Application of HPCAL1 in diagnosis and treatment of colon cancer

Abstract

The application belongs to the field of medicine and biotechnology, and discloses a molecular marker HPCAL1 gene or protein for auxiliary diagnosis of colon cancer. The expression level of HPCAL1 protein in cancer tissues of colon cancer is significantly higher than that in normal tissues adjacent to the cancer, and the expression level of HPCAL1 protein in colon cancer cell lines is significantly higher than that in normal intestinal epithelial cell strains. Knocking down the HPCAL1 gene has a significant inhibitory effect on the proliferation and migration of colon cancer cells, and can also inhibit the growth of colon cancer tumors. Therefore, HPCAL1 can be used as a molecular marker for auxiliary diagnosis of colon cancer to assist in the diagnosis of colon cancer. Meanwhile, HPCAL1 can be used as a drug, a drug target or a target gene in gene therapy, and applied to the prevention, alleviation or / and treatment of colon cancer, so as to provide a new strategy for the prevention and treatment of colon cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of HPCAL1 in diagnosis and treatment of colon cancer. BACKGROUND

[0002] Colorectal cancer (CRC) is the third most common malignant tumor in the world, accounting for 10% of the global annual cancer diagnosis and cancer-related death cases. Surgery and chemotherapy are still the first choice for patients with colon cancer. Early patients generally adopt surgical treatment, and the most common way for patients in the late stage is drug treatment. With the development of gene sequencing technology and precise targeted drugs, the survival period of patients with colon cancer has been improved. For example: the drugs targeting epidermal growth factor (EGFR), such as cetuximab and panitumumab, can significantly improve the survival rate of patients with advanced colorectal cancer; the inhibitor targeting BRAF, such as conifinib, also shows significant effect in prolonging the survival period of patients with colon cancer. However, there will still be a part of patients with acquired drug resistance who are insensitive or ineffective to the current treatment methods. The occurrence and development of colon cancer is a complex process, which is regulated by a variety of abnormal signal molecules. At present, there are still the following problems in the diagnosis and treatment of colon cancer in China: (1) low early diagnosis rate; (2) serious drug resistance of tumor seriously limits the efficacy of existing drugs; (3) high postoperative recurrence and metastasis rate; (4) most of the patients with stage Ⅲ and Ⅳ colon cancer in clinic still lack effective targeted and immunotherapy drugs, and most of the clinical mainstream anticancer drugs rely on import; (5) the current drug research and development cannot meet the clinical needs of colon cancer, and there are still potential effective drug action targets that have not been found, and there is still a huge space for the clinical research and development of new drugs for colon cancer. Therefore, it is urgent to find new tumor markers of colon cancer and to study the molecular mechanism, so as to provide new treatment targets and new treatment methods for the treatment of colon cancer.

[0003] Hippocalcin-Like Protein 1 (HPCAL1), also known as BDR1, HLP2, VILIP-3, is a member of the neuron-specific calcium-binding protein family, which was first found to be expressed in the retina and brain. Recently, researchers found that HPCAL1 can inhibit the development of liver cancer, but promote the development of glioma, indicating that HPCAL1 plays a differential role in different tumors, but the role of HPCAL1 in the occurrence and development of colon cancer has not been reported. The present application studies HPCAL1 as a target, and finds that overexpression of HPCAL1 can significantly promote the proliferation and migration ability of colon cancer cells; knockdown of HPCAL1 can significantly inhibit the proliferation and migration ability of colon cancer cells; knockdown of HPCAL1 can significantly inhibit the tumor formation rate and growth rate of colon cancer cells in nude mice. Therefore, HPCAL1 is a new tumor marker for colon cancer. The present application will provide a new tumor marker and kit for the diagnosis and treatment of colon cancer, and has important theoretical and clinical significance. SUMMARY

[0004] In view of the problems and deficiencies in the prior art, the purpose of the present application is to provide the application of HPCAL1 in the diagnosis and treatment of colon cancer.

[0005] To achieve the purpose of the application, the technical scheme adopted by the present application is as follows:

[0006] The present application provides a biomarker for assisting in the diagnosis of colon cancer, wherein the biomarker is HPCAL1 gene or protein.

[0007] The expression of HPCAL1 protein in colon cancer tissues and normal tissues adjacent to cancer was detected by Western Blot, and it was found that the expression level of HPCAL1 protein in the cancer tissues of colon cancer was significantly higher than that in the normal tissues adjacent to cancer; it was indicated that HPCAL1 protein was up-regulated in colon cancer tissues to different degrees.

[0008] The expression of HPCAL1 protein in colon cancer cell lines SW480, SW620, RKO, HCT116 and normal intestinal epithelial cells NCM460 was detected by Western Blot, and it was found that the expression level of HPCAL1 protein in colon cancer cell lines SW480, SW620, RKO, HCT116 was significantly higher than that in normal intestinal epithelial cells NCM460; it was indicated that HPCAL1 protein was up-regulated in colon cancer cell lines to different degrees.

[0009] The present application provides the application of a detection reagent for HPCAL1 gene or protein in the preparation of a product for assisting in the diagnosis of colon cancer.

[0010] According to the above-mentioned use, preferably, the product detects the expression level of HPCAL1 gene or protein in the sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, Western Blot, chip, high-throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay.

[0011] According to the above-mentioned use, preferably, the product contains an antibody specifically binding to HPCAL1 protein or a peptide segment of HPCAL1 protein, or contains primers specifically amplifying HPCAL1 gene, or contains a probe specifically detecting HPCAL1 gene.

[0012] According to the above-mentioned use, preferably, the antibody is a monoclonal antibody, a polyclonal antibody or a single-domain antibody.

[0013] According to the above-mentioned use, preferably, the sample detected by the product is a cell, a tissue or serum.

[0014] According to the above-mentioned use, preferably, the product is a chip, a preparation or a kit.

[0015] The third aspect of the present application provides the use of HPCAL1 gene or protein as a drug target for screening a drug for preventing, alleviating or / and treating colon cancer.

[0016] The fourth aspect of the present application provides the use of a substance inhibiting the expression and / or function of HPCAL1 gene or protein in any one of the following:

[0017] (A1) the use in preparing a product for preventing, alleviating or / and treating colon cancer;

[0018] (A2) the use in preparing a product for inhibiting tumor growth; the tumor is colon cancer;

[0019] (A3) the use in preparing a product for inhibiting the proliferation of colon cancer cells;

[0020] (A4) the use in preparing a product for inhibiting the migration of colon cancer cells.

