Colorectal cancer treatment target HSD17B6 gene and application thereof

Abstract

The invention relates to the field of medicine, in particular to a colorectal cancer treatment target HSD17B6 gene and application thereof, it is found that HSD17B6 can be used as a target for treating colorectal cancer, and results of low expression and overexpression experiments of HSD17B6 in corresponding colorectal cancer tissues and colorectal cancer cell lines prove that overexpression of the gene can be used as a target for treating colorectal cancer. The HSD17B6 can influence the proliferation, migration and invasion of colorectal cancer cells, so that the HSD17B6 can be used as a treatment target of the colorectal cancer, and the carrier developed according to the molecular target can effectively treat the colorectal cancer and has great clinical value and economic value.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a colorectal cancer treatment target HSD17B6 gene and applications thereof. Background Art

[0002] 17β-hydroxysteroid dehydrogenase type 6 (HSD17B6) is a microsomal enzyme, a member of the 17β-hydroxysteroid dehydrogenases (17β-HSDs). It influences biochemical metabolism and endocrine function by regulating steroid hormone levels, particularly androgen levels. HSD17B6 also participates in immune responses, influencing the development and progression of various metabolic and endocrine diseases and tumors, such as metabolic syndrome, obesity, inflammation, and hormone-dependent endometrial, prostate, and breast malignancies. Although the mechanism by which HSD17B6 inhibits tumor development remains unclear, HSD17B6 can synthesize DHT, and abnormal levels of DHT can affect tumor progression in various ways, such as in PCa, breast cancer, lung cancer, and colorectal cancer. Furthermore, HSD17B6 expression is closely linked to alterations in immune function.

[0003] Although it is expressed in various tissues, its specific role in colon cancer has not been fully elucidated. Therefore, it is urgent to verify the role of the HSD17B6 gene in colorectal cancer. Summary of the Invention

[0004] In order to solve the above problems, the first object of the present invention is to provide a colorectal cancer treatment target HSD17B6 gene, wherein the treatment target is the HSD17B6 gene.

[0005] The second object of the present invention is to provide an application of the HSD17B6 gene, a target for colorectal cancer treatment, wherein the application is a product for diagnosing colorectal cancer or screening drugs for treating colorectal cancer.

[0006] Preferably, the product is a kit, a chip, or a test paper.

[0007] Preferably, the drug is a vector containing an HSD17B6 gene overexpression vector.

[0008] The third object of the present invention is to provide a detection kit for colorectal cancer, which comprises a primer pair for detecting the HSD17B6 gene.

[0009] A fourth object of the present invention is to provide a test strip for detecting colorectal cancer, wherein the test strip comprises an antibody for detecting HSD17B6 protein.

[0010] A fifth object of the present invention is to provide a use of the above-mentioned kit or the above-mentioned test paper in detecting colorectal cancer or screening drugs for treating colorectal cancer, wherein the use is for non-diagnostic or non-therapeutic purposes.

[0011] Preferably, the application is scientific research.

[0012] The sixth object of the present invention is to provide a colorectal cancer treatment product, which contains an HSD17B6 gene overexpression vector.

[0013] The seventh object of the present invention is to provide an application of a vector containing an HSD17B6 gene overexpression in the preparation of a product for treating colorectal cancer.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects:

[0015] The present invention discovered that HSD17B6 is lowly expressed in corresponding colorectal cancer tissues and colorectal cancer cell lines. The results of overexpression experiments showed that overexpression of this gene can affect the proliferation, migration and invasion of colorectal cancer cells, indicating that HSD17B6 can be used as a therapeutic target for colorectal cancer. The overexpression vector developed based on this molecular target can effectively treat colorectal cancer and has significant clinical and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Volcano plot of gene differential expression analysis between colorectal cancer tissues and normal tissues (Log2 Fold Change vs. Log10 FDR).

[0017] Figure 2. Expression of HSD17B6 in colorectal cancer tissues and normal tissues; Figure 2A Display mRNA results; Figure 2B , Western blot results.

[0018] Fig. 3 Expression of HSD17B6 in colorectal cancer cell lines and normal cell lines. Figure 3A Display mRNA results; Figure 3B , Western blot results.

[0019] Figure 4 Validation of the HSD17B6 overexpression experiment.

