Application of LncRNA AC113143.1 as a diagnostic marker for colon cancer

Abstract

The present invention belongs to the technical field of colon cancer markers, and specifically relates to the application of LncRNA AC113143.1 as a diagnostic marker for colon cancer. The present invention shows through research results that the expression level of LncRNA AC113143.1 derived from serum Exosomes in colon cancer patients is significantly higher than the expression level in healthy people. Further, the inventors found that the high level expression of LncRNA AC113143.1 derived from serum Exosomes has good sensitivity and specificity when used to diagnose colon cancer, and therefore can be used as a biomarker for colon cancer diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of colon cancer markers, and particularly relates to the application of LncRNA AC113143.1 as a colon cancer diagnostic marker. 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. Although surgery-based comprehensive treatment has significantly improved the prognosis of patients with colon cancer, most patients with colon cancer are already in the late stage when they are first diagnosed due to the lack of obvious clinical symptoms in the early stage. They have distant metastasis and lose the opportunity for radical surgery. They have poor prognosis, high mortality and low 5-year survival rate. Therefore, exploring new diagnostic markers has important clinical value for improving the prognosis of patients with colon cancer.

[0004] 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 that are directly extracted from blood for tumor diagnosis is overcome, making the test results more real and accurate. These characteristics make Exosomes helpful for early diagnosis and prognosis of tumors.

[0005] At present, there are few reports on the role of serum exosomes-derived lncRNA in the occurrence of colon cancer. Therefore, the present invention studies the expression of serum exosomes-derived lncRNA AC113143.1 in colon cancer patients, in order to provide a breakthrough for the treatment of colon cancer. Summary of the invention

[0006] The present invention shows through research results that the expression level of serum exosomes-derived LncRNA AC113143.1 in colon cancer patients is significantly higher than that in healthy people. Furthermore, the inventors found that the high level expression of serum exosomes-derived LncRNA AC113143.1 has good sensitivity and specificity when used to diagnose colon cancer, and thus can be used as a biomarker for colon cancer diagnosis.

[0007] Based on the above research results, the present invention provides the following technical contents:

[0008] In a first aspect, the present invention provides a method for preparing a colon cancer diagnosis product using a reagent for detecting LncRNA AC113143.1.

[0009] The LncRNA AC113143.1 is derived from serum exosomes and is located on chromosome 8, and its nucleotide sequence is shown in SEQ ID NO.1. The present invention found that the expression of LncRNA AC113143.1 in clinical samples of patients with colon cancer was significantly upregulated, and there was a significant difference in the content of LncRNA AC113143.1 in normal samples, and the test results were highly sensitive and specific. The results of this study show that LncRNA AC113143.1 derived from serum Exosomes can be used as a screening and early diagnosis and evaluation marker for people at high risk of colon cancer.

[0010] Furthermore, the application includes any one of the following:

[0011] (1) Screening of active drugs for the treatment of colon cancer;

[0012] (2) Colon cancer diagnostic test reagents.

[0013] Furthermore, the detection reagent is used to detect the expression level of LncRNA AC113143.1 in a biological sample.

[0014] Furthermore, the detection reagent uses nucleic acid amplification technology to detect the expression level of LncRNA AC113143.1.

[0015] Furthermore, the detection method is a real-time fluorescence quantitative PCR method.

[0016] Furthermore, the colon cancer diagnostic product includes any one of a test strip, a test chip or a test kit.

[0017] Furthermore, the diagnosis of colon cancer comprises the following steps:

[0018] A1. Collect samples from test subjects and collect control samples;

[0019] A2. Detect and compare the expression level of LncRNA AC113143.1 in the test subject sample and the control sample;

[0020] If the expression level of LncRNA AC113143.1 in the sample of the tested subject is increased, the tested subject is diagnosed as having colon cancer or being at risk of having colon cancer.

