CircCHD6-216aa protein and application thereof in preparation of products for prognosis prediction or treatment of colorectal cancer

Abstract

The invention discloses circCHD6-216aa protein and application thereof in preparation of products for prognosis prediction or treatment of colorectal cancer, and belongs to the field of molecular biology. Specifically, it is found that the circCHD6-216aa protein is highly expressed in colorectal cancer tissue, the highly expressed circCHD6-216aa protein can promote migration, invasion and metastasis of tumor cells, meanwhile, inhibition of the circCHD6-216aa protein can inhibit migration and invasion of tumor cells, and it is prompted that the circCHD6-216aa protein can serve as a target for diagnosing colorectal cancer and serve as a therapeutic target spot.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically, relates to circCHD6-216aa protein and its application in preparing colorectal cancer prognosis detection or treatment products. Background Art

[0002] The occurrence and development of sporadic colorectal cancer has a typical linear evolution process of "normal intestinal mucosa → adenoma → adenocarcinoma → cancer metastasis" (also known as adenoma-adenocarcinoma sequence). However, it is still unclear whether there are specific mutations or metastasis-specific driver mutations in the metastasis process. Through whole exome sequencing of normal intestinal mucosal tissue, colorectal cancer primary lesion tissue and liver metastasis lesion cancer tissue, data analysis found that the mutation spectrum of metastatic lesions was similar to that of primary lesions, and no specific driver mutations of colorectal cancer liver metastasis were found. Therefore, distant metastasis of colorectal cancer may not be a genetic event, but is regulated by transcriptional levels and epigenetics. Therefore, it is necessary to find molecular targets for intervention in colorectal cancer metastasis from other angles.

[0003] Different from the traditional linear RNA splicing mode, the downstream 5' splice site of the precursor mRNA (pre-mRNA) is reversely connected to the upstream 3' splice site to form a phosphodiester bond during the back-splicing process, forming a closed circular structure. The circular structure of circRNA avoids its degradation by nuclease exonucleases, so compared with linear RNA, circular RNA has better stability. CircRNAs play an important role in tumor progression as epigenetic regulatory molecules. Most of the tumor-related circRNAs identified so far usually act as miRNA molecular sponges and RNA-binding protein (RBPs) molecular chaperones. CircRNAs can also use themselves as templates to encode functional peptides driven by ribosome entry sites (IRES) or N6-methylation modifications (m6A). These peptides can act as antigenic peptides in tumors to induce anti-tumor immune responses, affect tumor growth and metastasis, and affect the self-renewal of cancer cell stem cells (CSCs). Given the specificity and stability of circRNAs in tumors, the unique products they encode are expected to become new therapeutic targets and diagnostic biomarkers. In recent years, circular RNA vaccines targeting the novel coronavirus and tumor antigen peptides have been successfully developed. These studies have shown that protein-encoding circular RNA has great potential to become tumor biomarkers and new therapeutic targets. Summary of the invention

[0004] In view of this, the main purpose of the present invention is to provide a new colorectal cancer diagnostic marker and its application as a therapeutic target

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] The first aspect of the present invention is to provide a circCHD6-216aa protein, whose amino acid sequence is shown in SEQ ID NO: 1.

[0007] Furthermore, the circCHD6-216aa is the encoded protein of the alternative splicing body of the parent gene CHD6. Based on the special structure of the circular RNA of the alternative splicing body, when the ribosome moves to the back-splicing site of splicing, the open reading frame encoding circCHD6-216aa undergoes a frameshift and translates a completely new amino acid sequence.

[0008] Furthermore, the coding sequence of circCHD6-216aa is shown in SEQ ID NO:2.

[0009] The second aspect of the present invention is to provide biological materials that can encode the circCHD6-216aa protein described in the first aspect, and the biological materials include but are not limited to:

[0010] 1) Nucleic acid sequences encoding circCHD6-216aa protein or its alternative splicing variants;

[0011] 2) an expression cassette containing the nucleic acid sequence described in 1);

[0012] 3) A recombinant vector containing the nucleic acid sequence or expression cassette described in 1) or 2);

[0013] 4) A recombinant cell containing 1) the nucleic acid sequence, 2) the expression cassette, or 3) the recombinant vector.

[0014] Furthermore, the nucleic acid sequence encoding the open reading frame of the circCHD6-216aa protein or its alternative splicing form is shown in SEQ ID NO: 2.

[0015] The third aspect of the present invention is to provide the use of circCHD6-216aa protein or its encoding gene as a marker in the preparation of a cancer diagnostic reagent.

