A marker for evaluating the efficacy of astragalus polysaccharide intervention therapy in patients with colorectal cancer and its application

Abstract

The present invention belongs to the technical field of colorectal cancer efficacy evaluation, and particularly relates to a marker for evaluating the efficacy of colorectal cancer patients after astragalus polysaccharide intervention therapy and its application. The present invention first discovered that l ncRNA-CERNA3-201 can be used as an independent marker for predicting the efficacy of colorectal cancer patients after astragalus polysaccharide intervention therapy, and can be used as a drug development target to improve the therapeutic effect of colorectal cancer patients.

Description

Technical Field

[0001] The present invention relates to the technical field of colorectal cancer efficacy evaluation, and in particular to a marker for evaluating the efficacy of colorectal cancer patients after astragalus polysaccharide intervention therapy and an application thereof. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors. The latest epidemiological data show that in 2020, there were more than 1.9 million new cases of colorectal cancer and more than 900,000 deaths, accounting for about 10% of the total cancer cases and deaths. Its incidence rate ranked third and its mortality rate ranked second, and the incidence and mortality rates are increasing year by year.

[0003] At present, the main treatment for colorectal cancer is multidisciplinary combined treatment with surgery and radiotherapy and chemotherapy as the main treatment, especially the application of traditional Chinese medicine has gradually gained attention. The ultimate target of anti-tumor treatment and research is to inhibit tumor cell proliferation and induce tumor cell death. The Chinese medicine Astragalus takes "strengthening the body, replenishing the middle and replenishing qi" as the treatment principle, and is the main medicine in a variety of anti-tumor drug compounds. Astragalus polysaccharide (APS) is the main active ingredient of the Chinese medicine Astragalus. The applicant's previous research showed that Astragalus polysaccharide can significantly inhibit the growth and invasion of colorectal cancer ectopic transplanted tumors, inhibit tumor cell growth and proliferation, and induce tumor cell death in tumor tissue. However, how to monitor the effect of Astragalus polysaccharide intervention treatment is a current technical difficulty. Therefore, the purpose of the present invention is to provide a new method for predicting the prognosis of colorectal cancer patients using Astragalus polysaccharide intervention treatment by studying the prognosis-related biomarkers of colorectal cancer patients using Astragalus polysaccharide intervention treatment, which is conducive to selecting individualized treatment plans based on the differences between patients and improving the effect of traditional Chinese medicine treatment of colorectal cancer. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a marker for evaluating the efficacy of colorectal cancer patients after astragalus polysaccharide intervention therapy and its application. The present invention first discovered that lncRNA-CERNA3-201 can be used as an independent marker for predicting the efficacy of colorectal cancer patients after astragalus polysaccharide intervention therapy, and can be used as a drug development target to improve the treatment effect of colorectal cancer patients.

[0005] One of the purposes of the present invention is to provide a biomarker for evaluating the efficacy of astragalus polysaccharide intervention therapy in patients with colorectal cancer, characterized in that the biomarker is l ncRNA-CERNA3-201, and the sequence is shown in SEQ ID NO.6.

[0006] Preferably, the present invention also provides the use of a reagent for detecting l ncRNA-CERNA3-201 in the preparation of a product for evaluating the efficacy of astragalus polysaccharide intervention therapy in patients with colorectal cancer.

[0007] Further preferably, the product is in the form of a kit.

[0008] Further preferably, the reagent is a primer or probe for quantitatively detecting l ncRNA-CERNA3-201.

[0009] Further preferably, the primers are shown as SEQ ID NOs. 1 and 2.

[0010] Preferably, the present invention also provides a method for screening drugs for treating colorectal cancer, characterized in that the method uses l ncRNA-CERNA3-201 as a therapeutic target and screens agents that can inhibit its expression as potential drugs for treating colorectal cancer.

[0011] Further preferably, the agent includes but is not limited to si RNA.

[0012] More preferably, the sequence of the si RNA is as shown in SEQ ID NO.5.

[0013] Preferably, the present invention also provides a si RNA for treating colorectal cancer, characterized in that the sequence of the si RNA is as shown in SEQ ID NO.5.

