Application of LncRNA in the diagnosis and treatment of colon cancer

Abstract

The present invention belongs to the technical field of colon cancer markers, and specifically relates to the application of LncRNA in the diagnosis and treatment of colon cancer. The present invention proves for the first time that the expression level of LncRNA AC078860.2 in colon cancer patients is higher than that in normal people, and interfering with the expression of LncRNA AC078860.2 can inhibit the proliferation and migration of colon cancer cells. Therefore, LncRNA AC078860.2 can be used as an effective drug for the diagnosis and / or treatment of colon cancer, providing a more favorable means for the diagnosis and treatment of colon cancer, and is of great significance for the early diagnosis and treatment of colon cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of colon cancer markers, and specifically relates to the application of LncRNA in the diagnosis and treatment of colon cancer. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Colon cancer is one of the common digestive system tumors. Its morbidity and mortality have been increasing in recent years and has become an important public health issue. Currently, surgery, chemotherapy and radiotherapy are the main treatments for colon cancer. Despite improvements in diagnosis and treatment, most patients with colon cancer are already in the advanced stage when they are first diagnosed because they have no obvious clinical symptoms and have distant metastases, thus losing the opportunity for radical surgery. The morbidity and mortality of many patients with advanced colon cancer are still relatively high. Therefore, exploring new diagnostic markers has important clinical value for improving the prognosis of patients with colon cancer.

[0004] Non-coding RNA is a type of molecule that does not participate in protein coding but functions in the form of RNA. In recent years, it has become a research hotspot in the field of various diseases, especially in the field of tumor prevention and treatment. Non-coding RNAs such as miRNA, long non-coding RNA and circular RNA have been reported to be abnormally expressed in various tumors and participate in the occurrence and development of tumors. Among them, long non-coding RNA (LncRNA) is more than 200 nucleotide units in length and has no or limited protein coding ability. Depending on its location in the cell, LncRNA has the function of regulating chromatin and genes. In addition, existing studies have shown that abnormal expression of LncRNA is closely related to various tumors, such as gastric cancer, breast cancer and other types of tumors.

[0005] Exosomes are a type of vesicles with a diameter of 30-150nm, containing components such as RNA, lipids and proteins. Exosomes are present in a variety of body fluids such as blood, saliva, urine, cerebrospinal fluid and breast milk, and can shuttle back and forth in body fluids to transport genetic material and proteins between cells. Tumors will continuously release Exosomes into the surrounding environment during their growth. At the same time, Exosomes can be stored stably for a long time at ultra-low temperatures. By isolating Exosomes in serum for diagnosis, the influence of non-detectable substances in the blood when directly using blood to extract molecules for tumor diagnosis is overcome, making the test results more real and accurate. These characteristics make Exosomes helpful for the early diagnosis and prognosis of tumors.

[0006] In the field of colon cancer research, there is no literature report on the use of serum exosomes-derived LncRNA AC078860.2 as a detection marker. Therefore, the present invention studies the expression of serum exosomes-derived LncRNA AC078860.2 in colon cancer patients, in order to provide a breakthrough for the early diagnosis and treatment of colon cancer. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides the application of LncRNA in the diagnosis and treatment of colon cancer. The present invention first discovered that the expression of LncRNA AC078860.2 is abnormally high in colon cancer and is closely related to the diagnosis of colon cancer. Further studies have shown that interfering with the expression of LncRNA AC078860.2 can inhibit the proliferation and migration of colon cancer cells. Therefore, LncRNA AC078860.2 can be used as a diagnostic molecular marker for colon cancer, and can also be used as a potential therapeutic target for colon cancer, thereby completing the present invention.

[0008] Specifically, the present invention relates to the following technical solutions:

[0009] The first aspect of the present invention provides the use of a substance for detecting LncRNA AC078860.2 in the preparation of a colon cancer diagnosis or auxiliary diagnosis product:

[0010] (a1) Screening of active drugs for the treatment of colon cancer;

[0011] (a2) Diagnostic or auxiliary diagnostic test reagents for colon cancer.

[0012] Among them, LncRNA AC078860.2 is located on chromosome 12 (Gene: ENSG00000257761.3, Position: chr12 71587642-71594404), and its nucleotide sequence is shown in SEQ ID NO.1.