[0021] According to the above-mentioned use, preferably, the substance inhibiting the expression and / or function of HPCAL1 comprises shRNA specifically targeting HPCAL1 gene or siRNA specifically targeting HPCAL1 gene or an inhibitor of HPCAL1 protein.

[0022] According to the above-mentioned use, preferably, the shRNA specifically targeting HPCAL1 gene is shRNA1 or shRNA2, and both shRNA1 and shRNA2 are composed of a sense strand and an antisense strand.

[0023] The nucleotide sequence of the shRNA1 sense strand is:

[0024] 5'-CCGGCCCTGGACAGTTGCAGAGAGGATCCTCTCTGCAACTGTCCAGGGTTTTTG-3' (as shown in Sequence 1 in the sequence listing),

[0025] The nucleotide sequence of the shRNA1 antisense strand is:

[0026] 5'-AATTCAAAAACCCTGGACAGTTGCAGAGAGGATCCTCTCTGCAACTGTCCAGGG-3' (as shown in Sequence 2 in the sequence listing);

[0027] The nucleotide sequence of the shRNA2 sense strand is:

[0028] 5'-CCGGCGCAGCGAGATGCTGGAGAGGATCCTCTCCAGCATCTCGCTGCGTTTTTG-3' (as shown in Sequence 3 in the sequence listing),

[0029] The nucleotide sequence of the shRNA2 antisense strand is:

[0030] 5'-AATTCAAAAACGCAGCGAGATGCTGGAGAGGATCCTCTCCAGCATCTCGCTGCG-3' (as shown in Sequence 4 in the sequence listing).

[0031] The fifth aspect of the present application provides a medicine for preventing, alleviating or / and treating colon cancer, wherein the medicine contains a substance for inhibiting the expression and / or function of HPCAL1.

[0032] According to the above-mentioned medicine, preferably, the substance for inhibiting the expression and / or function of HPCAL1 includes shRNA specifically targeting the HPCAL1 gene or siRNA specifically targeting the HPCAL1 gene or an inhibitor of HPCAL1 protein.

[0033] According to the above-mentioned medicine, preferably, the shRNA specifically targeting the HPCAL1 gene is shRNA1 or shRNA2, and both shRNA1 and shRNA2 are composed of a sense strand and an antisense strand.

[0034] The nucleotide sequence of the shRNA1 sense strand is:

[0035] 5'-CCGGCCCTGGACAGTTGCAGAGAGGATCCTCTCTGCAACTGTCCAGGGTTTTTG-3' (as shown in Sequence 1 in the sequence listing),

[0036] The nucleotide sequence of the antisense strand of shRNA1 is:

[0037] 5'-AATTCAAAAACCCTGGACAGTTGCAGAGAGGATCCTCTCTGCAACT GTCCAGGG-3'(as shown in sequence 2 in the sequence listing);

[0038] The nucleotide sequence of the antisense strand of shRNA1 is:

[0039] 5'-CCGGCGCAGCGAGATGCTGGAGAGGATCCTCTCCAGCATCTCGCTG CGTTTTTG-3'(as shown in sequence 3 in the sequence listing),

[0040] The nucleotide sequence of the antisense strand of shRNA1 is:

[0041] 5'-AATTCAAAAACGCAGCGAGATGCTGGAGAGGATCCTCTCCAGCATC TCGCTGCG-3'(as shown in sequence 4 in the sequence listing).

[0042] According to the above-mentioned medicine, preferably, the medicine further contains a pharmaceutically acceptable carrier / auxiliary.

[0043] Compared with the prior art, the positive beneficial effects obtained by the present application are as follows:

[0044] (1) The present application first found that the expression amount of HPCAL1 in the tumor tissue of colon cancer was significantly up-regulated compared with the normal tissue adjacent to the cancer, and the expression level of HPCAL1 in the colon cancer cell line was significantly higher than that in the normal intestinal epithelial cells, therefore, HPCAL1 can be used as a molecular marker for the auxiliary diagnosis of colon cancer, to assist in the diagnosis of colon cancer. Moreover, the ROC curve AUC for diagnosing and distinguishing colon cancer tissue and normal colon tissue using HPCAL1 can reach 0.718, indicating that HPCAL1 has high diagnostic value for the auxiliary diagnosis of colon cancer.

[0045] (2) The present application also found that HPCAL1 is related to the proliferation and migration ability of colon cancer cells, and knocking down HPCAL1 gene has a significant inhibitory effect on the proliferation and migration of colon cancer cells, and can also inhibit the growth of colon cancer tumor, therefore, HPCAL1 can be used as a drug, a drug target or a target gene in gene therapy, and can be applied to the prevention, alleviation or / and treatment of colon cancer, which can provide a new strategy for the prevention and treatment of colon cancer, and also provide a new direction for further studying the etiology and corresponding prevention and treatment strategies of colon cancer.

[0046] (3) The shRNA sequence provided by the application can efficiently inhibit or knock down the expression of HPCAL1 in target cells, inhibit the growth of colon cancer tumors, and also inhibit the proliferation and migration of colon cancer cells, thereby inhibiting the malignant progression of colon cancer cells, and thus can be used for relieving or / and treating colon cancer, and has important significance in the treatment of colon cancer. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Fig. 10 is the RT-PCR detection result of HPCAL1 gene expression in 10 pairs of colon cancer tissues and adjacent normal tissues;

[0048] Figure 2 Fig. 11 is a Western Blot detection result diagram of HPCAL1 protein expression level in 10 pairs of colon cancer tissues and adjacent normal tissues; wherein, A is a representative Western Blot band, N represents adjacent normal tissue, and T represents colon cancer tissue; B is the Western Blot quantitative analysis result of HPCAL1 protein, blue represents adjacent normal tissue, and red represents colon cancer tissue;

[0049] Figure 3 Fig. 12 is the expression level detection result of HPCAL1 protein in human normal intestinal epithelial cells and colon cancer cell lines;

[0050] Figure 4 Fig. 13 is the ROC curve of clinical sample evaluation of HPCAL1 in diagnosis of colon cancer;

[0051] Figure 5 Fig. 14 is the ROC curve of bioinformatics analysis evaluation of HPCAL1 in diagnosis of colon cancer;