[0020] Figure 5 Effect of HSD17B6 overexpression on the proliferation of colorectal cancer cells.

[0021] Figure 6 Effect of HSD17B6 overexpression on the migration of colorectal cancer cells.

[0022] Figure 7 Effect of HSD17B6 overexpression on the invasion of colorectal cancer cells.

[0023] Figure 8 Effect of HSD17B6 overexpression on NRF2 expression level in colorectal cancer cells. DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1 Screening for genes associated with colorectal cancer

[0026] More than 50 colorectal cancer tissues and corresponding adjacent normal tissue samples were collected and sent to BGI for high-throughput sequencing and transcriptome analysis. All patients gave informed consent, and the acquisition of all the above specimens was approved by the organization's ethics committee. Figure 1 , MYC gene and KRAS gene are upregulated in colorectal cancer; CDH1 gene expression is downregulated in colorectal cancer. The differential expression of MYC gene, KRAS gene and CDH1 gene are common marker genes of rectal cancer tissue and can be used as positive controls; at the same time, it was shown that HSD17B6 was significantly downregulated in colorectal cancer tissue.

[0027] Example 2 Verification of the differential expression of the HSD17B6 gene in humans and cells

[0028] (1) Differential expression in humans:

[0029] (1.1) Thirty new patient cancer tissue samples and adjacent tissue samples were collected and validated by large-sample QPCR for differential expression of the HSD17B6 gene.

[0030] (1.2) RNA extraction: Tissue RNA extraction kit (QIAGEN) was used.

[0031] (1.3) Reverse transcription:

[0032] 1 μg of total RNA template was mixed with 2 μL of 10× buffer, 2 μL of dATP (10 mM), 0.5 μL of polyA polymerase, 0.5 μL of RNase inhibitor, and RNase-free water to a final volume of 20 μL and incubated at 37°C for 1 h. Then, 1 μL of 0.5 μg / μL Oligo(dT)-specific RT primer was added to the reaction tube, incubated at 70°C for 5 min, and immediately incubated on ice for at least 2 min. The reaction mixture was then mixed with 4 μL of 5× buffer, 1 μL of dNTPs (10 mM), 0.5 μL of M-MLV reverse transcriptase, 0.5 μL of RNase inhibitor, 10 μL of polyA reaction mix, and 4 μL of RNase-free water and incubated at 42°C for 1 h.

[0033] (1.4) QPCR amplification test

[0034] QPCR amplification primers were designed based on the coding sequences of the HSD17B6 gene and β-actin gene in Genebank and synthesized by Wuhan Qingke Biotechnology Co., Ltd. The specific primer sequences are as follows:

[0035] 1) Primer design:

[0036] The primer sequences for the HSD17B6 gene are:

[0037] Forward primer: 5′-GCTGGGACCTGAAGATGAAC-3′ (SEQ ID NO. 1);

[0038] Reverse primer: 5′-CAGCCAGGTCCAGCATACTC-3′ (SEQ ID NO. 2);

[0039] Primer sequence for β-actin:

[0040] Forward primer: 5′-CATGTACGTTGCTATCCAGGC-3′ (SEQ ID NO. 3);

[0041] Reverse primer: 5′-CTCCTTAATGTCACGCACGAT-3′ (SEQ ID NO. 4);

[0042] 2) Reaction system

[0043] SYBR Green polymerase chain reaction system 12.5 μL, forward and reverse primers (5 μM) 1 μL each, template cDNA 2.0 μL, ddH2O 8.5 μL.

[0044] 3) Reaction conditions

[0045] 95℃ for 10 min, (95℃ for 15 s, 60℃ for 60 s) × 45 cycles. SYBR Green was used as a fluorescent marker and PCR was performed on a Light Cycler fluorescent quantitative PCR instrument. β-actin was used as a reference gene. The target band was determined by melting curve analysis and electrophoresis. ΔΔ Relative quantification was performed by CT method.

[0046] (1.5) Results

[0047] The results are shown in Figure 2. Figure 2A The results showed that the expression level of HSD17B6 gene was down-regulated in colorectal cancer tissues (P<0.05), which was consistent with the results of Example 1. In addition, Western blot results showed that the protein level of HSD17B6 in colorectal cancer tissues was significantly decreased ( Figure 2B ).