[0013] The present invention found that the expression of LncRNA AC078860.2 in colon cancer tissues was significantly higher than that in normal tissues, suggesting that LncRNA AC078860.2 plays a role in the progression of colon cancer. At the same time, there is a significant difference between the content of LncRNA AC078860.2 in the serum of colon cancer patients and that in normal serum samples, and the test results have high sensitivity and good specificity. Therefore, LncRNA AC078860.2 can be used as a new marker for diagnosis or auxiliary diagnosis of colon cancer, providing a basis for early diagnosis of colon cancer.

[0014] The second aspect of the present invention provides the use of a substance that inhibits the expression of LncRNA AC078860.2 in the preparation of a product for treating colon cancer. The substance that inhibits the expression level of LncRNA AC078860.2 includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA and gene knockout substances targeting LncRNA AC078860.2.

[0015] Furthermore, the function of the product for treating colon cancer is any one or more of the following:

[0016] (b1) Inhibit the proliferation of colon cancer cells;

[0017] (b2) inhibiting colon cancer cell invasion;

[0018] (b3) Promote apoptosis of colon cancer cells;

[0019] (b4) Inhibit colon cancer metastasis.

[0020] Furthermore, the siRNA comprises a sequence as shown in SEQ ID NO.2-3; and the product is a drug.

[0021] The third aspect of the present invention provides a product for treating colon cancer, wherein the active ingredient is a substance for inhibiting the expression level of LncRNA AC078860.2, wherein the substance is siRNA, and the siRNA is a sequence as shown in SEQ ID NO.2-3.

[0022] Sense 5'-GAA GAC UAA CAA GCA UUC ATT-3' (SEQ ID NO.2)

[0023] Anti-sense 5'-UGA AUG CUU GUU AGU CUU CTT-3' (SEQ ID NO.3)

[0024] Furthermore, the functions of the product are any one or more of the following:

[0025] (b1) Inhibit colon cancer cell proliferation;

[0026] (b2) inhibiting colon cancer cell invasion;

[0027] (b3) Promote apoptosis of colon cancer cells;

[0028] (b4) inhibiting colon cancer metastasis;

[0029] The product described is a drug.

[0030] The beneficial effects of one or more of the above technical solutions are as follows:

[0031] The above technical scheme proves for the first time that the expression level of LncRNA AC078860.2 in patients with colon cancer is higher than that in normal people, and interfering with the expression of LncRNA AC078860.2 can inhibit the proliferation and migration of colon cancer cells. Therefore, LncRNA AC078860.2 can be used as an effective drug for the diagnosis and / or treatment of colon cancer.

[0032] The above technical means provide a more favorable means for the diagnosis and treatment of colon cancer, which is of great significance for the early diagnosis and treatment of colon cancer. At the same time, it also lays an experimental foundation for the development of efficient drugs for the treatment of colon cancer, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 Schematic diagram of the expression difference of LncRNA AC078860.2 in colon cancer tissue and adjacent tissue in Example 1;

[0035] Figure 2 Schematic diagram of the expression difference of LncRNA AC078860.2 in serum exosomes of normal people and colon cancer patients in Example 1, wherein (A) is the relative expression of LncRNA AC078860.2 in serum exosomes of normal people and colon cancer patients; (B) is the receiver operating characteristic curve drawn for the relative expression of LncRNA AC078860.2 in serum exosomes of normal people and colon cancer patients;

[0036] Figure 3 This is a schematic diagram of the relative content of LncRNA AC078860.2 in colon cancer cells after verifying the interference of LncRNA AC078860.2 expression in Example 2;

[0037] Figure 4 This is a schematic diagram of the effect of interfering with LncRNA AC078860.2 expression on the proliferation ability of colon cancer cells observed by CCK-8 experiment in Example 2. Cell viability is represented by the OD value at a wavelength of 450 nm;

[0038] Figure 5The scratch experiment in Example 2 observed the effect of interfering with LncRNA AC078860.2 expression on the migration ability of colon cancer cells, wherein (A) is the wound healing diagram of interfering with LncRNA AC078860.2 in colon cancer cells HT29 and the control group at 0h and 48h, and (B) is the wound healing rate (%) of interfering with LncRNA AC078860.2 in colon cancer cells HT29 and the control group. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0041] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit its contents. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art, and can all be obtained from commercial sources.

