TsRNA marker related to colorectal cancer and application of tsRNA marker

Abstract

The invention discloses a tsRNA marker related to colorectal cancer and application of the tsRNA marker. The nucleotide sequence of the tsRNA marker is as shown in SEQ ID NO: 1. By knocking down the tsRNA provided by the scheme of the invention, proliferation, invasion and metastasis of colorectal cancer cells can be inhibited, apoptosis can be promoted, and a new and powerful molecular biological tool is provided for treatment of colorectal cancer. The method has profound clinical significance and important popularization and application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a tsRNA marker associated with colorectal cancer and an application thereof. Background Art

[0002] Colorectal cancer (CRC) is a malignant lesion of the colon mucosal epithelium caused by a variety of carcinogenic factors such as the environment or genetics. It is a malignant tumor with a very high mortality rate in the world. Globally, colorectal cancer remains one of the tumors with the highest incidence and mortality. Common sites of onset include the rectum, sigmoid colon, cecum, ascending colon, descending colon and transverse colon. According to the site of onset, colorectal cancer is generally divided into three types: right colon (cecum, ascending colon, transverse colon) cancer, left colon (descending colon, sigmoid colon) cancer and rectal cancer.

[0003] tRNA-derived small RNAs (tsRNAs) are a class of multifunctional small RNAs with a length of about 14-30nt, which play an important role in the occurrence and development of tumors and drug resistance. The generation process of tsRNAs is highly conserved. Under the action of nuclease, tsRNAs are generated by cleavage of tRNA precursors or mature bodies. According to the cleavage position, tsRNAs derived from mature tRNAs are divided into three categories: 5'-tRFs, 3'-tRFs and i-RFs. The functions of tsRNAs are closely related to their subcellular localization. In the nucleus, tsRNAs affect gene transcription by regulating epigenetic modifications. In the cytoplasm, the functions of tsRNAs are mainly summarized into three types: ① RNA silencing: tsRNAs bind to mRNA sequences complementary to each other and play a role similar to miRNA. ② Translation regulation: tsRNAs affect protein synthesis by regulating translation initiation and elongation. ③ Function by binding to RBP: tsRNAs interact with RNA-binding proteins, affecting their post-translational modifications and biological functions.

[0004] In the related technologies, tsRNAs are rarely used in the research of colorectal cancer, so there is an urgent need to provide tsRNAs related to colorectal cancer. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides tsRNAs related to colorectal cancer.

[0006] The present invention also provides a reagent for detecting the above tsRNAs.

[0007] The present invention also provides the application of the above tsRNAs reagent.

[0008] The present invention also provides a tsRNAs inhibitor.

[0009] The present invention also provides the application of the tsRNAs inhibitor.

[0010] The invention also provides a medicine for treating colorectal cancer.

[0011] According to the first aspect of the present invention, a tsRNAs related to colorectal cancer is proposed, and the nucleic acid sequence of the tsRNAs is shown in SEQ ID NO: 1. The tsRNAs of the present invention, namely the tsRNA-Ala-3-0055 molecule, can be used as a marker for diagnosing colorectal cancer and for detecting colorectal cancer. At the same time, after inhibiting the expression of the tsRNA-Ala-3-0055 molecule, it has the effect of inhibiting the proliferation, migration and invasion of colorectal cancer cells and promoting cell apoptosis.

[0012] According to a second aspect of the present invention, a reagent for detecting the above-mentioned tsRNAs is provided, wherein the reagent comprises a PCR detection reagent.

[0013] In some embodiments of the present invention, the PCR detection reagent is a real-time fluorescence quantitative PCR detection reagent.

[0014] In some embodiments of the present invention, the PCR detection reagent includes primers for amplifying tsRNA-Ala-3-0055.

[0015] In some embodiments of the present invention, the sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0016] In some embodiments of the present invention, the PCR detection reagent also includes a reverse transcription primer.

[0017] In some embodiments of the present invention, the sequence of the reverse transcription primer is shown as SEQ ID NO:2.