[0016] Furthermore, the reagents include but are not limited to antibodies, probes, and aptamers.

[0017] Furthermore, the cancer is colorectal cancer.

[0018] The fourth aspect of the present invention is to provide the use of circCHD6-216aa protein or its encoding gene as a marker in the treatment and diagnosis of cancer.

[0019] Furthermore, the reagents include specific antibodies, siRNA, shRNA, and gRNA in CRISPR gene editing.

[0020] Furthermore, the reagent is siRNA, and preferably, the positive strand of the siRNA is shown in SEQ ID NO:3 or SEQ ID NO:5.

[0021] Furthermore, the cancer is colorectal cancer.

[0022] The fifth aspect of the present invention is to provide the circCHD6-216aa protein described in the first aspect, or the second aspect is to provide the use of a biological material encoding the circCHD6-216aa protein described in the first aspect in the preparation of an agent that promotes tumor cell metastasis.

[0023] Furthermore, the application is an in vitro cytological application.

[0024] Furthermore, the tumor cells are colorectal cancer cell lines.

[0025] The beneficial effects of the present invention include:

[0026] 1) circCHD6-216aa protein was found to be highly expressed in colorectal cancer tissues;

[0027] 2) High expression of circCHD6-216aa protein can promote the metastasis of tumor cells.

[0028] 3) Inhibition of circCHD6-216aa protein can inhibit the migration of tumor cells, suggesting that it can be used as a target for diagnosis of colorectal cancer and as a target for treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The circular RNA circCHD6 sequence was input into the coding capacity prediction website to obtain the prediction results of circCHD6 coding potential and the start position of the open reading frame. The results showed that circCHD6 started coding from the 24th base, bypassed the entire circular RNA for a week, and terminated translation at the 97th base.

[0030] Figure 2 The amino acid sequence of the protein encoded by the circular RNA circCHD6 sequence was predicted, and the red marked part is the unique amino acid sequence produced after crossing the circCHD6 back-splicing site.

[0031] Figure 3 A translation diagram based on the coding situation of circular RNA circCHD6. Below is a schematic diagram comparing the structures of circCHD6-216aa and the CHD6 protein encoded by the circCHD6 parent gene CHD6.

[0032] Figure 4Protein antibodies specifically targeting circCHD6-216aa were used to detect the expression of circCHD6-216aa in colorectal cancer cell lines HT29, SW620, HCT8, SW480 and normal intestinal epithelial cell line NCM460, as well as the expression of circCHD6-216aa in normal-colorectal cancer paired tissues; Western Blot experiments verified that circCHD6-216aa was more highly expressed in colorectal cancer cell lines and colorectal cancer tissues.

[0033] Figure 5 After circCHD6-216aa protein was overexpressed in colorectal cancer cell lines, the migration and invasion abilities of colorectal cancer cell lines SW480 and HCT8 were significantly enhanced; this result was achieved using Transwell chambers; the bar graph on the right of the photo was obtained by counting the percentage of crystal violet-stained cells in the entire field of view using ImageJ software.

[0034] Figure 6 After knocking down the expression of circCHD6-216aa protein template in colorectal cancer cell lines using siRNA, the migration and invasion abilities of colorectal cancer cell lines SW620 and HT29 were inhibited; this result was achieved using Transwell chambers; the bar graph on the right of the photo was obtained by counting the percentage of crystal violet-stained cells in the entire field of view using ImageJ software. DETAILED DESCRIPTION

[0035] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0036] Example 1 Prediction of circCHD6 coding capacity

[0037] 1. Methods

[0038] 2.1 Prediction of circCHD6 coding capacity

[0039] 1) In the circular RNA database circRNADb ( http: / / reprod.njmu.edu.cn / circrnadb ) to predict the open reading frame (ORF). Figure 1 ) and the amino acid sequence of circCHD6-encoded protein circCHD6-216aa is as follows

[0040] MKMKIQKKEKQLSNLKVLNHSPMSDASVNFDYKSPSPFDCSTDQEEKI

[0041]

[0042] ( Figure 2 ).

[0043] 2) CircCHD6 encodes circCHD6-216aa protein, and its coding pattern is shown in the figure Figure 3 As shown in the figure. circCHD6 is an alternative splicing product of the parent gene CHD6, and their translation start sites are consistent. Due to the special structure of circular RNA, when the ribosome moves to the splicing splicing site (back-splicing site), the open reading frame encoding circCHD6-216aa undergoes a frameshift and translates a completely new amino acid sequence. Therefore, the N-terminus of the circCHD6-216aa protein is consistent with part of the sequence of the parent gene, and the C-terminus has a unique sequence.