[0042] It will be understood by the skilled artisan that the term "expression level" refers to the amount of a gene product present in vivo or in a sample at a specific point in time. Expression levels can be measured / quantified / detected, for example, by the protein or mRNA expressed by the gene. Expression levels can be quantified, for example, by normalizing the amount of the target gene product present in the sample with the total amount of the same type of gene product (total protein or mRNA) in the same sample or a reference sample (e.g., a sample obtained from the same individual at the same time or a portion of the same size (weight, volume) of the same sample), or by determining the amount of the target gene product / defined sample size (weight, volume, etc.). Expression levels can be measured or detected by any method known in the art, such as methods for direct detection and quantification of the target gene product (e.g., mass spectrometry), or typically by combining the target gene product with one or more different molecules or detection devices (e.g., primers, probes, antibodies, proteins) that are specific for the target gene product.

[0043] Methods for indirect detection and measurement of target gene products that work by combining with a scaffold. It is also known to the skilled person to determine the level of gene copies, which also includes determining the absence or presence of one or more fragments (e.g., by nucleic acid probes or primers, such as quantitative PCR, multiplex ligation-dependent probe amplification (Multiplex ligation-dependent probe amplification, MLPA) PCR).

[0044] The terms "indicator" and "marker" are used interchangeably in the present invention and refer to a sign or signal of a disease or are used to monitor a disease. Such a "disease" refers to a biological state of a cell, tissue or organ, or refers to the health and / or disease state of an individual. An indicator can be the presence or absence of a molecule including but not limited to peptides, proteins and nucleic acids, or can be a change in the expression level or pattern of such a molecule in a cell, or tissue, organ or individual. An indicator can be a sign of the occurrence, development or presence of a disease in an individual or the further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0045] The terms "up-regulated", "increased" or "increased" in the level of an indicator refer to an increase in the level of such indicator in a sample compared to a reference or reference sample.

[0046] The terms "down-regulated", "reduced" or "decreased" in the level of an indicator refer to a decrease in the level of such indicator in a sample compared to a reference or reference sample.

[0047] The inventors previously screened LncRNA members specifically expressed in colon cancer using gene sequencing technology, among which LncRNA AC078860.2 was selected as a candidate gene with great variability and no reports in colon cancer, suggesting that it may be used as a new molecular biomarker for the diagnosis and treatment of colon cancer.

[0048] In view of this, in one embodiment of the present invention, there is provided a use of a substance for detecting LncRNA AC078860.2 in the preparation of any one or more of the following products:

[0049] (a1) Screening of active drugs for the treatment of colon cancer;

[0050] (a2) Detection reagents for the diagnosis or auxiliary diagnosis of colon cancer.

[0051] The present invention found that the expression of LncRNA AC078860.2 in colon cancer tissues was significantly higher than that in normal tissues, suggesting that LncRNA AC078860.2 plays a role in the progression of colon cancer. At the same time, there is a significant difference between the content of LncRNA AC078860.2 in the serum of colon cancer patients and that in normal serum samples, and the test results have high sensitivity and good specificity. Therefore, LncRNA AC078860.2 can be used as a new marker for diagnosis or auxiliary diagnosis of colon cancer, providing a basis for early diagnosis of colon cancer.

[0052] In the application (a2), the detection reagent is used to detect the expression level of LncRNA AC078860.2 in a biological sample.

[0053] In another specific embodiment of the present invention, the detection reagent uses nucleic acid amplification technology to detect the expression level of LncRNAAC078860.2.

[0054] In another specific embodiment of the present invention, the colon cancer diagnosis or auxiliary diagnosis product includes but is not limited to primers, probes, nucleic acid membrane strips, preparations or kits for detecting the expression level of LncRNA AC078860.2 in the sample to be tested.

[0055] In another specific embodiment of the present invention, the kit comprises a reagent for detecting the expression of LncRNA AC078860.2.

[0056] Specifically, the kit contains specific PCR primers for LncRNA AC078860.2 and GAPDH internal reference, and the nucleotide sequences of the specific PCR primers are as follows:

[0057] LncRNA AC078860.2:

[0058] Primer F 5'-CCTCATCTGTGACCTTCGCA-3' (SEQ ID NO.4),

[0059] Primer R 5'-CCCCTCTTTTGACAGCCTTATG-3' (SEQ ID NO. 5);

[0060] GAPDH internal reference specific PCR primers:

[0061] Primer F 5'-TGGTCACCAGGGCTGCTT-3' (SEQ ID NO.6),

[0062] Primer R 5'-AGCTTCCCGTTCTCAGCCTT-3' (SEQ ID NO. 7).