[0018] According to the third aspect of the present invention, the use of the above tsRNAs or reagents in any one of the following a1) to a3) is proposed:

[0019] a1) Preparation of products for the treatment or adjuvant treatment of colorectal cancer;

[0020] a2) preparing reagents for detecting or assisting in detecting colorectal cancer;

[0021] a3) Preparation and screening of products for the treatment or adjuvant treatment of colorectal cancer.

[0022] In some embodiments of the invention, the product comprises a medicament.

[0023] In some embodiments of the present invention, the treatment or adjuvant treatment of colorectal cancer includes inhibiting cancer cell proliferation, inhibiting cancer cell migration, invasion and / or promoting cancer cell apoptosis.

[0024] According to a fourth aspect of the present invention, an inhibitor of tsRNAs is provided, wherein the inhibitor is at least one of a substance that inhibits the activity of tsRNAs, a substance that degrades tsRNAs, or a substance that reduces the expression level of tsRNAs;

[0025] The nucleic acid sequence of the tsRNAs is shown in SEQ ID NO:1.

[0026] In some embodiments of the present invention, the substance that reduces the expression level of tsRNAs is at least one of b1)-b3):

[0027] b1) siRNA, dsRNA, miRNA, ribozyme or shRNA targeting tsRNAs;

[0028] b2) a nucleic acid molecule encoding the siRNA, dsRNA, miRNA, ribozyme or shRNA targeting tsRNAs described in b1);

[0029] b3) An expression cassette, a vector or a transgenic cell line comprising the nucleic acid molecule described in b1).

[0030] In some embodiments of the present invention, the substance that reduces the expression level of tsRNAs is at least one of c1)-c3):

[0031] c1) siRNA targeting tsRNAs;

[0032] c2) a nucleic acid molecule encoding the siRNA targeting tsRNAs described in c1);

[0033] c3) An expression cassette, a vector or a transgenic cell line comprising the nucleic acid molecule described in c1).

[0034] In some embodiments of the present invention, the nucleotide sequence of the substance that reduces the expression level of tsRNAs is shown in SEQ ID NO:7.

[0035] According to the fifth aspect of the present invention, the use of the above-mentioned tsRNAs inhibitor in the preparation of a product for the treatment or adjuvant treatment of colorectal cancer is proposed.

[0036] In some embodiments of the invention, the product comprises a pharmaceutical product.

[0037] In some embodiments of the present invention, the treatment or adjuvant treatment of colorectal cancer includes inhibiting cancer cell proliferation, inhibiting cancer cell migration, invasion and / or promoting cancer cell apoptosis.

[0038] According to a sixth aspect of the present invention, a drug for treating colorectal cancer is provided, comprising the above-mentioned tsRNAs inhibitor.

[0039] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention provides a new tsRNAs molecule tsRNA-Ala-3-0055, which can be used as a diagnostic marker for colorectal cancer and is effectively used for detecting colorectal cancer. At the same time, knocking down tsRNA-Ala-3-0055 can inhibit the proliferation, invasion, and metastasis of colorectal cancer cells and promote apoptosis, providing a new and powerful molecular biological tool for the treatment of colorectal cancer. It has far-reaching clinical significance and important prospects for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0041] Figure 1 This is a schematic diagram of the molecular structure of tsRNA-Ala-3-0055 in Example 1 of the present invention;

[0042] Figure 2 This is a diagram showing the analysis and comparison results of the normal colorectal epithelial tissue and colorectal cancer tissue database in Example 2 of the present invention;

[0043] Figure 3 This is a graph showing the results of tsRNA-Ala-3-0055 molecule expression detection in normal colorectal cancer adjacent tissues and colorectal cancer tissues in Example 2 of the present invention, wherein “*” indicates p<0.05;

[0044] Figure 4 is a ROC curve diagram in Example 2 of the present invention;