[0044]

[0045] Example 2 Verification of circCHD6 Coding Capability

[0046] Materials and methods

[0047] 1. Materials

[0048] Colorectal cancer cell lines HT29, SW620, HCT8, and SW480 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Normal intestinal epithelial cell line NCM460 was provided by EK bioscience. RPMI1640, DMEM, and 0.05% Trypsin were purchased from Gibco; fetal bovine serum was purchased from Gibco. CircCHD6-216aa antibody was prepared by ABclonal; GAPDH antibody was purchased from Immunoway. RIPA lysis buffer and protease inhibitor PMSF were purchased from Beyotime. BCA quantitative kit was purchased from Thermo Fisher.

[0049] 2. Methods

[0050] 2.1 Preparation of specific antibodies targeting circCHD6-216aa protein

[0051] 1) Synthesize artificial peptide-coupled proteins as antigens based on predicted specific peptide segments;

[0052] 2) Immunize 8-12 SPF mice using synthetic artificial peptide-coupled proteins as antigens;

[0053] 3) The antiserum ELISA titer must be at least 1:50,000, and the serum must be used for WB experiments to verify that the endogenous sample has a band of the correct size;

[0054] 4) Screening and testing of monoclonal antibodies in ascites, and purifying ascites that meet the two conditions of antigen ELISA positive and able to recognize circCHD6-216aa protein in WB experiment;

[0055] 5) Obtain a specific monoclonal antibody targeting circCHD6-216aa protein and store it under appropriate conditions.

[0056] 2.2 Detection of circCHD6-216aa protein expression in colorectal cancer cell lines and paired normal-cancer tissue samples from colorectal cancer patients

[0057] 1) Prepare protein lysis buffer on ice, RIPA lysis buffer + PMSF (1 mL RIPA + 10 μL PMSF), and vortex evenly.

[0058] 2) Discard the culture medium of the cells in the culture plate, rinse with ice PBS 2-3 times, discard the PBS, add protein lysis solution and let it stand on ice for 5 minutes, then scrape all the cells with a cell scraper and transfer them to the corresponding EP tube. Tissue samples are pre-cut into smaller pieces with scissors, liquid nitrogen is added to the mortar to keep the tissue low temperature, and the lysis solution is added and quickly ground to obtain a tissue suspension, which is transferred to the corresponding EP tube.

[0059] 3) Place on ice for 10 minutes and vortex every 3 minutes to fully lyse the cells / tissues.

[0060] 4) Use a contact ultrasonic disruptor at 20-30% power, 10s on 10s off for 4-6 cycles to disrupt and lyse cells / tissues on ice. Clean the probe with ddH2O each time you change the sample and wipe it dry with dust-free paper. Note: Try to avoid bubbles when the probe is placed under the liquid.

[0061] 5) After sonication, the sample was centrifuged at maximum speed at 4°C for 15 minutes. After centrifugation, the supernatant was transferred to a new EP tube for protein quantification and sample preparation.

[0062] 6) Sample preparation: 5× loading buffer: protein sample according to the ratio of 1:4. After mixing, heat in a metal bath at 100°C for 5 minutes to denature and then store at -20 to -40°C.

[0063] 7) Protein concentration quantification by BCA method

[0064] ① Prepare the assay working solution: A (transparent): B (blue) = 50:1, 200 μL per well, mix well and set aside for use. Prepare and use immediately.

[0065] ②Prepare standard curve: Prepare BCA standard with ddH2O, with a total volume of 10 μL, and add to the wells in sequence.

[0066] BCA 0 2 4 6 8 10 μL

[0067] ddH2O 10 8 6 4 2 0μL

[0068] ③ Protein concentration determination: add 9ul ddH20 and 1ul protein sample to each sample well, then add 200ul assay working solution, gently tap the 96-well plate to mix, react in a 60℃ oven for 10 minutes, and then use an ELISA reader to detect, setting the absorbance to 562 for detection. Calculate the protein concentration based on the standard curve fitting straight line. Note that the original protein concentration needs to be calculated *10.

[0069] 8) Western blot detection

[0070] ① Electrophoresis: 70V voltage electrophoresis, after the sample runs into the separation gel, adjust the voltage to 120V until the indicator reaches the bottom of the gel.

[0071] ② Transfer: Place the sponge pad - 3 layers of filter paper - gel - membrane - 3 layers of filter paper - sponge pad in the order from negative electrode to positive electrode, and transfer the membrane at a constant voltage of 100V for 100 minutes.

[0072] ③ Blocking: Place the membrane in TBST blocking solution containing 5% skimmed milk powder and shake for 1 hour.