[0063] The kit also includes RNA extraction reagent and reverse transcription reagent.

[0064] Specifically, the RNA extraction reagent includes Trizol reagent, chloroform, isopropanol and DEPC-treated water;

[0065] The reverse transcription reagent includes gDNA Clean Reaction Mix, Evo M-MLV RT Reaction Mix and DEPC treated water.

[0066] In another specific embodiment of the present invention, the sample to be tested is a human sample, and more specifically, the sample to be tested includes serum of a subject.

[0067] In another specific embodiment of the present invention, there is provided use of a substance that inhibits the expression of LncRNA AC078860.2 in the preparation of a product for treating colon cancer;

[0068] The functions of the product for treating colon cancer are any one or more of the following:

[0069] (b1) Inhibit colon cancer cell proliferation;

[0070] (b2) inhibiting colon cancer cell invasion;

[0071] (b3) Promote apoptosis of colon cancer cells;

[0072] (b4) Inhibit colon cancer metastasis.

[0073] The substances that inhibit the expression level of LncRNA AC078860.2 include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA and gene knockout substances targeting LncRNA AC078860.2.

[0074] In another specific embodiment of the present invention, the siRNA is a sequence as shown in SEQ ID NO.2-3.

[0075] The product described is a drug.

[0076] In another specific embodiment of the present invention, a product for treating colon cancer is provided, wherein the active ingredient is a substance for inhibiting the expression level of LncRNA AC078860.2, wherein the substance is siRNA, and the siRNA is a sequence as shown in SEQ ID NO.2-3.

[0077] In another specific embodiment of the present invention, the functions of the product are any one or more of the following:

[0078] (b1) Inhibit colon cancer cell proliferation;

[0079] (b2) inhibiting colon cancer cell invasion;

[0080] (b3) Promote apoptosis of colon cancer cells;

[0081] (b4) inhibiting colon cancer metastasis;

[0082] The product described is a drug.

[0083] According to the present invention, the above-mentioned medicine further comprises at least one inactive pharmaceutical ingredient.

[0084] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy. Moreover, according to the usual methods, the drug can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0085] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0086] The carriers, excipients and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0087] The drugs of the present invention can be administered to the body in known ways. For example, they can be delivered systemically by intravenous delivery or local injection into the tissue of interest. They can be administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention can vary greatly depending on a variety of factors,

[0088] Such as target cells, biological type or its tissue, general condition of the subject to be treated, route of administration, mode of administration, etc.

[0089] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0090] Example 1: Difference in expression of LncRNA AC078860.2 in serum exosomes of colon cancer patients and normal subjects

[0091] 1. Expression differences between colon cancer tissue and adjacent tissues in patients with colon cancer

[0092] (1) Sample collection: The present invention collected colon cancer tissues and adjacent normal tissues from 50 patients who underwent colon cancer resection in the Department of General Surgery of the First Affiliated Hospital of Shandong First Medical University (Shandong Qianfoshan Hospital). Each patient was informed of the use of the samples and signed an informed consent form. The tissue samples were stored in liquid nitrogen. This study was approved by the Ethics Committee of the First Affiliated Hospital of Shandong First Medical University, Shandong Qianfoshan Hospital.

[0093] (2) Extraction of RNA from tissues: Take an appropriate amount of specimens in a 1.5 mL EP tube, add 1 mL Trizol and grind into liquid with a handheld tissue grinder, then add 200 μL chloroform, shake vigorously by hand for 15-30 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4°C 12000 g for 15 minutes; take the supernatant in a 1.5 mL RNase-free EP tube, add an equal volume of isopropanol and mix well, let stand at room temperature for 10 minutes, and centrifuge at 4°C 12000 g for 10 minutes; discard the supernatant, add 1 mL 75% ethanol (diluted with DEPC-treated water), shake and wash, centrifuge at 4°C 7500 g for 5 minutes, discard the supernatant, open the EP tube and dry at room temperature, and add 10-30 μL DEPC-treated water to dissolve RNA. The concentration and quality of RNA were measured by UV spectrophotometer. When the OD260 / 280 ratio was between 1.8 and 2.0, the RNA quality was good and the extraction was qualified.