[0045] Figure 5 This is a graph showing the results of the detection of tsRNA-Ala-3-0055 molecule expression in normal colorectal cells and colorectal cancer cells in Example 4 of the present invention, wherein "**" indicates p<0.01 and "***" indicates p<0.001;

[0046] Figure 6 This is a graph showing the effect of the inhibitor in Example 4 of the present invention on the expression of tsRNA-Ala-3-0055 molecules, where “**” indicates p<0.01;

[0047] Figure 7 This is a graph showing the detection results of the effect of overexpression of tsRNA-Ala-3-0055 molecules on the expression of tsRNA-Ala-3-0055 molecules in Example 4 of the present invention, wherein "**" indicates p<0.01;

[0048] Figure 8 This is a diagram showing the results of a scratch healing experiment in Example 4 of the present invention;

[0049] Fig. 9 This is a graph showing the results of the apoptosis experiment in Example 4 of the present invention;

[0050] Fig.10 This is a statistical analysis diagram of the apoptosis test results in Example 4 of the present invention, wherein "**" indicates p < 0.01;

[0051] Fig.11 This is a diagram showing the results of a clone-forming cell proliferation experiment in Example 4 of the present invention;

[0052] Fig.12 This is a graph showing the results of the CCK8 cell proliferation assay in Example 4 of the present invention, wherein “***” indicates p<0.001. DETAILED DESCRIPTION

[0053] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0054] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0055] Example 1

[0056] This embodiment provides a tsRNAs marker associated with colorectal cancer, and the screening process is as follows: through the tsRNA expression online database, tsRNAs molecules are analyzed and screened from the tRF explorer sequencing data, and the tsRNAs are named: tsRNA-Ala-3-0055, the sequence is: 5'-UCCCCGGCAUCUCCACCA-3' (as shown in SEQ ID NO: 1), the molecule length is 18nt, and the structure is as follows Figure 1 shown.

[0057] Example 2 Application of tsRNA-Ala-3-0055 in the preparation of reagents for diagnosing colorectal cancer

[0058] 1. Cell expression detection

[0059] The results of the database analysis and comparison of normal colorectal epithelial tissue and colorectal cancer tissue are shown in the figure below. Figure 2 As shown, real-time fluorescence quantitative PCR was performed in normal colorectal epithelial cells and colorectal cancer cells to detect the expression of tsRNA-Ala-3-0055 molecules. The specific experimental process is as follows:

[0060] (1) RNA was extracted from normal colorectal epithelial cells and colorectal cancer cells using the Trizol method. The specific process is as follows:

[0061] 1) Add 1 mL of Trizol and pipette down the cells in a six-well plate;

[0062] 2) Add 0.2 mL of chloroform, vortex for 15 seconds, place on ice for 5 minutes, and centrifuge at 12,000 g at 4°C for 15 minutes;

[0063] 3) Place the upper aqueous phase in a new enzyme-free EP tube, add 0.5 mL of isopropanol, place on ice for 10 min, and centrifuge at 12,000 g at 4°C for 10 min;

[0064] 4) Discard the supernatant, add 1 mL of 75% ethanol to wash, vortex to mix, centrifuge at 7500 g (2°C-8°C) for 5 min, discard the supernatant, and repeat once;

[0065] 5) Allow the precipitated RNA to dry at room temperature for 6-10 minutes;

[0066] 6) Add 15-30 μL of DECP water to each tube of sample, measure the concentration and then proceed with the subsequent reverse transcription experiment.

[0067] (2) Reverse transcription reaction

[0068] The reverse transcription reaction was carried out using the stem-loop method. The reverse transcription primer was: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGGTGGAG-3' (as shown in SEQ ID NO: 2), wherein the first 44 bases were the stem-loop sequence and the last 8 bases were the 8 bases on the tsRNA-Ala-3-0055 molecule that were reverse complementary to its 3' residue.