[0052] Figure 6 Fig. 15 is a result diagram of Western blotting detection of HPCAL1 protein expression level after knockdown (left) and overexpression (right) of colorectal cancer cells;

[0053] Figure 7 Fig. 16 is a result diagram of CCK-8 experiment for detecting the influence of HPCAL1 expression level on the proliferation ability of colorectal cancer cells; wherein, A is the influence result of HPCAL1 knockdown on the proliferation ability of colorectal cancer cells, NC represents the knockdown control group, KD1 represents the shRNA1 knockdown group, and KD2 represents the shRNA2 knockdown group; B is the influence result of HPCAL1 overexpression on the proliferation ability of colorectal cancer cells, NC represents the pLVX empty control group, and OE represents the HPCAL1 overexpression group;

[0054] Figure 8Figure of results of plate cloning experiment for detecting the influence of HPCAL1 expression level on the colony formation ability of colorectal cancer cells; wherein, A is the influence of HPCAL1 knockdown on the colony formation ability of colorectal cancer cells, the upper graph NC indicates the knockdown control group, KD1 indicates the shRNA1 knockdown group, and KD2 indicates the shRNA2 knockdown group; B is the influence of HPCAL1 overexpression on the colony formation ability of colorectal cancer cells, NC indicates the pLVX empty control group, and OE indicates the HPCAL1 overexpression group;

[0055] Figure 9 Figure of results of Transwell migration experiment for detecting the influence of HPCAL1 expression level on the migration ability of colorectal cancer cells; wherein, A is the influence of HPCAL1 knockdown on the migration ability of colorectal cancer cells, NC indicates the knockdown control group, KD1 indicates the shRNA1 knockdown group, and KD2 indicates the shRNA2 knockdown group; B is the influence of HPCAL1 overexpression on the migration ability of colorectal cancer cells, NC indicates the pLVX empty control group, and OE indicates the HPCAL1 overexpression group;

[0056] Figure 10 Figure of results of nude mouse subcutaneous tumor formation experiment; wherein, A is the subcutaneous tumor photos of different groups of nude mice; B is the statistical columnar graph of tumor weights of different groups; C is the growth curve of tumors of different groups, NC indicates the knockdown control group, and KD indicates the shRNA1 knockdown group. DETAILED DESCRIPTION

[0057] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0058] The following detailed description is exemplary in nature and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0059] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, components, and / or groups thereof.

[0060] The experimental methods in the following examples, unless otherwise specified, adopt the conventional techniques in the art, or follow the conditions suggested by the manufacturers; the reagents or instruments used, unless otherwise specified, are conventional products that can be obtained by purchase in the market.

[0061] Example 1: Study on expression of HPCAL1 gene in colon cancer tissues and adjacent normal tissues by mRNA level detection

[0062] 1. Experimental samples

[0063] Ten pairs of colon cancer tissues and adjacent normal tissues were collected. The colon cancer tissues and adjacent normal tissues were confirmed by postoperative pathological diagnosis. To prevent RNA degradation, the specimens were immediately cut off after surgery, washed twice with PBS, and then placed into a cryogenic tube and immediately placed into liquid nitrogen. Finally, they were transported to a -80 °C refrigerator for long-term storage. All patients were first diagnosed patients and had not received radiotherapy or chemotherapy before surgery. Surgery was the preferred treatment option. According to the system stipulated by the Ethics Review Committee, each patient signed an informed consent form before sampling. The collected colon cancer tissue and its corresponding adjacent normal tissue samples were taken out of the liquid nitrogen and placed on ice. A green bean-sized tissue was cut into small pieces with a sterile scissors and placed into a 2 mL grinding tube for RNA extraction.

[0064] 2. Experimental method

[0065] Real-Time PCR was used to detect the expression level of HPCAL1 gene in colon cancer tissues and adjacent normal tissues. The specific experimental method is as follows:

[0066] (1) Total RNA extraction:

[0067] 1) Tissue homogenate: Add 1 mL of TRIzol to each tube, then add two small steel balls, tighten the cap, and place it into the rack of a multi-sample tissue grinder. Tighten the screw and adjust the parameters to 60 Hz and 1 min to grind the tissue thoroughly. If the grinding is not sufficient, cool it on ice and grind it again until it is in a homogenate state. Place the ground sample symmetrically into a centrifuge at 4 °C and 12000 rpm for 10 min.

[0068] 2) Separation: Take out the sample from the centrifuge and use a pipette to transfer the supernatant to a new labeled nuclease-free 1.5 mL EP tube. Add 0.2 times chloroform (1 mL of TRIzol plus 0.2 mL of chloroform), tighten the cap, and shake vigorously for 30 s. Let it stand at room temperature for 3 min. Place the sample into a 4 °C centrifuge at 12000 rpm for 15 min. Then carefully pipette the upper aqueous phase (about 500 μL) into a new nuclease-free 1.5 mL EP tube.

[0069] 3) Precipitation: Add 500 μL of isopropanol, mix well, and precipitate at room temperature for 10 min. Centrifuge at 12000 g and 4 °C for 10 min. The RNA precipitate can be seen at the bottom of the tube.

[0070] 4) Wash: Discard the supernatant, add 1 ml 75% ethanol per ml TrQuick Reagent, mix gently by inverting the tube, and centrifuge at 12,000 g for 2 minutes at 4°C. Discard the liquid, being careful not to lose the RNA pellet. Let the tube sit at room temperature for 5-10 minutes to dry.

[0071] 5) Dissolve: Add 50 μL Nuclease-Free Water to dissolve the RNA pellet. Use NanoDrop 2000 to detect the concentration and purity of the RNA.

[0072] (2) Reverse transcription to synthesize cDNA:

[0073] According to the measured concentration, calculate the volume corresponding to 1000 ng of RNA. Take out the All-in-One Script RT premix (with dsDNase) reagent and thaw it on ice. Configure the reaction solution required for reverse transcription in the RNase-Free centrifuge tube according to the formula in Table 1. After a brief centrifugation, place it in the PCR instrument for reverse transcription. Reverse transcription reaction program: 37°C for 2 min (random primer pairing with RNA template and removing genomic DNA), 55°C for 15 min (reverse transcription reaction and dsDNase rapid inactivation), 85°C for 5 min (reverse transcriptase inactivation). The above reverse transcription kit is provided by Kehua Meiya Company.