[0094] (3) LncRNA AC078860.2 reverse transcription: Use the reverse transcription premix kit of Acori Biotechnology Co., Ltd.

[0095] Step 1: Remove genomic DNA. The reaction system is shown in Table 1.

[0096] Table 1 Reaction system for removing genomic DNA

[0097]

[0098] Reaction conditions: 42°C, 2 min; 4°C, hold.

[0099] Step 2: Reverse transcription reaction, the system is shown in Table 2.

[0100] Table 2 Reverse transcription system

[0101]

[0102] Reaction conditions: 37°C, 15 min; 85°C, 5 min, 4°C, hold.

[0103] (4) Real-time quantitative PCR was performed using the AC078860.2 specific primers synthesized by Ascenta (Nanjing) Life Science Technology Co., Ltd. The 10 μL reaction system is shown in Table 3.

[0104] Table 3 PCR reaction system

[0105]

[0106] Real-time fluorescence quantitative PCR reaction procedure:

[0107]

[0108] (5) Determination of indicators: The experimental data were analyzed by relative quantitative analysis method, GAPDH was used as the internal reference gene, and its nucleotide sequence is shown in SEQ ID NO.6-7. The data were analyzed using Graph Pad Prism 8 software. The results are shown in Figure 1 As shown in the figure, compared with the expression of LncRNA AC078860.2 in adjacent tissues, the expression of LncRNA AC078860.2 in colon cancer tissues was significantly upregulated, and the difference was significant ( P <0.001).

[0109] 2. Expression differences of LncRNA AC078860.2 derived from exosomes in patients with colon cancer and healthy subjects

[0110] Peripheral blood samples from 50 patients diagnosed with colon cancer and 50 normal subjects were collected from the First Affiliated Hospital of Shandong First Medical University (Qianfoshan Hospital of Shandong Province). Blood coagulation tubes were used to collect 2 mL of blood from the individual to be tested. After blood collection, the blood was centrifuged at 3500 rpm for 10 min, and the serum was drawn and placed in a 1.5 mL EP tube and stored in a -80°C refrigerator. Each patient was informed of the use of the sample and signed an informed consent form. This study was approved by the Ethics Committee of Qianfoshan Hospital of Shandong Province, the First Affiliated Hospital of Shandong First Medical University.

[0111] The SBI brand exosome extraction kit (Cat. No.: EXOQ5A-1) was used to separate the exosomes in the serum, and the Thermo Fisher brand exosome RNA extraction kit (Cat. No.: 4478545) was used to extract and purify the RNA in the exosomes. The specific experimental steps are as follows:

[0112] (1) Isolation of Exosomes in Serum

[0113] Isolation of Exosomes in serum: Add 100 μL of Total Exosome Isolation Reagent to 500 μL of each serum sample, vortex to mix, and react at 4°C for 30 min. Centrifuge at 10,000 g for 10 min at room temperature. Exosomes are present at the bottom of the EP tube, and resuspend the Exosomes with 200 μL PBS.

[0114] (2) Extraction and purification of RNA from exosomes

[0115] Extraction of RNA from Exosomes: Add 200 μL of 2X Denaturing Solution and mix well, incubate on ice for 5 min, then add 400 μL of acid-Phenol: Chloroform and vortex for 60 s. Centrifuge at 12000 g for 10 min at room temperature. The supernatant contains RNA.

[0116] Purification of RNA: Pipette 300 μL of supernatant into an enzyme-free EP tube, add 375 μL of anhydrous ethanol, and mix the two. Add the mixed solution to the filter column, centrifuge at 10,000 g for 15 seconds, and pour out the mixed solution in the collection tube. Add 700 μL of miRNA Wash Solution 1, centrifuge at 10,000 g for 15 seconds at room temperature, and pour out the mixed solution in the collection tube. Add 500 μL of Wash Solution 2 / 3, centrifuge at 10,000 g for 15 seconds at room temperature, and repeat this step. Place the filter column in the collection tube and centrifuge at 10,000 g for 1 min. Place the filter column in a new collection tube, add 35 μL of Elution Solution, and centrifuge at 10,000 g for 30 seconds at room temperature to obtain purified RNA, and test the RNA purity and concentration.

[0117] (3) The reverse transcription reaction method is the same as step (3) in “I. Expression differences between colon cancer tissues and adjacent tissues in colon cancer patients”.