[0069] The reverse transcription reaction system was as follows: RNA: 1 μg, reverse transcription primer (10 μM) 0.5 μL, U6 downstream primer (10 μM) 0.5 μL, 5×miRNA RT (Stem-loop) Buffer 4 μL, miRNA RT Enzyme Mix 2 μL, and enzyme-free water to make up to 20 μL.

[0070] The reverse transcription reaction procedure is shown in Table 1 below.

[0071] Table 1

[0072] temperature Reaction time 25℃ 5min 42℃ 15min 85℃ 5sec 4℃ —

[0073] (3) Real-time quantitative PCR reaction

[0074] The primers for real-time quantitative PCR detection of tsRNA-Ala-3-0055 are:

[0075] Upstream primer: 5'-GCGATATCCCCGGCATCT-3' (as shown in SEQ ID NO: 3);

[0076] Downstream primer: 5'-AGTGCAGGGTCCGAGGTATT-3' (as shown in SEQ ID NO: 4).

[0077] The total system of real-time fluorescence quantitative PCR reaction was: 2×SYBR Green MIX 10 μL, upstream and downstream primers (10 mM): 1 μL each, cDNA template 2 μL, and supplemented to 20 μL with enzyme-free water;

[0078] The reaction procedure was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 5 s, and extension at 70°C for 10 s, for a total of 40 cycles, followed by melting curve analysis: fluorescence signals were collected at temperatures of 70°C-95°C.

[0079] The internal reference gene is U6, and the primer sequence for detecting the internal reference gene is:

[0080] Upstream primer: 5'-CTCGCTTCGGCAGCACA-3' (as shown in SEQ ID NO: 5);

[0081] Downstream primer: 5'-AACGCTTCACGAATTTGCGT-3' (as shown in SEQ ID NO: 6).

[0082] The results are as follows Figure 2 As shown in the figure, it can be seen that the tsRNA-Ala-3-0055 molecule is significantly highly expressed in colorectal cancer cells compared with normal colorectal cells.

[0083] 2. Clinical testing and verification

[0084] Sample information: 12 samples of cancerous tissues and adjacent tissues from patients diagnosed with colorectal cancer and 12 samples of colorectal tissues from healthy subjects were collected, all with informed consent.

[0085] Real-time fluorescence quantitative PCR was used to detect the expression of tsRNA-Ala-3-0055 molecules in colorectal cancer tissues and adjacent tissues of colorectal cancer patients and colorectal tissues of healthy subjects. The specific experimental process is as follows:

[0086] (1) RNA extraction was performed using the Trizol method. The specific process is as follows:

[0087] 1) Grind the tissue with a grinder filled with liquid nitrogen, add 1 mL of Trizol, and collect the tissue homogenate;

[0088] 2) Add 0.2 mL of chloroform, vortex for 15 seconds, place on ice for 5 minutes, and centrifuge at 12,000 g at 4°C for 15 minutes;

[0089] 3) Place the upper aqueous phase in a new enzyme-free EP tube, add 0.5 mL of isopropanol, place on ice for 10 min, and centrifuge at 12,000 g at 4°C for 10 min;

[0090] 4) Discard the supernatant, add 1 mL of 75% ethanol to wash, vortex to mix, centrifuge at 7500 g (2°C-8°C) for 5 min, discard the supernatant, and repeat once;

[0091] 5) Allow the precipitated RNA to dry at room temperature for 6-10 minutes;

[0092] 6) Add 15-30 μL of DECP water to each tube of sample, measure the concentration and then proceed with the subsequent reverse transcription experiment.

[0093] (2) Reverse transcription reaction

[0094] The reverse transcription reaction was performed using the stem-loop method, and the reverse transcription primer was: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC TGGTGGAG -3' (as shown in SEQ ID NO: 2), wherein the first 44 bases are a stem-loop sequence, and the last 8 bases are the 8 bases that are reverse complementary to the 3' sequence on the tsRNA-Ala-3-0055 molecule.