[0074] Table 1 Reverse transcription reaction system

[0075] Reagent Amount Used Total RNA 1 μg 5x Reaction Mix 2 μL KREnzyme Mix 1.5 μL Nuclease-Free Water qPCR Premix (Universal)

[0076] (3) Fluorescence quantitative detection:

[0077] Take out the 2x SYBR Green qPCR Premix reagent and the required primers and dissolve them on ice. Add 90 μL Nuclease-Free Water to the above reverse transcription cDNA product to dilute the cDNA product by 10 times.

[0078] Using ACTIN as the internal reference, detect the relative expression of HPCAL1 in colon cancer patients and normal tissues adjacent to cancer by Real Time PCR reaction, and determine the expression difference between the two.

[0079] The nucleotide sequence of the specific amplification primer of HPCAL1 is as follows:

[0080] Upstream primer: 5'-CCTTCAGCATGTACGACCTGGA-3';

[0081] Downstream primer: 5'-GATCTTGTCTGTGCGCTTCTCC-3'.

[0082] The nucleotide sequences of the ACTIN-specific amplification primers are as follows:

[0083] Upstream primer: 5'-GACAGGATGCAGAAGGAGATTACT-3';

[0084] Downstream primer: 5'-TGATCCACATCTGCTGGAAGGT-3'.

[0085] The specific amplification primers for HPCAL1 and ACTIN were designed and synthesized by General Biotechnology (Anhui) Co., Ltd.

[0086] Prepare the Real-time PCR reaction system (10 μL system) according to the proportions in Table 2.

[0087] Table 2 Real-time PCR reaction system

[0088] 5 μL 10 μM Forward Primer 0.2 μL 10 μM Reverse Primer 0.2 μL cDNA Template 1 μL Nuclease-Free Water qPCR Premix (Universal) 5 μL 10 μM Forward Primer 0.2 μL

[0089] Real-Time PCR was performed using a two-step method, and melting curves were generated. The program settings are shown in Table 3.

[0090] Table 3 Two-step Real-Time PCR reaction procedure

[0091]

[0092] 3. Data processing and analysis

[0093] Based on the original Real-Time PCR test results, according to 2 -ΔΔCт The relative expression level of HPCA1 was calculated, which is the difference in the transcription level of the target gene HPCA1 between cancer tissue samples and adjacent normal tissue control samples.

[0094] 4. Experimental Results

[0095] The results of real-time PCR detection of HPCA1 expression levels in 10 pairs of colon cancer tissues and adjacent normal tissues are as follows: 10 μM Reverse Primer As shown.

[0096] Depend on 0.2 μL It can be seen that the expression level of HPCA1 in colorectal cancer tissue is significantly higher than that in adjacent normal tissue, and the difference is statistically significant (P<0.05).

[0097] Example 2: Study on the expression of HPCA1 protein in colon cancer tissue and adjacent normal tissue

[0098] 1. Experimental Samples

[0099] Ten pairs of colon cancer tissues and adjacent normal tissues were collected, which were confirmed by postoperative pathological diagnosis. To prevent RNA degradation, the specimens were immediately cut off after surgery, washed twice with PBS, and then placed in a cryogenic tube and immediately placed in liquid nitrogen. Finally, they were transported to a -80°C refrigerator for long-term storage. All patients were first diagnosed patients and had not received radiotherapy or chemotherapy before surgery. Surgery was the preferred treatment option. According to the system stipulated by the Ethics Review Committee, each patient signed an informed consent form before sampling. The collected colon cancer tissue and its corresponding adjacent normal tissue samples were taken out of the liquid nitrogen and placed on ice. A green bean-sized tissue was cut into two parts using a sterile scissors and placed in a 2 mL grinding tube for protein extraction.

[0100] 2. Experimental method

[0101] The expression level of HPCAL1 in the colon cancer tissue and adjacent normal tissue of the experimental sample was detected by Western Blot. The specific experimental method is as follows:

[0102] (1) Total protein extraction:

[0103] 1) Tissue homogenate: Add 200 μL RIPA lysis buffer (containing protease inhibitors) to each tube, then add two small steel balls, tighten the cover, and place it in the rack prepared by the multi-sample tissue grinder. Tighten the screw, adjust the parameters to 60 Hz and 1 min, and grind the tissue thoroughly. If the grinding is not sufficient, cool it on ice and grind it again until it is homogenized.

[0104] 2) Place the ground sample symmetrically in the centrifuge, 4°C, 12000 rpm, centrifuge for 10 min.

[0105] 3) Take the supernatant and use the BCA detection kit to detect the protein concentration.

[0106] (2) Western Blot detection:

[0107] 1) Sample preparation: Add RIPA lysis buffer (containing protease inhibitors) to adjust the protein concentration, add SDS-Loading Buffer, and boil at 95°C for 10 min. 4°C, 12000 rpm, centrifuge for 10 min.

[0108] 2) Protein electrophoresis: Select a 4-20% gradient gel, load 20 μg protein per well, 120 V, 80 min.

[0109] 3) Membrane transfer: Take out the protein gel and place the gel and 0.2 μm NC membrane between the buffer-soaked filter paper to make a transfer "sandwich" and add NCM fast transfer buffer, 400 mA, 30 min for membrane transfer.

[0110] 4) Take out the transferred membrane, add an appropriate amount of eosin dye, shake at room temperature for 1 min, wash the membrane with a large amount of water until the water is clear and the protein band is clear, take a picture to record the transfer efficiency.

[0111] 5) Blocking: Add an appropriate amount of 5% skim milk powder, slowly shake at room temperature for 1 h. Wash with TBST for 3 times, 10 min each time.

[0112] 6) Primary antibody incubation: Dilute HPCAL1 primary antibody with blocking solution at a dilution ratio of 1:1000 according to the antibody instruction; dilute GAPDH primary antibody with blocking solution at a dilution ratio of 1:10000 according to the antibody instruction. Add the diluted primary antibody to the antibody incubation, evenly cover the membrane, and shake at 4°C overnight. Wash with TBST for 3 times, 10 min each time.

[0113] 7) Secondary antibody incubation: Dilute the secondary antibody with TBST, add it to the antibody incubation, evenly cover the membrane, and shake at room temperature for 1 h. After incubation, remove the secondary antibody and add TBST for washing 3 times, 10 min each time.