[0118] (4) The qPCR reaction method and primers were the same as step (4) in “I. Expression differences between colon cancer tissues and adjacent tissues in colon cancer patients”.

[0119] (5) Detection and calculation

[0120] A relative quantitative analysis method was used, GAPDH was used as the internal reference gene, and its nucleotide sequence was shown in SEQ ID NO.6-7. The data were analyzed using GraphPad Prism 8 software. The test results are shown in Figure 2 As shown in (A) in .

[0121] (6) Receiver operating characteristic curve analysis

[0122] SPSS Statistics26 was used to calculate the experimental data, and the receiver operating characteristic curve was drawn. The area under the curve (AUC) was calculated to evaluate the diagnostic ability of LncRNA AC078860.2 in colon cancer serum Exosomes. The closer the AUC is to 1, the stronger the diagnostic ability is, and when it is equal to 0.5, it is considered to have no diagnostic ability. After further calculation of the Youden index, the sensitivity and specificity of the optimal critical value were obtained. The ROC curve is shown in Figure 2 As shown in (B), the AUC can reach 0.9812, the specificity can reach 96%, and the sensitivity can reach 92%. From the results, it can be seen that LncRNA AC078860.2 has high sensitivity and specificity in diagnosing colon cancer. Therefore, LncRNA AC078860.2 derived from serum exosomes can be used as a marker for diagnosing colon cancer.

[0123] Example 2: Interfering with LncRNA AC078860.2 expression and exploring its functional impact in colon cancer

[0124] (1) Cell culture

[0125] The revived HT29 colon cancer cells were inoculated into a culture dish containing an appropriate amount of RPMI-1640 complete culture medium (10% FBS, penicillin concentration 100 U / mL, streptomycin 0.1 mg / mL), and the culture dish was moved into a 37°C cell culture incubator with 5% CO2 for routine culture. After 24 hours, the culture medium was replaced. Depending on the growth status of the cells, the medium was replaced every 1-2 days. When the cell fusion reached 70%-80%, the cell was subcultured.

[0126] (2) siRNA transfection of cells

[0127] siRNA sequence: Sense 5'-GAAGACUAACAAGCAUUCATT-3' (SEQ ID NO.2), Anti-sense5'-UGA AUG CUU GUU AGU CUU CTT-3' (SEQ ID NO.3);

[0128] siNC sequence: Sense 5'-UUC UCC GAA CGU GUC ACG UTT-3' (SEQ ID NO. 8), Anti-sense 5'-ACG UGA CAC GUU CGG AGA ATT-3' (SEQ ID NO. 9).

[0129] Transfection was performed according to the instructions of Lipofectamine 2000 transfection kit.

[0130] When the cell confluence in the six-well plate reached 80%, the culture medium without antibiotics was replaced two hours before transfection, and Lipofectamine 2000-mediated cell transfection was performed.

[0131] Set up the experimental group, control group, and negative control group, and prepare the transfection solution in each well according to the proportion:

[0132] Experimental group: 5 μL siRNA (20 μM) was added to 250 μL serum-free and antibiotic-free culture medium and gently mixed; Lipofectamine 2000 was gently mixed, 5 μL liposomes were added to 250 μL serum-free and antibiotic-free culture medium, gently mixed and incubated at room temperature for 5 min, after 5 min, Lipofectamine 2000 and diluted siRNA were mixed, gently mixed, and incubated at room temperature for 20 min to allow the complex to form;

[0133] Control group: 5 μL si-NC (20 μM) was added to 250 μL serum-free and antibiotic-free culture medium and gently mixed; Lipofectamine 2000 was gently mixed, 5 μL liposomes were added to 250 μL serum-free and antibiotic-free culture medium, gently mixed and incubated at room temperature for 5 min, after 5 min, Lipofectamine 2000 and diluted si-NC were mixed, gently mixed, and incubated at room temperature for 20 min to allow the complex to form;

[0134] Negative control group: add 5 μL Lipofectamine RNAi to 250 μL serum-free and antibiotic-free culture medium and mix gently; gently mix Lipofectamine 2000, add 5 μL liposomes to 250 μL serum-free and antibiotic-free culture medium, mix gently and incubate at room temperature for 5 min. After 5 min, mix Lipofectamine 2000 and diluted Lipofectamine RNAi, mix gently, and incubate at room temperature for 20 min to allow the complex to form.