[0073] ④ Primary antibody incubation: Incubate the membrane in the corresponding primary antibody working solution prepared with primary antibody diluent at 4°C overnight.

[0074] ⑤ Wash the membrane: Take out the membrane and wash it three times with TBST, 5 minutes each time.

[0075] ⑥ Secondary antibody incubation: incubate the membrane in the corresponding secondary antibody at 4℃ for 1h.

[0076] ⑦ Wash the membrane: Take out the membrane and wash it three times with TBST, 5 minutes each time.

[0077] ⑧ Scanning the membrane: Take out the membrane and detect it using the Odyssey (Li-COR) two-color infrared laser system.

[0078] Western blot analysis confirmed that the expression level of circCHD6-216aa in colorectal cancer cell lines was significantly higher than that in normal intestinal epithelial cell lines; in paired colorectal cancer tissues, the expression level of circCHD6-216aa in cancer tissues was also significantly higher than that in normal tissues. Figure 4 .

[0079] Example 3: Promotion of circCHD6-216aa on the migration and invasion ability of colorectal cancer cells

[0080] Materials and methods

[0081] 1. Materials

[0082] Colorectal cancer cell lines SW480 and HCT8 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640, 0.05% Trypsin, and fetal bovine serum were purchased from Gibco. Transwell double-layer plates were purchased from COSTAR, Matrigel was purchased from BD, and crystal violet was purchased from Beyotime. Other drugs were domestic analytical grade.

[0083] 2. Methods

[0084] 1) Migration experiment

[0085] ① Apply 20 μL of 2 μg / mL FN glue to the back of the Transwell chamber in advance, scrape it flat with the side of the pipette tip, and dry it naturally in the clean bench.

[0086] ② Digest the cells with trypsin, discard the trypsin and resuspend in 10% FBS complete medium, centrifuge at 800g for 5 minutes, wash once with PBS, collect the cells, and resuspend in serum-free medium.

[0087] ③After counting with a cell counter, add the cell suspension evenly to the upper chamber of the Transwell according to the specific number, add 600 μl of 10% FBS complete culture medium to the lower chamber, gently put the chamber in, and place it in a 37℃ 5% CO2 incubator for the corresponding time.

[0088] ④ After a certain period of time, take out the chamber, discard the liquid in the lower chamber, use a cotton swab to absorb the culture medium in the upper chamber, rinse with PBS 3 times, 5 minutes each time, place the chamber in 4% paraformaldehyde solution for 10 minutes, and rinse with PBS 3 times, 5 minutes each time. Put the chamber in 0.1% crystal violet for staining for 15 minutes, rinse with PBS once, and finally use a cotton swab to gently wipe off the cells that have not passed through the upper chamber, taking care not to touch the lower surface of the chamber.

[0089] ⑤After the chamber is dry, take a photo under a microscope.

[0090] 2) Invasion assay

[0091] ① Apply 20 μl of 2 μg / ml FN glue to the back of the Transwell chamber in advance, scrape it flat with the side of the pipette tip, and dry it naturally in the clean bench.

[0092] ② Dilute Matrigel 1:20 with culture medium without FBS and add it to the upper chamber. Let it stand in a 37°C incubator for 30 minutes to wait for it to solidify.

[0093] ③Digest the cells with trypsin, discard the trypsin and resuspend them with 10% FBS complete medium, centrifuge at 800g for 5 minutes, wash once with PBS, collect the cells, and resuspend them in serum-free medium.

[0094] ④ After counting with a cell counter, add the cell suspension evenly to the upper chamber of the Transwell according to the specific number, add 600 μl of 10% FBS complete culture medium to the lower chamber, gently put the chamber in, and place it in a 37℃ 5% CO2 incubator for the corresponding time.

[0095] ⑤ After a certain period of time, take out the chamber, discard the liquid in the lower chamber, use a cotton swab to absorb the culture medium in the upper chamber, rinse with PBS 3 times, 5 minutes each time, fix the chamber in 4% paraformaldehyde solution for 10 minutes, and rinse with PBS 3 times, 5 minutes each time. Stain the chamber in 0.1% crystal violet for 15 minutes, rinse with PBS once, and finally use a cotton swab to gently wipe off the cells that have not passed through the upper chamber, taking care not to touch the lower surface of the chamber.

[0096] ⑥After the chamber is dry, take a photo under a microscope.

[0097] The results of the Transwell experiment to detect cell migration and invasion ability are as follows Figure 5 The results showed that the migration and invasion ability of colorectal cancer cells transfected with circCHD6-216aa was significantly increased compared with the negative control group.