[0114] 8) Development: Prepare the luminescent solution (A and B) at a ratio of 1:1 (note: avoid light), use a pipette to take an appropriate amount of luminescent solution to cover the PVDF membrane, expose it to ECL luminescence instrument and collect the image.

[0115] 3. Data processing and analysis

[0116] Use Image J software to calculate the gray value of each band, and use prism software to statistically analyze the data of three independent repeated experiments. P<0.05 is considered statistically significant. Adobe illustrator is used to arrange and combine the Western Blot exposure pictures.

[0117] 4. Experimental results

[0118] The Western Blot detection results are shown in cDNA Template .

[0119] As shown in 1 μL , the protein expression level of HPCAL1 in colon cancer tissue was significantly higher than that in normal tissue adjacent to cancer, and the difference was statistically significant (P<0.05). Therefore, the expression level of HPCAL1 is related to the occurrence of colon cancer, and can be used as a molecular marker for colon cancer for clinical auxiliary diagnosis of colon cancer.

[0120] Example 3: Study on the expression of HPCAL1 protein in colon cancer cells and normal intestinal epithelial cells

[0121] 1. Cell selection and culture

[0122] Cultured human normal intestinal epithelial cells NCM460, colon cancer cell lines SW480, SW620, RKO, HCT116. NCM460 cells were routinely cultured in 1640 cell culture medium containing 10% FBS, 1% penicillin and streptomycin, SW480, SW620, RKO, HCT116 cells were routinely cultured in DMEM cell culture medium containing 10% FBS, 1% penicillin and streptomycin. All were cultured at 37℃, 5% CO 2 and saturated humidity. The cells were replaced every 2 days, and were passaged at 1:3.

[0123] 2. Experimental method

[0124] Western Blot was used to detect the expression of HPCAL1 in human colon cancer cells and normal intestinal epithelial cells. The specific experimental method is as follows:

[0125] (1) Cell total protein extraction: collect each 6cm dish of NCM460, SW480, SW620, RKO, HCT116 in logarithmic growth phase, wash with PBS for two times, add 500μL RIPA lysis buffer (containing protease inhibitor), lyse on ice for 30min, scrape the cells with cell scraper, collect into 1.5mL EP tube. Symmetrically place the sample into centrifuge, 4℃, 12000rpm, centrifuge for 10min, take the supernatant into a new EP tube.

[0126] (2) Protein sample preparation: BCA detection kit was used to detect the protein concentration; SDS-Loading Buffer was added to the protein sample, and the sample was boiled at 95℃ for 10min; 4℃, 12000rpm, centrifuge for 10min.

[0127] (3) Western Blot detection: Western Blot detection was performed on the protein sample obtained in step (2), and the specific operation of Western Blot detection was the same as that of Example 2, which will not be repeated here.

[0128] 3. Data processing and analysis

[0129] Image J software was used to calculate the gray value of each band, and prism software was used to statistically analyze the data of three independent repeated experiments, and p<0.05 was considered to have statistical significance. Adobe illustrator was used to arrange and combine the Western Blot exposure pictures.

[0130] 4. Experimental results

[0131] The results of Western Blot detection are shown in Nuclease-Free Water .

[0132] From qPCR Premix (Universal)As can be seen, the protein expression level of HPCAL1 in colon cancer cells SW480, SW620, RKO, HCT116 is significantly higher than that in normal intestinal epithelial cells NCM460. This shows that the expression level of HPCAL1 in colon cancer cells is significantly up-regulated.

[0133] Example 4: Evaluation of the value of HPCAL1 for colon cancer diagnosis

[0134] In order to further verify the ability of HPCAL1 for colon cancer diagnosis, colon cancer patient and healthy control samples were collected, and according to the expression amount of HPCAL1 in the experimental samples, the ROC curve was made according to the expression amount, and the value of HPCAL1 for colon cancer diagnosis was evaluated.

[0135] 1. Evaluate the value of HPCAL1 for colon cancer diagnosis using clinical experimental samples:

[0136] (1) Experimental samples:

[0137] 20 cases of colon cancer tissues and 20 cases of healthy control samples were collected, and the samples were from the Department of Gastroenterology, Henan Provincial People's Hospital.

[0138] (2) Experimental method:

[0139] The expression amount of HPCAL1 in colon cancer tissues and healthy control tissues was detected by Real-Time PCR method. The specific detection method of Real-Time PCR is the same as that of Example 1, which is not repeated here.

[0140] According to the expression amount of HPCAL1, the ROC curve (as shown in 5 μL ) was made to evaluate the value of HPCAL1 for colon cancer diagnosis.

[0141] As can be seen from 10 μM Forward Primer , the AUC of the ROC curve for diagnosing and distinguishing colon cancer tissues and healthy controls using HPCAL1 reached 0.708, which can effectively distinguish colon cancer and healthy controls. This shows that HPCAL1 can be used for the auxiliary diagnosis of colon cancer and has high diagnostic value.

[0142] 2. Evaluate the value of HPCAL1 for colon cancer diagnosis by bioinformatics data analysis:

[0143] The RNAseq data of TCGA-COAD and TCGA-READ project STAR process were downloaded and sorted from TCGA database (https: / / portal.gdc.cancer.gov), and the data in TPM format was extracted, a total of 647 cases of colon cancer tissues and 51 cases of normal colon tissues were obtained, pROC package was used for ROC analysis of the data, and the results were visualized by ggplot2. The results are as follows0.2 μL As shown.

[0144] Depend on 10 μM Reverse Primer The results show that the AUC of the ROC curve for distinguishing colon cancer tissue from healthy controls using HPCA1 reached 0.718, indicating that it can effectively differentiate between colon cancer and healthy controls. This demonstrates that HPCA1 can be used for the auxiliary diagnosis of colon cancer and has high diagnostic value.

[0145] Example 5: Effects of HPCA1 on cell proliferation, colony formation, and migration in colon cancer cells

[0146] 1. Silencing of the HPCA1 gene

[0147] (1) Cell culture:

[0148] Human colon cancer cell lines RKO and HCT116 were selected as HPCAL1 knockdown cell lines for this study. Human embryonic kidney cells 293T were used as tool cells for lentivirus packaging. All cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C, 5% CO2, and 90% relative humidity.