[0021] Furthermore, the control sample is derived from a healthy population or a healthy tissue of a test subject; the sample of the test subject includes a clinical biological sample of the subject, including but not limited to a blood sample, body fluid, tissue, organ or secretion, the blood sample includes serum, plasma or whole blood; the secretion includes a cotton swab and pus; in a preferred embodiment, the sample of the test subject is a blood sample, the tissue sample of the test subject includes a colon tissue of the test subject, such as a colon biopsy sample, and the control sample is derived from a colon tissue of a healthy subject, such as a colon biopsy sample, or a healthy tissue of the test subject, such as a cancer-adjacent tissue.

[0022] The third aspect of the present invention provides a kit for diagnosing colon cancer, wherein the kit comprises a reagent for detecting the expression of LncRNA AC113143.1.

[0023] Furthermore, the kit contains specific PCR primers for LncRNA AC113143.1 and GAPDH internal reference, and the nucleotide sequences of the specific PCR primers are as follows:

[0024] LncRNA AC113143.1:

[0025] Primer F 5'-CCATCTCTGGGGTCACTAACA-3' (SEQ ID NO.2),

[0026] Primer R 5'-ACGGTGAGTTGTTGTCCTTGTA-3' (SEQ ID NO. 3);

[0027] GAPDH internal reference specific PCR primers:

[0028] Primer F 5'-TGGTCACCAGGGCTGCTT-3'(SEQ ID NO.4),

[0029] Primer R 5'-AGCTTCCCGTTCTCAGCCTT-3' (SEQ ID NO. 5).

[0030] Furthermore, the kit includes RNA extraction reagents, reverse transcription reagents and a quantitative PCR reaction system.

[0031] Furthermore, the RNA extraction reagent includes Trizol reagent, chloroform, isopropanol and DEPC treated water;

[0032] Further, the reverse transcription reagent includes gDNA Clean Reaction Mix, Evo M-MLV RT Reaction Mix and DEPC treated water;

[0033] Furthermore, the reagents used for the quantitative PCR include the specific PCR primers of the LncRNA AC113143.1 and GAPDH internal reference, and also include SYBR Green Pro Taq HS Premix and DEPC-treated water.

[0034] Furthermore, the kit is based on real-time fluorescence quantitative PCR to detect the expression level of LncRNA AC113143.1.

[0035] The beneficial effects of the present invention are as follows:

[0036] The present inventors have found that the expression level of LncRNA AC113143.1 in patients with colon cancer is significantly higher than that in healthy people. Furthermore, the present inventors found that the high level of expression of LncRNA AC113143.1 derived from serum exosomes has good sensitivity and specificity when used to diagnose colon cancer, and therefore can be used as a biomarker for colon cancer diagnosis. In addition, the present invention also found that the expression level of LncRNA AC113143.1 can be detected by serum exosomes of subjects to diagnose and predict colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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.

[0038] Figure 1 The expression difference of LncRNA AC113143.1 in colon cancer tissues and adjacent tissues;

[0039] Figure 2 Real-time fluorescence quantitative PCR analysis of the expression difference of LncRNA AC113143.1 in colon cancer tissues and normal colon tissues and ROC curve analysis;

[0040] Figure 3Expression difference and ROC curve analysis of LncRNA AC113143.1 derived from Exosomes in colon cancer patients and normal people. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In one or more embodiments of the present invention, the nucleotide sequences of primers for detecting the expression of LncRNA AC113143.1 are shown in SEQ ID NOs. 2 and 3.

[0045] Example 1: LncRNA AC113143.1 is highly expressed in colon cancer

[0046] 1. Collect 50 samples of colon cancer tissue and adjacent cancer tissue preserved in 2023 from a hospital in Jinan, place them in cryopreservation tubes, and store them in liquid nitrogen.