[0098] Example 4: Inhibition of migration and invasion ability of colorectal cancer cells by knockout of circCHD6-216aa template

[0099] Materials and methods

[0100] 1. Materials

[0101] Colorectal cancer cell lines HT29 and SW620 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640, 0.05% Trypsin, and fetal bovine serum were purchased from Gibco. Transfection reagent Genemut was purchased from SignaGen. Transwell double-layer plates were purchased from COSTAR, Matrigel was purchased from BD, and crystal violet was purchased from Beyotime. Other drugs were domestic analytical grade.

[0102] 2. Methods

[0103] 1) Experiment of knocking down circCHD6 by small interfering siRNA

[0104] ① Use small interfering siRNA to knock down circCHD6 in colorectal cancer cells. The small interfering siRNA was commissioned to be synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. The sequence of the small interfering siRNA is:

[0105] si1 positive strand: 5'-GUCCAGGAAGGCCAGAAAUAATT-3' (SEQ ID NO: 3)

[0106] si1 antisense strand: 5'-UUAUUUCUGGCCUUCCUGGACTT-3' (SEQ ID NO: 4)

[0107] si2 positive strand: 5'-GCCAGAAAUAAAUAUCUUCCUTT-3' (SEQ ID NO: 5)

[0108] si2 antisense strand: 5'-AGGAAGAUAUUUAUUUCUGGCTT-3' (SEQ ID NO: 6)

[0109] ② Digested cells were plated to ensure that the cells reached a density of 40-50% after 12-18 hours.

[0110] ③ Before transfection (30-60min), replace 1mL / well complete culture medium and prepare transfection system:

[0111] ●Dissolve siRNA. Centrifuge 1OD siRNA at 8000×g for 1 min, add 125μL DEPC water to dissolve, and place on ice for later use.

[0112] ● Dilute 5×Genemut buffer to 1× with DEPC water

[0113] Transfection system: 100μL 1×Genemut buffer dilution + 5μL siRNA (added to the liquid) + 4μL Genemut transfection reagent (added to the tube wall). Mix the siRNA first and then blow the transfection reagent into the liquid to mix; let stand at room temperature for 15 minutes, slowly add the mixed system drop by drop into the cells and shake well.

[0114] ④ Add 1 mL of fresh complete culture medium 12-18 hours after transfection.

[0115] ⑤ 48 hours after transfection, cells were collected for small interference knockdown efficiency detection.

[0116] 2) Migration and invasion experiments. See Example 3 for specific protocols.

[0117] The results are as follows Figure 6 As shown in the results, knockdown of circCHD6 significantly reduced the migration and invasion abilities of colorectal cancer cells.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention.

Claims

1. A circCHD6-216aa protein, whose amino acid sequence is shown in SEQ ID NO:

1.

2. The circCHD6-216aa protein according to claim 1, characterized in that The circCHD6-216aa is the encoded protein of the alternative splicing body of the parent gene CHD6. Based on the special structure of the circular RNA of the alternative splicing body, when the ribosome moves to the back-splicing site of splicing, the open reading frame encoding circCHD6-216aa undergoes a frameshift and translates a completely new amino acid sequence.

3. Biomaterials encoding the circCHD6-216aa protein according to claim 1 or 2, wherein the biomaterials include but are not limited to: 1) Nucleic acid sequences encoding circCHD6-216aa protein or its alternative splicing variants; 2) an expression cassette containing the nucleic acid sequence described in 1); 3) A recombinant vector containing the nucleic acid sequence or expression cassette described in 1) or 2); 4) A recombinant cell containing 1) the nucleic acid sequence, 2) the expression cassette, or 3) the recombinant vector.

4. Use of the circCHD6-216aa protein or its encoding gene as claimed in claim 1 as a marker in the preparation of a cancer prognosis prediction reagent.

5. The use according to claim 4, characterized in that The reagents include but are not limited to antibodies, probes, and aptamers.

6. The use according to claim 5, characterized in that The cancer is colorectal cancer.

7. Use of the circCHD6-216aa protein or its encoding gene as claimed in claim 1 as a marker in the preparation of drugs for the treatment and diagnosis of cancer.

8. The use according to claim 7, characterized in that The reagents include specific antibodies, siRNA, shRNA, and gRNA in CRISPR gene editing.

9. The use according to claim 8, characterized in that The cancer is colorectal cancer.

10. Use of the circCHD6-216aa protein according to claim 1 or 2 or the biomaterial according to claim 3 in the preparation of an agent for promoting tumor cell migration and / or migration, wherein the application is an in vitro cytological application.

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