[0014] Preferably, the present invention also provides the use of an agent for targeted inhibition of l ncRNA-CERNA3-201 in the preparation of a drug for preventing or treating colorectal cancer.

[0015] Preferably, the agent that targets and inhibits l ncRNA-CERNA3-201 includes but is not limited to si RNA.

[0016] More preferably, the sequence of the si RNA is as shown in SEQ ID NO.5.

[0017] The advantages of the present invention are as follows: The present invention first discovered that l ncRNA-CERNA3-201 can be used as an independent marker for predicting the efficacy of astragalus polysaccharide intervention treatment in patients with colorectal cancer, and can be used as a target for drug development to improve the therapeutic effect of patients with colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .l Expression of ncRNA-CERNA3-201 in patients with colorectal cancer treated with astragalus polysaccharide intervention;

[0019] Figure 2 .Prognostic analysis of patients with colorectal cancer treated with Astragalus polysaccharide intervention;

[0020] Figure 3 . Analysis of l ncRNA-CERNA3-201 expression in colorectal cancer cells after siRNA treatment;

[0021] Figure 4 .Analysis of the proliferation ability of colorectal cancer cells after siRNA treatment. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0023] Example 1

[0024] 1. Preparation and processing of experimental materials:

[0025] A total of 100 patients with primary colorectal cancer confirmed by colonoscopy and pathology were collected from the Second Department of General Surgery (General Laparoscopic Minimally Invasive Surgery) of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine. They were divided into an experimental group (Arastragalus polysaccharide) and a control group according to the principle of random control, with 50 cases in each group. In addition, 30 healthy volunteers were randomly selected as the healthy group. There were no statistical differences in age and gender between the normal control groups and the colorectal cancer groups.

[0026] Among them, the experimental group (astragalus polysaccharide): patients with primary colorectal cancer confirmed by colonoscopy and pathology, intravenous infusion of astragalus polysaccharide (Bern 250mg, national medicine standard Z20040086, Tianjin Sino Pharmaceutical Co., Ltd.) every day within 1 week from the date of diagnosis, 10ml of normal saline is drawn with a syringe and added to the vial, and immediately shaken until the drug is completely dissolved, and then added to 500mL of 0.9% sodium chloride injection or 5-10% glucose injection, and the infusion time is not less than 2.5 hours. 250mg once, once a day. The course of treatment is 7 days. The control group: patients with primary colorectal cancer confirmed by colonoscopy and pathology, no injection of astragalus polysaccharide is given after diagnosis. The healthy group: healthy volunteers.

[0027] 2. Test methods

[0028] qRT-PCR technology was used to detect the expression of lncRNA-CERNA3-201. Peripheral blood samples from each treatment group were collected and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScript First-Strand cDNA Synthesis Kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. cDNA was amplified using SYBR Premix Ex Taq (Takara Biotechnology Co.) in the ABI 7900 Fast System (Thermo Fisher Scientific). The expression of lncRNA-CERNA3-201 in colorectal cancer tissues was calculated using the standardized 2-ΔΔCt method, and all reactions were performed three times independently.

[0029] Among them, l ncRNA-CERNA3-201 real-time fluorescence quantitative PCR specific primers

[0030] Upstream primer: 5'-TAGTGGAGTTGGCGGGAAAG-3' (SEQ ID NO. 1);

[0031] Downstream primer: 5'-ACCAGAGTTCGTGTGCACTT-3' (SEQ ID NO. 2);

[0032] Internal reference GAPDH real-time fluorescence quantitative PCR

[0033] Upstream primer: 5′-GTCACCAGGGCTGC-3′ (SEQ ID NO. 3);

[0034] Downstream primer: 5′-TTCCCGTTCTCAGCC-3′ (SEQ ID NO. 4).