[0048] (2) Differential expression in colorectal cancer cell lines

[0049] (2.1) Cell culture

[0050] Human colorectal cancer cell line SW620 and human normal colon epithelial cells FHC were cultured in RPMI-1640 medium containing 10% FBS and placed in a 5% CO2, 37°C cell culture incubator for routine culture. The medium was changed every 2-3 days. Cells were passaged whenever the cell confluence reached 90%, and cells in the logarithmic growth phase were used for subsequent experiments.

[0051] (2.2) RNA extraction, reverse transcription, and QPCR were the same as step (1) of Example 2.

[0052] The results are shown in Figure 3. Figure 3A The results showed that the expression level of HSD17B6 gene was down-regulated in colorectal cancer cells (P<0.05), which was consistent with the results of Example 1. In addition, Western blot results showed that the protein level of HSD17B6 in colorectal cancer cells was significantly decreased ( Figure 3B ).

[0053] Example 3: Overexpression of the HSD17B6 gene

[0054] 1. Overexpression vector construction steps

[0055] 1. Carrier selection and design

[0056] 1.1. Select a commercial overexpression vector containing a strong CMV promoter (pLVX-IRES-ZsGreen1 or pCDH-CMV-MCS-EF1-copGFP). The vector must contain a resistance gene (such as a puromycin resistance gene) and a fluorescent marker (to facilitate transfection efficiency verification).

[0057] 1.2. When designing primers for HSD17B6 (Gene ID: 55702), include restriction enzyme sites (such as XhoI and NotI) and ensure that the primer annealing temperature matches the PCR enzyme.

[0058] Primer sequence (including XhoI and NotI restriction sites):

[0059] F:5'-CCGCTCGAGATGGCGGACAGTACAG-3' (SEQ ID NO.5);

[0060] R: 5'-GCGGCCGCTTACAGCTCCAGTGTAG-3' (SEQ ID NO. 6);

[0061] 2. Target gene amplification

[0062] 2.1. Total RNA was extracted from colorectal cancer cell lines by RT-PCR, reverse transcribed into cDNA, and then the CDS sequence of HSD17B6 (about 1.2 kb) was amplified.

[0063] 2.2 PCR system:

[0064] 2× KOD buffer 10 μL

[0065] dNTPs 2 μL

[0066] 1 μL each primer

[0067] 2 μL cDNA template

[0068] KOD enzyme 0.2 μL

[0069] ddH2O 4.8μL

[0070] 2.3. Verify the amplified product by electrophoresis and excise the gel for recovery and purification.

[0071] 3. Double enzyme digestion and ligation

[0072] The vector and target gene fragment were digested with XhoI and NotI (37°C, 1 h), and the linearized vector and target gene were recovered by agarose gel electrophoresis.

[0073] Ligation system (16°C, 4h or overnight):

[0074] Linearized vector 50ng

[0075] Target gene 100ng

[0076] 10×T4 ligase buffer 1 μL

[0077] T4 DNA ligase 1 μL

[0078] Add ddH2O to 10 μL

[0079] Transform into DH5α competent cells (ice bath for 30 min → heat shock at 42°C for 90 s → ice bath for 2 min), and spread on LB plates containing ampicillin.

[0080] 4. Sequencing Verification and Plasmid Extraction

[0081] Single clones were picked for PCR screening of bacterial solution, and positive clones were sent for sequencing to confirm the insertion direction and sequence accuracy.

[0082] The recombinant plasmid was extracted using a plasmid extraction kit and stored at -80°C after concentration determination.

[0083] 2. Transformation into Colorectal Cancer Cell Lines

[0084] 1. Cell Preparation

[0085] A commonly used colorectal cancer cell line (SW620) was selected and cultured in a culture medium containing 10% fetal bovine serum to the logarithmic growth phase.

[0086] 2. Transfection operation

[0087] The recombinant plasmid was transfected into cells using Lipofectamine 2000 transfection reagent according to the instructions (plasmid amount 2 μg / well).

[0088] 48-72 h after transfection, the transfection efficiency was confirmed by observing the fluorescent marker (such as ZsGreen1) under a fluorescence microscope.