[0047] 2. Extraction of RNA from tissues: Take an appropriate amount of specimens in a 1.5mL EP tube, add 1mL Trizol and grind into liquid with a handheld tissue grinder, then add 200μL chloroform, shake vigorously by hand for 15-30s, let stand at room temperature for 3min, centrifuge at 4℃12000g for 15min; take the supernatant in a 1.5mL RNase-free EP tube, add an equal volume of isopropanol to mix, let stand at room temperature for 10min, centrifuge at 4℃12000g for 10min; discard the supernatant, add 1mL 75% ethanol (diluted with DEPC-treated water) and shake to wash, centrifuge at 4℃7500g for 5min, discard the supernatant, open the EP tube and dry at room temperature, add 10-30μL DEPC-treated water to dissolve RNA. Determine the concentration and quality of RNA with a UV spectrophotometer. When the OD260 / 280 ratio is between 1.8-2.0, the RNA quality is good and the extraction is qualified.

[0048] 3. LncRNA AC113143.1 reverse transcription: Use the reverse transcription premix kit of Acori Biotechnology Co., Ltd.

[0049] Step 1: Removal of genomic DNA

[0050]

[0051] Reaction conditions: 42°C, 2 min;

[0052] 4℃, hold.

[0053] Step 2: Reverse transcription reaction

[0054]

[0055] Reaction conditions: 37°C, 15 min;

[0056] 85℃, 5min,

[0057] 4℃, hold.

[0058] 4. Real-time quantitative PCR was performed using the AC113143.1 specific primers synthesized by Anshengda (Nanjing) Life Science Technology Co., Ltd. The 10 μL reaction system is as follows:

[0059]

[0060]

[0061] Real-time fluorescence quantitative PCR reaction procedure:

[0062] 5. -2 ΔΔCTDetermination of indicators: The experimental data were analyzed by relative quantitative analysis method, GAPDH was used as the internal reference gene, and the data were analyzed by GraphPad Prism 8. The analysis found that compared with the expression of LncRNA AC113143.1 in adjacent tissues, the expression of LncRNA AC113143.1 in 50 patients with colon cancer was significantly upregulated, and the difference was significant (P<0.001) (e.g. Figure 1 shown).

[0063] Example 2: Analysis of expression differences between colon cancer tissue and normal colon tissue of colon cancer patients

[0064] Colon cancer tissues from 50 colon cancer patients preserved in 2024 and 20 normal human colon tissue samples were collected from a hospital in Jinan, placed in cryopreservation tubes, and stored in liquid nitrogen.

[0065] The experimental method was the same as that in Example 1. The results showed that compared with the expression of LncRNA AC113143.1 in normal colon tissue, the expression of LncRNA AC113143.1 in colon tissue of 50 patients with colon cancer was significantly upregulated, and the difference was significant (P<0.001) ( Figure 2 A), process the result data and draw the ROC curve. The result is as follows Figure 2 As shown in B, the AUC can reach 0.9600, the specificity can reach 94%, and the sensitivity can reach 95%. From the results, it can be seen that LncRNA AC113143.1 has high sensitivity and specificity in diagnosing colon cancer. Therefore, LncRNA AC113143.1 is used to diagnose colon cancer.

[0066] Example 3: Expression differences of LncRNA AC113143.1 derived from Exosomes in colon cancer patients and normal subjects

[0067] Peripheral blood samples from 50 patients diagnosed with colon cancer and 50 normal subjects were collected from a hospital in Jinan and the following experiment was performed.

[0068] 1. Isolation of Exosomes from Serum

[0069] 1.1 Separation of peripheral blood serum: Use a blood coagulation tube to collect 2 mL of blood from the individual to be tested. After blood collection, centrifuge at 3500 rpm for 10 minutes, draw serum and place it in a 1.5 mL EP tube and store it in a -80℃ refrigerator.

[0070] 1.2 Separation 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 minutes. Centrifuge at 10000g for 10 minutes at room temperature. Exosomes are stored at the bottom of the EP tube, and resuspend the Exosomes with 200 μL PBS. (Choose a commercialized Exosomes separation kit)

[0071] 2. Extraction and purification of RNA from Exosomes (select a commercially available Exosomes RNA isolation and purification kit)

[0072] 2.1 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, vortex for 60 s. Centrifuge at 12000 g for 10 min at room temperature. The supernatant contains RNA.

