] The cells were seeded in 6-well plates, 200 cells per well, and three replicate wells were set up. The plates were placed in a cell culture incubator and incubated for 14 days, and stained with 0.5% crystal violet prepared with methanol.

[0070] Example 7 Cell Migration

[0071] The cell suspension without FBS was added to the Transwell chamber, and the complete medium containing FBS was added to the lower chamber. The cells were incubated in a cell culture incubator for 16 h, and stained with 0.5% crystal violet prepared with methanol.

[0072] Example 8 Proteomics

[0073] DLD-1 cells were infected with control and KLHL23-silencing lentiviruses, and the cells were collected 48 hours later according to the requirements of proteomics. The cells were sent to Zhongke New Life Science Company for proteomics detection, and the differential genes and differential pathways were analyzed by bioinformatics.

[0074] Experimental results analysis

[0075] Analysis using the GSE9348 database revealed that KLHL23 is highly expressed in colorectal cancer, such as Figure 1 As shown in Figure 2, qRT-PCR was performed on 20 paired colon cancer tissues and adjacent normal mucosa samples, and the relative KLHL23 mRNA levels were shown in Figure 2. Compared with normal tissues, KLHL23 was highly expressed in most tumors, such as Figure 2 As shown (the corresponding samples and reagents are as shown in Example 2).

[0076] Survival analysis (Kaplan-Meier analysis) based on the expression of KLHL23 in colorectal cancer in the GSE39582, GSE29621, and GSE17538 databases Figure 3 The results showed that high KLHL23 levels were associated with poor recurrence-free survival, indicating that high KLHL23 levels were associated with the patient's malignant prognosis.

[0077] In order to prove the role of KLHL23 in the occurrence and development of colorectal cancer, the present invention detects cell proliferation by silencing KLHL23, such as Figure 4 As shown, qPCR experiments proved that the shRNA sequence could silence KLHL23, and real-time cell imaging detection showed that silencing KLHL23 could inhibit the proliferation of colorectal cancer cells (samples, reagents and experimental methods are as in Examples 2, 3 and 5).

[0078] The results of silencing KLHL23 in the colony formation of colorectal cancer cells were further verified. Figure 5 As shown, silencing fragment No. 1 of KLHL23 can inhibit the colony formation of colorectal cancer cells (samples, reagents and experimental methods are as in Examples 2, 3 and 6).

[0079] Transwell experiments demonstrated that silencing KLHL23 also inhibited the migration of colorectal cancer cells. Figure 6 As shown (samples, reagents and experimental methods are as in Examples 2, 3 and 7). Figure 6 In the figure, HA-KLHL23 indicates KLHL23 transfected with the PCDNA3.1 plasmid.

[0080] In order to determine the contribution of KLHL23 to tumor growth in vivo, the present invention conducted a CRC mouse subcutaneous tumor model experiment (see Examples 2, 3 and 4 for implementation methods). Specifically, HCT116 cells were infected with lentiviruses of Scramble, shKLHL23-1 and shKLHL23-2, and virus puromycing was performed to screen positive HCT116 cells for subcutaneous xenografting in mice. The experimental results are shown in Figure 2. Figure 7 As shown, silencing KLHL23 can inhibit the growth of subcutaneous tumors in mice. In particular, silencing the second fragment of KLHL23 has the best effect.

[0081] In addition, Figure 8 As shown in b, through proteomic detection, it was found that silencing the No. 1 fragment of KLHL23 significantly inhibited the amount of proteins related to the colorectal cancer cell cycle pathway (see Example 2 and Example 8 for implementation methods). After silencing KLHL23, proteomic sequencing was performed to enrich differentially expressed genes (the number of differentially expressed genes is shown in Figure 2). Figure 8 As shown in a), through bioinformatics analysis, the cell cycle pathway proteins were reduced.

[0082] In summary, the present study showed that KLHL23 is highly expressed in CRC and is associated with poor survival. Its positive effects on cell proliferation, cloning and migration will increase its carcinogenicity. In in vivo experiments, silencing KLHL23 significantly inhibited the growth rate of colorectal cancer. Further proteomics proved that KLHL23 has an important regulatory role in tumor cell cycle pathways, providing new ideas for personalized medication for tumor patients.

[0083] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of a KLHL23 inhibitor in the preparation of a drug for treating colorectal cancer, wherein the KLHL23 inhibitor is a lentiviral vector carrying a shRNA with a hairpin structure shown in SEQ ID NO.1 or SEQ ID NO.2 targeting the KLHL23 transcript.

2. A drug for treating colorectal cancer, characterized in that: The invention comprises an inhibitor of KLHL23, wherein the KLHL23 inhibitor is a lentiviral vector carrying a shRNA with a hairpin structure shown in SEQ ID NO.1 or SEQ ID NO.2 targeting the KLHL23 transcript.

3. A KLHL23 inhibitor, characterized in that The invention comprises a lentiviral vector carrying a shRNA with a hairpin structure shown in SEQ ID NO.1 or SEQ ID NO.2 targeting the KLHL23 transcript.

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