[0089] 3. Screening and verification

[0090] The culture medium containing puromycin (2 μg / mL) was switched to screen stable cell lines and the screening was continued for 1-2 weeks.

[0091] Verify the expression results. Figure 4 , Figure 4 The results showed that the HSD17B6 gene expression level in transfected colorectal cancer cells was higher than that in untransfected cells (empty vector), indicating that the HSD17B6 gene was successfully overexpressed.

[0092] 4. Cell proliferation assay: Repeat the following experimental steps three times:

[0093] 1) With a cell density of 5x10 3 / well, inoculated into 96-well plates, 100 μL per well, and 3 replicates per group.

[0094] 2) After the cells are fully adhered, transfection was performed according to the above steps. CCK8 assay was performed at 0 h, 24 h, 48 h, 72 h, and 96 h after transfection. The CCK8 solution was replaced, and the cells were incubated at 37°C in the dark for 2 h. The absorbance at a wavelength of 450 nm was measured using a microplate reader.

[0095] See the results Figure 5 The transfected colorectal cancer cells proliferated slower than the untransfected cells (empty vector), indicating that overexpression of HSD17B6 can significantly inhibit the proliferation of colorectal cancer cells.

[0096] 5. Cell migration and invasion assay

[0097] Transwell chambers were used for cell migration analysis, and Matrigel-coated Transwell chambers were used for invasion analysis. The pore size was 0.8 μm. The cell lines of the control and experimental groups were resuspended in serum-free culture medium and seeded into the chambers (approximately 3×10 4 cells / well) and cultured for the appropriate time according to the characteristics of the cells. The cells in the upper layer of the chamber that did not pass through the pores were removed with a cotton swab. The cells in the lower layer that had passed through the membrane were fixed with methanol for 15 minutes, stained with 0.1wt% crystal violet for 20 minutes, and observed under a microscope. Five fields of view were randomly observed and the number of cells that had passed through the membrane was counted. The results are shown in Figure 2. Figure 6-7 As shown in the results, it was shown that overexpression of HSD17B6 could significantly inhibit the migration and invasion of colorectal cancer cells.

[0098] Example 4: Confirmation of the mechanism of action of HSD17B6 in inhibiting the occurrence and development of colorectal cancer

[0099] Nuclear factor Erythroid2-related factor 2 (NRF2) is an important transcription factor for cells to resist oxidative stress and is involved in oxidative damage, inflammatory response and cell apoptosis. The latest research found that colorectal cancer tissues highly express NRF2, and colorectal cancer with high NRF2 expression is more invasive / metastatic (see Jin Peiyu et al., The relationship between NRF2 expression and tumor-associated macrophages in colorectal cancer and its clinical significance, Chinese Journal of Histochemistry and Cytochemistry, February 2024, Vol. 33, No. 1). We found that the expression of the antioxidant protein NRF2 was decreased in colorectal cancer SW620 cells with overexpression of HSD17B6 ( Figure 8 Therefore, we speculate that the mechanism by which HSD17B inhibits the occurrence and development of colorectal cancer may be closely related to NRF2.

[0100] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A colorectal cancer therapeutic target HSD17B6 gene, characterized in that: The therapeutic target is the HSD17B6 gene.

2. An application of the HSD17B6 gene as a target for colorectal cancer treatment, characterized in that: The application is a product for diagnosing colorectal cancer or screening drugs for treating colorectal cancer.

3. The use according to claim 2, characterized in that The product is a test kit, a chip, or a test paper.

4. The use according to claim 2, characterized in that The drug is a vector containing the HSD17B6 gene overexpression vector.

5. A colorectal cancer detection kit, characterized in that: The kit comprises a primer pair for detecting the HSD17B6 gene.

6. A test strip for colorectal cancer, characterized in that: The test strip contains antibodies for detecting HSD17B6 protein.

7. Use of the kit according to claim 5 or the test strip according to claim 6 in detecting colorectal cancer or screening drugs for treating colorectal cancer, wherein the use is for non-diagnostic or non-therapeutic purposes.

8. The use according to claim 7, characterized in that The application described is scientific research.

9. A colorectal cancer treatment product, characterized in that: The product contains an HSD17B6 gene overexpression vector.

10. Use of a vector containing an HSD17B6 gene overexpression vector in the preparation of a product for treating colorectal cancer.

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