[0149] (2) shRNA design and vector construction:

[0150] 1) shRNA design: shRNAs targeting HPCAL1 (shHPCAL1-1, shHPCAL1-2) and negative control shRNA (shNC) were synthesized by General (Anhui) Biotechnology Co., Ltd.

[0151] The nucleotide sequence of the positive strand of shRNA1 is as follows:

[0152] 5'-CCGGCCCTGGACAGTTGCAGAGAGGATCCTCTCTGCAACTGTCCAG GGTTTTTG-3' (as shown in sequence 1 of the sequence listing),

[0153] The nucleotide sequence of the shRNA1 antisense strand is as follows:

[0154] 5'-AATTCAAAAACCCTGGACAGTTGCAGAGAGGATCCTCTCTGCAACT GTCCAGGG-3' (as shown in sequence 2 of the sequence listing);

[0155] The nucleotide sequence of the positive strand of shRNA2 is as follows:

[0156] 5'-CCGGCGCAGCGAGATGCTGGAGAGGATCCTCTCCAGCATCTCGCTGCGTTTTTG-3' (as shown in sequence 3 in the sequence listing),

[0157] The nucleotide sequence of the antisense strand of shRNA2 is:

[0158] 5'-AATTCAAAAACGCAGCGAGATGCTGGAGAGGATCCTCTCCAGCATCTCGCTGCG-3' (as shown in sequence 4 in the sequence listing).

[0159] The nucleotide sequence of the antisense strand of shRNA2 is:

[0160] 5'-CCGGCAACAAGATGAAGAGCACCAAGGATCCTTGGTGCTCTTCATCTTGTTGTTTTTG-3',

[0161] The nucleotide sequence of the antisense strand of shRNA2 is:

[0162] 5'-AATTCAAAAACAACAAGATGAAGAGCACCAAGGATCCTTGGTGCTCTTCATCTTGTTG-3'.

[0163] (3) Construction of shRNA vector:

[0164] 1) Double enzyme digestion of the vector: 1 μg of pLKO.1 vector was added with 1 μL of restriction enzymes Age1 and EcoR, respectively, and incubated at 37°C for 6 h and then inactivated at 80°C for 20 min. The enzyme-digested pLKO.1 vector was added to a 1% agarose gel, and electrophoresed at 80 V for 30 min, and then the cut vector was recovered.

[0165] 2) Annealing of oligonucleotide fragments: each pair of shRNA sense and antisense strands was mixed according to Table 4, and the annealing program was set in the PCR instrument as follows: 37°C for 30 min, 95°C for 5 min, 90°C for 1 min, 85°C for 1 min, and then decreased by 5°C every 1 min until 25°C, and then annealed and recombined.

[0166] Table 4 is the reaction system of annealing oligonucleotide fragments

[0167] 0.2 μL cDNA Template 1 μL Nuclease-Free Water ddH2O qPCR Premix (Universal) 5 μL 10 μM Forward Primer

[0168] 3) Ligation: the above shRNA after return was diluted 200 times with ddH2O. The ligation reaction solution was configured according to Table 5, and the ligation was performed at room temperature for 4 h.

[0169] Table 5 is the ligation reaction system

[0170]

[0171]

[0172] 4) Transformation: add the ligated shRNA to competent cells, incubate on ice for 30 min, heat shock at 42°C for 50 s, add 500 μL LB medium, incubate at 37°C for 1 h, spread on LB plates with ampicillin, and incubate at 37°C overnight.

[0173] 5) Pick single colonies: pick single colonies into 500 μL LB liquid medium with ampicillin, and incubate at 37°C for 5 h.

[0174] 6) Sequencing verification: take part of the bacterial solution and send to GENEWIZ for sequencing. Compare the sequencing results to find the correct clone.

[0175] Extract the vector of the correct clone using a plasmid extraction kit, i.e., shRNA-pLKO.1. This can be used for subsequent lentivirus packaging.

[0176] (4) Lentivirus packaging

[0177] 1) Seed 293T cells into 6-well plates at 0.5 x 10 6 cells per well, and incubate overnight so that the cell density can reach about 70-80% the next day.

[0178] 2) Before transfection, replace the culture medium in each well of the 6-well plate with 2 mL of fresh culture medium.

[0179] 3) Prepare the shNC, shHPCAL1-1, and shHPCAL1-2 lentivirus packaging systems according to Table 6. For each well of cells in the 6-well plate to be transfected, prepare Solution A: 100 μL Opti- Medium, 1 μg shRNA, 1 μg packaging plasmid psPAX2, and 0.5 μg packaging plasmid VSVG2. Prepare Solution B: 100 μL Opti- Medium, and 5 μL Lipo2000 TM transfection reagent. After incubation at room temperature for 5 min, mix Solution A and Solution B, incubate at room temperature for 20 min, and add to the corresponding well.

[0180] 4) After incubation at 37°C in a 5% CO 2 incubator for 24 h, collect the culture supernatant, which is designated as virus P1. Add fresh medium, continue incubation for another 24 h, collect the culture supernatant, which is designated as virus P2.

[0181] (5) Lentivirus infection of colon cancer cells

[0182] 1) Inoculate appropriate amount of HCT116, RKO cells into 6-well plates, and culture overnight so that the cell density can reach about 70-80% the next day.

[0183] 2) Discard the old culture medium, add 1 mL of fresh culture medium and 1 mL of the packaged lentivirus in the previous step, mix gently and evenly, and place in a 37°C, 5% CO2 incubator for 24 h.

[0184] 3) Discard the old culture medium, add 2 mL of fresh culture medium, and culture in a 37°C, 5% CO2 incubator for 24 h.

[0185] 4) Add 1 μg / mL puromycin to screen positive cells. About 2 to 3 days, all uninfected cells are killed, leaving positive cells, which are HPCAL1 knockdown cells and control cells.

[0186] (6) Detection of knockdown efficiency

[0187] 1) Extract cell RNA and perform fluorescent quantitative PCR to quantify the mRNA expression level of HPCAL1. The specific steps are the same as above and will not be repeated.

[0188] 2) Extract total protein from cells and perform Western Blot to detect changes in the protein expression level of HPCAL1. The specific method of Western Blot is the same as in Example 2 and will not be repeated here. The Western Blot detection of HPCAL1 knockdown efficiency in HCT116, RKO is shown in 0.2 μL . As can be seen from 10 μM Reverse Primer , using shRNA1 or shRNA2 can significantly knock out HPCAL1.