[0095] The reverse transcription reaction system was as follows: RNA: 1 μg, reverse transcription primer (10 μM) 0.5 μL, U6 downstream primer (10 μM) 0.5 μL, 5×miRNA RT (Stem-loop) Buffer 4 μL, miRNA RT Enzyme Mix 2 μL, and enzyme-free water to make up to 20 μL.

[0096] The reverse transcription reaction procedure is shown in Table 2 below.

[0097] Table 2

[0098] temperature Reaction time 25℃ 5min 42℃ 15min 85℃ 5sec 4℃ —

[0099] (3) Real-time quantitative PCR reaction

[0100] The primers for real-time quantitative PCR detection of tsRNA-Ala-3-0055 are:

[0101] Upstream primer: 5'-GCGATATCCCCGGCATCT-3' (as shown in SEQ ID NO: 3);

[0102] Downstream primer: 5'-AGTGCAGGGTCCGAGGTATT-3' (as shown in SEQ ID NO: 4).

[0103] The total system of real-time fluorescence quantitative PCR reaction was: 2×SYBR Green MIX 10 μL, upstream and downstream primers (10 mM): 1 μL each, cDNA template 2 μL, and supplemented to 20 μL with enzyme-free water;

[0104] The reaction procedure was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 5 s, and extension at 70°C for 10 s, for a total of 40 cycles, followed by melting curve analysis: fluorescence signals were collected at temperatures of 70°C-95°C.

[0105] The internal reference gene is U6, and the primer sequence for detecting the internal reference gene is:

[0106] Upstream primer: 5'-CTCGCTTCGGCAGCACA-3' (as shown in SEQ ID NO: 5);

[0107] Downstream primer: 5'-AACGCTTCACGAATTTGCGT-3' (as shown in SEQ ID NO: 6).

[0108] The results are as follows Figure 3-4 As shown in the figure, it can be seen that the tsRNA-Ala-3-0055 molecule is significantly more highly expressed in colorectal cancer tissues compared with normal colorectal tissues, and can accurately distinguish colorectal cancer tissues from normal tissues. The ROC diagnostic curve AUC = 0.875, CI = 0.735-1, the sensitivity is 1, and the specificity is 0.66667, indicating that the tsRNA-Ala-3-0055 molecule has diagnostic significance in colorectal cancer.

[0109] Example 3 An expression inhibitor of tsRNA-Ala-3-0055 molecule

[0110] This example provides an expression inhibitor of tsRNA-Ala-3-0055 molecule, the sequence of which is as follows:

[0111] tsRNA-Ala-3-0055 inhibitor sequence: TGGTGGAGATGCCGGGGA (such as SEQ ID NO: 7

[0112] shown).

[0113] Example 4 Application of the expression inhibitor of tsRNA-Ala-3-0055 molecule in the preparation of tumor-inhibiting products

[0114] This example provides an application of an expression inhibitor of a tsRNA-Ala-3-0055 molecule in the preparation of a product for inhibiting tumors. The specific verification process is as follows:

[0115] (1) The sequences of mimics and inhibitors (based on reverse complementation and binding to tsRNA-Ala-3-0055 to inhibit its function) of artificially synthesized tsRNA-Ala-3-0055 molecules are as follows:

[0116] The mimic sequence is: sense:TCCCCGGCATCTCCACCA (as shown in SEQ ID NO: 8);

[0117] Antisense sequence: TGGTGGAGATGCCGGGGA (as shown in SEQ ID NO: 9).

[0118] (2) Cell transfection:

[0119] 1) Take human colorectal cancer cells HCT8 with good growth status and healing degree of about 80% and inoculate them into a well plate, culture them in an incubator at 37°C and 5% CO2 for 12-20 hours, and start transfection when the cells grow to a confluence of 40%-50%;

[0120] 2) Add 5 μL of Lipofectamine 3000 reagent and 50 nM mimics or inhibitor to a sterile EP tube and mix well in 200 μL DMEM medium and let stand for 20 minutes;

[0121] 3) While the liposome transfection reagent is still, wash the cells twice with PBS, add 1.80 mL of complete medium (without double antibody), add the above liposome mixture to the well plate, shake well, and culture in a 37°C, 5% CO2 incubator for 36-72 hours.