[0073] 2.2 RNA purification: 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 minute. 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. To detect the purity and concentration of RNA, the reverse transcription and real-time quantitative method in step 3 of Example 1 was used to detect LncRNA AC113143.1. The results are as follows: Figure 3 As described in A, it was confirmed that LncRNA AC113143.1 was highly expressed in serum Exosomes. The result data was processed and the ROC curve was drawn. The results are shown in Figure 3 As shown in Figure B, the AUC can reach 0.9652, the specificity can reach 94%, and the sensitivity can reach 94%. From the results, it can be seen that LncRNA AC113143.1 has high sensitivity and specificity in diagnosing colon cancer. Therefore, LncRNA AC113143.1 derived from serum exosomes can be used to diagnose colon cancer.

[0074] Example 4: Kit for screening and early diagnosis of high-risk groups for colon cancer

[0075] In this embodiment, a kit is provided for screening and early diagnosis of a high-risk population for colon cancer. The kit is used to detect the expression level of long non-coding RNA AC113143.1 in a clinical sample of a subject. The kit specifically comprises the following detection reagents (100 reactions):

[0076] 1. Trizol reagent 110mL

[0077] 2. Chloroform 22mL

[0078] 3. Isopropyl alcohol 55mL

[0079] 4. 80 mL of anhydrous ethanol

[0080] 5. DEPC treated water 30mL

[0081] 6.5x gDNA Clean Reaction Mix 220μL

[0082] 7.5x Evo M-MLV RT Reaction Mix420μL

[0083] 8.2x SYBR Green Pro Taq HS Premix 520μL

[0084] 9.10μM LncRNA AC113143.1 real-time fluorescence quantitative PCR specific primers 110μL

[0085] AC113143.1Primer F 5'-CCATCTCTGGGGTCACTAACA-3' (SEQ ID NO.2);

[0086] AC113143.1Primer R 5'-ACGGTGAGTTGTTGTCCTTGTA-3' (SEQ ID NO.3);

[0087] 10.10μM GAPDH real-time fluorescence quantitative PCR specific primer 110μL

[0088] GAPDH Primer F 5'-TGGTCACCAGGGCTGCTT-3',

[0089] GAPDH Primer R 5'-AGCTTCCCGTTCTCAGCCTT-3'.

[0090] 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 reagent for detecting LncRNA AC113143.1 in preparing a colon cancer diagnostic product, wherein the nucleotide sequence of the LncRNA AC113143.1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The application includes any of the following: (1) Screening of active drugs for the treatment of colon cancer; (2) Colon cancer diagnostic test reagents.

3. The use according to claim 2, characterized in that The detection reagent is used to detect the expression level of LncRNAAC113143.1 in a biological sample.

4. The use according to claim 2, characterized in that The detection reagent uses nucleic acid amplification technology to detect the expression level of LncRNAAC113143.

1.

5. The use according to claim 1, characterized in that The colon cancer diagnostic product includes any one of a test strip, a test chip or a test kit.

6. The use according to claim 1, characterized in that The diagnosis of colon cancer comprises the following steps: A1. Collect samples from test subjects and control samples; A2. Detect and compare the expression level of LncRNA AC113143.1 in the test subject sample and the control sample; If the expression level of LncRNA AC113143.1 in the sample of the tested subject is increased, the tested subject is diagnosed as having colon cancer or being at risk of having colon cancer.

7. The use according to claim 6, characterized in that The control sample is derived from healthy people or healthy tissues of the test subject; the sample of the test subject is a blood sample or tissue, and the blood sample includes serum, plasma or whole blood.

8. The use according to claim 7, characterized in that The sample of the subject to be tested is a blood sample.

Images