[0189] 2, Overexpression of HPCAL1 gene

[0190] (1) Cell culture:

[0191] Human colon cancer cell lines RKO and HCT116 were selected as HPCAL1 overexpression cell lines for research. Human embryonic kidney cells 293T were used as tool cells for lentivirus packaging. All were cultured in DMEM cell culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, in a 37°C, 5% CO2, relative humidity 90% incubator.

[0192] (2) Construction of HPCAL1 overexpression vector

[0193] 1) Obtain the HPCAL1 gene sequence from NCBI.

[0194] 2) Synthesize the HPCAL1 gene sequence through Anhui General Biotechnology, and construct it into the mammalian cell overexpression plasmid pLVX-puro.

[0195] (3) Construction of HPCAL1 overexpression colon cancer cells

[0196] 1) Lentivirus packaging: The specific steps of lentivirus packaging are the same as those in step 1 HPCAL1 gene silencing above, and are not repeated here.

[0197] 2) Lentivirus infection of colon cancer cells: The packaged virus was added to HCT116 and RKO cells, respectively, and then positive cells were selected with DMEM medium containing 1 μg / mL puromycin. Lentivirus infection of colon cancer cells is the same as that in step 1 HPCAL1 gene silencing above, and is not repeated here.

[0198] (4) Detection of overexpression efficiency

[0199] Total protein was extracted and Western Blot was performed to detect the change in the protein expression amount of HPCAL1. The specific method of Western Blot is the same as that in Example 2, and is not repeated here.

[0200] The overexpression efficiency of HPCAL1 in HCT116 and RKO was detected by Western Blot as shown in 0.2 μL . It can be known from cDNA Template that the expression of HPCAL1 is significantly increased by using HPCAL1-pLVX overexpression vector.

[0201] 3, CCK8 experiment to detect the effect of HPCAL1 on colon cancer cell proliferation

[0202] (1) Cell culture:

[0203] sh-HPCAL1 cells and shNC cells, HPCAL1-pLVX overexpression cells and pLVX empty control group cells were cultured in DMEM medium containing 1 μg / mL puromycin, respectively.

[0204] (2) CCK8 experiment:

[0205] 1) Take cells in good growth state and in logarithmic growth phase, count after digestion, and dilute cells to 10000 cells / mL with fresh culture medium.

[0206] 2) Prepare 96-well plates, add 100 μL cells to each well, i.e. 1000 cells / well. According to the design of 3 replicates per day for each group of cells for 5 days, a total of 15 wells of each group of cells were seeded, and blank control wells were set. Incubate in a 37℃, 5% CO2 incubator.

[0207] 3) Every 24h, take out the 96-well plate, suck off the old culture medium, add fresh culture medium containing 10% CCK8 reagent, and place in a 37°C, 5% CO2 incubator for 2h.

[0208] 4) Place the incubated 96-well plate into an enzyme marker for detection, and measure the absorbance at 450nm.

[0209] 5) Draw the growth curve of each group of cells.

[0210] (3) Experimental results:

[0211] The experimental results of CCK8 experiment for detecting cell proliferation are shown in 1 μL .

[0212] As can be seen from Nuclease-Free Water , compared with shNC group cells, the proliferation ability of HPCAL1-knocked-down colon cancer cells was significantly inhibited (P<0.05); compared with pLVX empty control group, the proliferation ability of HPCAL1-overexpressed colon cancer cells was significantly enhanced (P<0.05).

[0213] 4. Plate clone experiment for detecting the influence of HPCAL1 on the clone formation ability of colon cancer cells

[0214] (1) Experimental method:

[0215] 1) Cell culture:

[0216] DMEM culture medium containing 1 μg / mL puromycin was used to culture sh-HPCAL1 cells and shNC cells, and HPCAL1-pLVX overexpression cells and pLVX empty control group cells, respectively.

[0217] 2) Take cells in good growth state and in logarithmic growth phase, count after digestion, and dilute the cells to 1000 cells / mL with fresh culture medium.

[0218] 3) Prepare a 6-well plate, design 3 replicate holes for each group of cells, and add 2 mL of diluted cells to each hole, i.e. 2000 cells per hole.

[0219] 4) Culture in a 37°C, 5% CO2 incubator for 7 days, and replace the fresh culture medium in between.

[0220] 5) Discard the culture medium, wash with PBS for 2 times, fix with 4% paraformaldehyde for 15 min, stain with crystal violet for 15 min, rinse with water until the background is clean, dry and take a photo.

[0221] (2) Data processing and analysis:

[0222] Use Image J software to calculate the number of clone formation, and use Primer statistical analysis.

[0223] (3) Experimental results

[0224] The experimental results of detecting the colony formation ability of the plate clone cells are shown in qPCR Premix (Universal) .

[0225] It can be seen from 5 μL that the colony formation ability of the colon cancer cells with HPCAL1 knockdown was significantly inhibited compared with the shNC group cells (P<0.05); and the colony formation ability of the colon cancer cells with HPCAL1 overexpression was significantly enhanced compared with the pLVX empty control group (P<0.05).

[0226] 5. Tranwell chamber migration experiment for detecting the effect of HPCAL1 on the migration ability of colon cancer cells

[0227] (1) Experimental method:

[0228] 1) Cell culture:

[0229] The sh-HPCAL1 cells and shNC cells, and the HPCAL1-pLVX overexpression cells and pLVX empty control group cells were cultured in DMEM medium containing 1 μg / mL puromycin, respectively.

[0230] 2) The cells in good growth state and in logarithmic growth phase were counted after digestion, and the cell suspension was prepared at 25000 cells / mL using the medium without fetal bovine serum.

[0231] 3) 700 μL of medium containing 10% fetal bovine serum was added to the lower chamber of the 24-well plate, and the Transwell chamber was placed in each well. 200 μL of the above prepared cell suspension was added to the upper chamber, i.e. 50000 cells / well.

[0232] 4) Incubate in a 37°C, 5% CO2 incubator for 24-48 h.

[0233] 5) Take out the Transwell chamber, wash with PBS for 3 times, fix with 4% paraformaldehyde for 15 min, add crystal violet for staining for 15 min, then observe the coloring intensity under a microscope, discard the crystal violet solution, wash with pure water, place under a fluorescence microscope for observation, dry and take a photo.