[0122] (3) Reverse transcription reaction

[0123] Prepare the reverse transcription reaction system: RNA: 1 μg, reverse transcription primer (sequence as shown in SEQ ID NO: 2) (5 μM) 1 μL, 5×RT Mix Buffer 2 μL, RT Enzyme Mix 2 μL, enzyme-free water to 10 μL.

[0124] The reverse transcription reaction procedure is shown in Table 1. After the reaction is completed, the product is taken out and placed on ice for subsequent real-time fluorescence quantitative PCR reaction, and the excess sample is stored at -20°C.

[0125] (4) Real-time quantitative PCR (qRT-PCR)

[0126] The total reaction system was prepared as follows: 10 μL of 2×SYBR Green MIX, 1 μL of each of the upstream and downstream primers (10 mM), 2 μL of cDNA template, and the mixture was made up to 20 μL with enzyme-free water;

[0127] The reaction procedure was: 95℃5min pre-denaturation, 95℃30s denaturation, 60℃5s annealing, 70℃10s extension, 40 cycles in total, and then melting curve analysis: the temperature was 70℃-95℃ to collect fluorescence signals. After the reaction was completed, the amplification curve and melting curve of qRT-PCR were confirmed, and the expression intensity of each gene was standardized according to the CT value (threshold cycle values) and the internal reference gene (U6) and then 2 -△△ct Computation of gene expression.

[0128] The results are as follows Figure 5-7 As shown, NC is the blank control result. It can be seen from the figure that the tsRNA-Ala-3-0055 molecule was successfully overexpressed or knocked down in colorectal cancer cells.

[0129] (5) Scratch healing assay and cell apoptosis assay verified the effect of tsRNA-Ala-3-0055 on the invasion and migration of transfected colorectal cancer cells

[0130] The steps of the scratch test are as follows:

[0131] 1) Incubate the treated cells with a healing rate close to 100% in a medium containing 2% FBS for 12 hours in advance;

[0132] 2) Use a 10 μL pipette tip to make a “+” scratch in each well of a 6-well plate, and wash the cells five times with PBS until there are almost no suspended cells under a microscope;

[0133] 3) Remove PBS and add 2 mL of culture medium containing 2% serum to each well;

[0134] 4) Observe the healing of the scratches under an inverted microscope at the same time every day, take photos, and keep records;

[0135] 5) Use Image-Pro Plus image analysis software to analyze the scratch distance and obtain the average width of the scratch.

[0136] Cell apoptosis experiment:

[0137] 1) HCT8 cells (1×10 5 Cells) were transfected with inhibitor and mimics for 36 h;

[0138] 2) Digest the cells with EDTA-free trypsin and collect them into 1.5 ml EP tubes;

[0139] 3) After centrifugation, discard the supernatant, resuspend the cells with PSB, and repeat the wash twice;

[0140] 4) Single staining and double staining of HCT8 cells using Annexin v-fitc / PI double staining cell apoptosis detection kit;

[0141] 5) Place the cell plate in a cell culture incubator at 37°C with 5% CO2 and incubate for 15 minutes;

[0142] 6) Remove the cells and directly perform flow cytometry detection.

[0143] The results are as follows Figure 8-10 As shown, from Figure 8 As can be seen, the scratch healing experiment showed that overexpression of tsRNA-Ala-3-0055 promoted the invasion and migration of colorectal cancer cells, and NC was the blank control; inhibition of tsRNA-Ala-3-0055 molecule expression inhibited the invasion and migration of colorectal cancer cells;

[0144] from Figure 9-10 It can be seen that inhibiting the expression of tsRNA-Ala-3-0055 molecules can promote the apoptosis of colorectal cancer cells.