[0135] Add the above mixture (total volume 500 μL) to the culture wells, shake the culture plate back and forth to make it evenly distributed, and place it in the incubator for culture. After 6 hours of culture, replace it with complete culture medium.

[0136] After culturing for 48 hours, 1 mL of Trizol reagent was added to the cells to denature the cells and then RNA was extracted. After reverse transcription, the transfection efficiency was determined by fluorescent quantitative PCR. The primer sequences were shown in SEQ ID NO.4-5, and the internal reference GAPDH nucleotide sequence was shown in SEQ ID NO.6-7.

[0137] (3) CCK8 experiment

[0138] The siRNA cell transfection was performed using the method in step (2), and a control group was set up at the same time. 24 hours after cell transfection, the transfected cells were collected and resuspended after trypsin digestion in a 6-well plate, and 100 μL of cell suspension (about 2000 cells) was added to each well of a 96-well plate. Six duplicate wells and three blank wells (only culture medium was added, no cells) were set up, and the edge wells were filled with PBS solution. The plates were placed in an incubator and cultured for 24 hours.

[0139] Add 10 μL of CCK-8 solution to each well and incubate in the incubator for 4 h.

[0140] The absorbance at 450 nm was measured with an enzyme-labeled instrument at 24 h, 48 h, and 72 h.

[0141] (4) Scratch test

[0142] Cell culture was performed according to the method in step (1). When the cell confluence in the six-well plate reached 80%, five straight lines were drawn behind the six-well plate, with the interval between two straight lines being at least 0.5 cm.

[0143] The siRNA cell transfection was performed using the method in step (2), and a control group was set up at the same time. 24 h after cell transfection, a 10 μL pipette tip was used to scratch the bottom of the cell vertically.

[0144] 3) After washing three times with PBS solution, add 2 mL of dual antibody-free culture medium / well and place in the incubator.

[0145] 4) Take photos under a microscope at 0 h and 48 h.

[0146] 5) Calculate the scratch area using Photoshop and ImageJ software.

[0147] The present invention uses siRNA to knock down LncRNA AC078860.2, and verifies the knockdown efficiency by qPCR. Figure 3 The results of the CCK8 experiment are shown in Figure 4 As shown in Figure 2, knocking down LncRNA AC078860.2 in colon cancer cells significantly inhibited the proliferation rate of colon cancer cells. Figure 5 As shown in (A) and (B), knockdown of LncRNA AC078860.2 can inhibit the migration ability of colon cancer cells. The above results indicate that LncRNA AC078860.2 may be involved in the occurrence and development of colon cancer and can be used as a new therapeutic target.

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a detection reagent for detecting the expression level of LncRNA AC078860.2 in a biological sample in the preparation of a colon cancer diagnostic product, wherein the nucleotide sequence of the LncRNA AC078860.2 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The detection reagent uses nucleic acid amplification technology to detect the expression level of LncRNAAC078860.

2.

3. The use according to claim 1, characterized in that The colon cancer diagnostic product includes primers, probes, nucleic acid membrane strips or kits for detecting the expression level of LncRNA AC078860.2 in a sample to be tested.

4. Use of a substance that inhibits the expression of LncRNA AC078860.2 in the preparation of a product for treating colon cancer, wherein the substance that inhibits the expression of LncRNA AC078860.2 is an siRNA targeting LncRNA AC078860.2, and the sequence of the siRNA is as follows: Sense 5'-GAA GAC UAA CAA GCA UUC ATT-3', and, Anti-sense 5'-UGA AUG CUU GUU AGU CUU CTT-3'; The product described is a drug.

5. The use according to claim 4, characterized in that: The functions of the product for treating colon cancer are any one or more of the following: (b1) Inhibit colon cancer cell proliferation; (b2) Inhibit colon cancer cell migration.

6. A product for treating colon cancer, characterized in that: The active ingredient is a substance for inhibiting the expression level of LncRNA AC078860.2, and the substance is siRNA, and the siRNA sequence is as follows: Sense 5'-GAA GAC UAA CAA GCA UUC ATT-3', and, Anti-sense 5'-UGA AUG CUU GUU AGU CUU CTT-3'; The product described is a drug.

7. The product according to claim 6, characterized in that The functions of the product are any one or more of the following: (b1) Inhibit colon cancer cell proliferation; (b4) Inhibit colon cancer cell migration.

Images