[0234] (2) Data processing and analysis:

[0235] Use Image J software to calculate the number of colony formation, and use Primer statistical analysis.

[0236] (3) Experimental results:

[0237] The experimental results of detecting the migration ability of the cells in the Tranwell chamber are shown in 10 μM Forward PrimerAs shown in the figure.

[0238] As shown in the figure. 0.2 μL It can be seen that the migration ability of colon cancer cells with HPCAL1 knockdown was significantly inhibited compared with shNC group (P<0.05); the migration ability of colon cancer cells with HPCAL1 overexpression was significantly enhanced compared with pLVX empty control group (P<0.05).

[0239] Example 6: Effect of HPCAL1 silencing on the formation ability of colon cancer subcutaneous tumor

[0240] 1. Experimental method

[0241] 1) Preparation of nude mice: 8 female nude mice of 4-5 weeks old were purchased from Jisuiyaoke and cultured in Zhengda animal house for about 1 week until the nude mice were in good condition.

[0242] 2) Cell preparation: control group and HPCAL1 knockdown group HCT116 cells in good growth condition and in logarithmic growth phase were taken, counted after trypsin digestion, resuspended with PBS to prepare cell suspension, 100 million cells / mL.

[0243] 3) Subcutaneous inoculation: 100 μL of the prepared cell suspension was inoculated into the back of the forelimb armpit of the nude mice at a distance of 0.3 cm with a 1 mL syringe, and shNC-HCT116 was inoculated on the right side and shHPCAL1-HCT116 was inoculated on the left side.

[0244] 4) Observation and measurement: from the 3rd day after tumor formation, the length, width and height of the tumor were measured every other day with a vernier caliper, the body weight of the nude mice was measured, and the state of the nude mice was observed and recorded.

[0245] 5) End of collection: after about 2 weeks of inoculation, the tumor tissue was collected, the volume and weight of the tumor were calculated, and the photograph was taken. Then it was divided into two parts for preservation: ① 4% paraformaldehyde fixation, ②-80℃ low temperature preservation.

[0246] 2. Data processing and analysis

[0247] The tumor volume was calculated using the formula length* width 2 / 2. The experimental data was expressed as mean ± standard error (Mean ± SEM), and all data was processed and statistically analyzed using GraphPad Prism 6. The comparison between the means of two groups of samples was performed by t test, and P<0.05 indicated that the difference was statistically significant.

[0248] 3. Experimental results

[0249] The results of the nude mouse subcutaneous transplantation tumor experiment are shown in the figure. 10 μM Reverse Primer

[0250] As shown in the figure. 0.2 μL cDNA Template 1 μL Nuclease-Free Water qPCR Premix (Universal) 5 μL 10 μM Forward Primer 0.2 μL 10 μM Reverse Primer 0.2 μL cDNA Template 1 μL Nuclease-Free Water qPCR Premix (Universal) 5 μL 10 μM Forward Primer 0.2 μL 10 μM Reverse Primer 0.2 μL cDNA Template 1 μL Nuclease-Free Water qPCR Premix (Universal) 5 μL 10 μM Forward Primer 0.2 μL 10 μM Reverse Primer 0.2 μL cDNA Template 1 μL Nuclease-Free Water qPCR Premix (Universal) 5 μL 10 μM Forward Primer 0.2 μL 10 μM Reverse Primer 0.2 μL cDNA Template 1 μL Nuclease-Free Water qPCR Premix (Universal ​It can be seen that compared with the shNC group cells, the tumor formation ability of the HPCAL1 knockdown colon cancer cells is obviously inhibited (P<0.05).

[0251] Finally, it should be noted that: the above examples are merely the preferred embodiments of the present application, not other forms of the limitations of the present application, any skilled in the art can use the above technical content as inspiration to change or modify. The equivalent embodiments of such equivalent changes. However, any simple modification, equivalent change and modification made according to the technical essence of the present application without departing from the technical concept of the present application, still belongs to the protection scope of the claims of the present application.

Claims

1. Use of a detection reagent for the expression level of HPCAL1 gene or protein in the preparation of a product for the auxiliary diagnosis of colon cancer.

2. Use according to claim 1, characterized in that, The product detects the expression level of HPCAL1 gene or protein in the sample by real-time quantitative PCR, Western Blot, high-throughput sequencing platform or immunohistochemistry.

3. Use according to claim 2, characterized in that, The product contains an antibody specifically binding to HPCAL1 protein or a peptide segment of HPCAL1 protein, or contains primers specifically amplifying HPCAL1 gene, or contains probes specifically detecting HPCAL1 gene.

4. Use according to claim 3, characterized in that, The antibody is a monoclonal antibody, a polyclonal antibody or a single domain antibody.

5. Use according to any one of claims 1 to 4, characterized in that, The product is a chip, a preparation or a kit.

6. Use of a substance inhibiting the expression of HPCAL1 gene or protein in any of the following: (A1) the preparation of a product for relieving or / and treating colon cancer; (A2) the preparation of a product for inhibiting the growth of a tumor; the tumor is colon cancer. The substance inhibiting the expression of HPCAL1 gene or protein includes shRNA specifically targeting HPCAL1 gene, which is shRNA1 or shRNA2, both of which consist of a sense strand and an antisense strand; the nucleotide sequence of the sense strand of shRNA1 is shown in sequence 1 of the sequence listing, and the nucleotide sequence of the antisense strand of shRNA1 is shown in sequence 2 of the sequence listing; the nucleotide sequence of the sense strand of shRNA2 is shown in sequence 3 of the sequence listing, and the nucleotide sequence of the antisense strand of shRNA2 is shown in sequence 4 of the sequence listing.

7. A medicament for alleviating or / and treating colon cancer, characterized by, The drug contains a substance inhibiting the expression of HPCAL1 gene or protein; the substance inhibiting the expression of HPCAL1 gene or protein includes shRNA specifically targeting HPCAL1 gene, which is shRNA1 or shRNA2, both of which consist of a sense strand and an antisense strand; the nucleotide sequence of the sense strand of shRNA1 is shown in sequence 1 of the sequence listing, and the nucleotide sequence of the antisense strand of shRNA1 is shown in sequence 2 of the sequence listing; the nucleotide sequence of the sense strand of shRNA2 is shown in sequence 3 of the sequence listing, and the nucleotide sequence of the antisense strand of shRNA2 is shown in sequence 4 of the sequence listing.

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