[0035] The real-time fluorescence quantitative PCR reaction system includes: 6 μL 10× PCR Buffer, 2 μL SYBR Green I fluorescent dye, 2 μL dNTP, 22 μl MgCl, 1 μL each of upstream and downstream primers, 2 μL Taq DNA polymerase and 2 μL cDNA;

[0036] The real-time fluorescence quantitative PCR reaction program included: pre-denaturation at 95°C for 5 min; 95°C for 50 s, 56°C for 60 s, and extension at 72°C for 50 s, for a total of 40 cycles; extension at 72°C for 5 min;

[0037] The relative expression of ncRNA-CERNA3-201 was calculated according to the formula 2-ΔΔCt, and the results are shown in Figure 1 .Depend on Figure 1 It can be seen that compared with the healthy population, the expression of l ncRNA-CERNA3-201 in the blood samples of the experimental group patients was not significantly different from that of the healthy population, while compared with the control group, the expression of l ncRNA-CERNA3-201 in the blood of the experimental group patients was significantly decreased, and the difference was significant. This result suggests that l ncRNA-CERNA3-201 can be used as a prognostic indicator for the intervention treatment of colorectal cancer patients with astragalus polysaccharide.

[0038] Example 2

[0039] Patients in the experimental group and control group of Example 1 were followed up and monitored. Survival curves were drawn to show that l ncRNA-CERNA3-201 was significantly correlated with the prognosis of patients with colorectal cancer treated with astragalus polysaccharide intervention (p<0.05), and patients with low expression of l ncRNA-AB007962 had a longer survival period. This result confirms that l ncRNA-CERNA3-201 is closely related to the prognosis of patients with colorectal cancer treated with astragalus polysaccharide intervention, and can be used as an independent predictive factor.

[0040] Example 3

[0041] Artificially synthesize si RNA targeting and inhibiting l ncRNA-CERNA3-201, whose sequence is 5′-UCCAGAAGGACGAGAAGCCA-3′ (SEQ ID NO.5). Human colorectal cancer cell SW480 cells were cultured, and the cells after 3 generations of culture were used for experiments. Among them, the experimental group was intervened with 10μg / mL si RNA, the control group was not intervened, and the blank group was DMEM culture medium. qPCR was used to analyze the cell expression of l ncRNA-CERNA3-201 and CCK8 was used to detect changes in cell proliferation.

[0042] The results are as follows Figure 3 and 4 As shown in the figure, after siRNA treatment, the expression of l ncRNA-CERNA3-201 in the cells decreased significantly, and the proliferation level of SW480 cells in the experimental group decreased significantly, while the l ncRNA-CERNA3-201 in the control group still maintained a high level and maintained a high proliferation activity. The above results further confirmed that l ncRNA-CERNA3-201 is an independent factor involved in the prognosis of colorectal cancer, and the prognosis effect can be improved by correcting the expression level in colorectal cancer.

[0043] In summary, the inventors can conclude that astragalus polysaccharides regulate the prognosis of colorectal cancer patients by targeted inhibition of l ncRNA-CERNA3-201, the prognosis of patients can be predicted by detecting the expression level of l ncRNA-CERNA3-201 in patients, and the treatment effect of colorectal cancer patients can be further improved by developing reagents that inhibit the expression of l ncRNA-CERNA3-201.

[0044] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Use of a reagent for detecting the expression of lncRNA-CERNA3-201 in the preparation of a product for evaluating the efficacy of astragalus polysaccharide intervention therapy in patients with colorectal cancer, characterized in that: The sequence of lncRNA-CERNA3-201 is shown as SEQ ID NO.

6.

2. The use as claimed in claim 1, characterized in that The product is in the form of a kit.

3. The use as claimed in claim 1, characterized in that The reagent is a primer or probe for quantitatively detecting lncRNA-CERNA3-201.

4. The use as claimed in claim 3, characterized in that The primers are shown in SEQ ID NOs. 1 and 2.

5. A siRNA for treating colorectal cancer, characterized in that: The sequence of the siRNA is shown in SEQ ID NO.

5.

6. Use of an agent for targeted inhibition of lncRNA-CERNA3-201 in the preparation of a drug for preventing or treating colorectal cancer, characterized in that: The reagent for targeted inhibition of lncRNA-CERNA3-201 is siRNA, the sequence of the siRNA is shown in SEQ ID NO.5, and the sequence of lncRNA-CERNA3-201 is shown in SEQ ID NO.6.

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