[0145] (5) Cloning experiment verifies the effect of tsRNA-Ala-3-0055 on the invasion and migration of transfected colorectal cancer cells

[0146] CCK8 cell proliferation assay:

[0147] 1) Inoculate a 96-well plate with the cells transfected with inhibitor and mimics prepared in step (4) above, with 1×10 3 Each well was filled with 200 μL of cell suspension. Four replicate wells were set up for each group. The cells were evenly seeded in a 96-well plate to prevent the cells from aggregating and growing and affecting the subsequent determination of absorbance. The 96-well plate with cells was placed in a cell culture incubator at 37°C containing 5% CO2 for culture.

[0148] 2) 12 hours after the plate is laid, when the cells are completely attached, the measurement on day 0 can be performed. Take out the 96-well plate from the cell culture incubator and add 10 μL of CCK-8 working solution;

[0149] 3) Place the 96-well plate with CCK-8 working solution in a cell culture incubator and incubate for 2 hours, then use an ELISA reader to detect the absorbance of each well at a wavelength of 450 nm;

[0150] 4) CCK-8 was added at the same time point on days 1, 2, 3, and 4 after culture for measurement;

[0151] 5) Collect the absorbance values ​​every day and draw a growth curve according to the OD values.

[0152] The CCK8 cell proliferation assay detected the effect of overexpression or knockdown of tsRNA-Ala-3-0055 on the proliferation ability of colorectal cancer cells. Figure 11-12 As shown, the results showed that the CCK8 cell proliferation experiment showed that overexpression of tsRNA-Ala-3-0055 promoted the proliferation of colorectal cancer cells, and NC was the blank control; knocking down the tsRNA-Ala-3-0055 molecule inhibited the invasion and migration of colorectal cancer cells.

[0153] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A tsRNAs associated with colorectal cancer, characterized in that: The nucleic acid sequence of the tsRNAs is shown in SEQ ID NO:

1.

2. A reagent for detecting tsRNAs according to claim 1, characterized in that: The reagents include PCR detection reagents; Preferably, the PCR detection reagent includes primers for amplifying tsRNA-Ala-3-0055; More preferably, the sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:

4.

3. Use of the tsRNAs according to claim 1 or the reagent according to claim 2 in any one of the following a1) to a3): a1) Preparation of products for the treatment or adjuvant treatment of colorectal cancer; a2) preparing reagents for detecting or assisting in detecting colorectal cancer; a3) Prepare and screen products for the treatment or adjuvant treatment of colorectal cancer.

4. A tsRNAs inhibitor, characterized in that The inhibitor is at least one of a substance that inhibits the activity of tsRNAs, a substance that degrades tsRNAs, and a substance that reduces the expression level of tsRNAs; The nucleic acid sequence of the tsRNAs is shown in SEQ ID NO:

1.

5. The tsRNAs inhibitor according to claim 4, characterized in that The substance that reduces the expression level of tsRNAs is at least one of b1)-b3): b1) siRNA, dsRNA, miRNA, ribozyme or shRNA targeting tsRNAs; b2) a nucleic acid molecule encoding the siRNA, dsRNA, miRNA, ribozyme or shRNA targeting tsRNAs described in b1); b3) An expression cassette, a vector or a transgenic cell line comprising the nucleic acid molecule described in b1).

6. The tsRNAs inhibitor according to claim 4, characterized in that The nucleotide sequence of the substance that reduces the expression level of tsRNAs is shown in SEQ ID NO:

7.

7. Use of the tsRNAs inhibitor according to any one of claims 4 to 6 in the preparation of a product for the treatment or adjuvant treatment of colorectal cancer.

8. The use according to claim 7, characterized in that: The products include pharmaceutical products.

9. The use according to claim 7, characterized in that: The treatment or adjuvant treatment of colorectal cancer includes inhibiting cancer cell proliferation, inhibiting cancer cell migration, invasion and / or promoting cancer cell apoptosis.

10. A drug for treating colorectal cancer, characterized in that: Containing the tsRNAs inhibitor according to any one of claims 4-6.

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