CN108998519A - Predict the biomarker long-chain non-coding RNA LINC02413 and kit of colon cancer prognosis - Google Patents

Abstract

The invention provides a biomarker long-chain non-coding RNA LINC02413 and a kit for predicting the prognosis of colon cancer, which are used to predict the prognosis and survival of colon cancer patients. The preparation for predicting the prognosis of colon cancer is a real-time fluorescent quantitative PCR detection kit. The real-time fluorescent quantitative PCR detection kit contains specific primers for detecting the expression of the target gene LINC02413 and specific primers for the expression of the internal reference gene TUBA1A, and also includes real-time fluorescent quantitative SYBR Dye, RNase-free water. The present invention finds that the LINC02413 derived from colon cancer tissue is correlated with the survival rate of patients through fluorescent quantitative PCR and survival curve analysis, and the higher the content, the higher the survival rate. This method provides strong technical support for predicting the prognosis and survival of colon cancer patients, and is helpful to improve the postoperative life quality of colon cancer patients, formulate postoperative treatment plans, and improve survival rate, which has far-reaching clinical significance.

Description

Long non-coding RNA LINC02413 and biomarkers predicting the prognosis of colon cancer Reagent test kit

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a biomarker long-chain non-coding RNA LINC02413 and a kit for predicting the prognosis of colon cancer.

Background technique

Colon cancer refers to the malignant lesion of the colon mucosal epithelium under the action of various carcinogenic factors such as environment or heredity. It is one of the common malignant tumors. Its morbidity is high, and its morbidity and mortality are still on the rise. So far, the early detection, early diagnosis and early treatment of colon cancer are far from satisfactory. Because there are no obvious and specific early clinical symptoms, it is not easy to be found, so the situation of misdiagnosis and missed diagnosis is very serious. It takes a long time for most patients to be diagnosed correctly, so that quite a few patients lose the chance of cure. Colorectal cancer ranks third in the incidence of tumors, ranks second in the incidence of all tumors in urban areas, and ranks fourth in the mortality rate of all malignant tumors. At present, surgical resection is still the main treatment for the long-term survival of colon cancer patients, but most of them still cannot escape the fate of recurrence and metastasis. Tumor recurrence and metastasis are still the main cause of death of patients. At present, there is no ideal molecular index to evaluate the prognosis and the risk of metastasis clinically. Therefore, finding new prognostic biomarkers is an urgent problem to be solved.

Noncoding RNA refers to the RNA in the genome of an organism that does not code for protein. According to the length of the transcript, non-coding RNA can be roughly divided into two categories: one is small molecule non-coding RNA with a length of less than 200 nucleotides. At present, there are many researches on microRNAs, small interfering RNAs, and RNAs interacting with piwi proteins (piRNAs), which mediate gene silencing, participate in RNA splicing and protein modification, and finely regulate gene transcription and translation in vivo; The other category is long non-coding RNAs larger than 200nt, which account for a considerable proportion of non-coding RNAs. Long non-coding RNAs were previously considered to be the transcriptional noise of the genome. In recent years, studies have shown that they play an important role in chromatin remodeling, transcriptional regulation, post-transcriptional processing, and protein metabolism. Long-chain non-coding RNA classification Long-chain non-coding RNAs are a type of functional RNA with generally low conservation. A large number of studies have shown that the expression of long non-coding RNAs in the genome has obvious spatiotemporal specificity and tissue specificity, and its content increases with the increase of species complexity. The broad functional composition of long noncoding RNAs in the genome includes a series of roles in chromosome dynamics, telomeres, and subcellular structural organization. Studies have shown that long non-coding RNAs are widely involved in many basic processes of gene regulation, including chromatin modification, transcriptional regulation, post-transcriptional regulation and other regulatory processes.

LINC02413 is a newly identified non-coding RNA with a transcript length greater than 200 nucleotides. At present, the important role of LINC02413 in the process of normal cell development or tumor development is still under further study. Whether LINC02413 can be used as a new tumor marker to predict the prognosis of patients with colon cancer has not been reported yet. Therefore, for the first time, we elucidated the feasibility of using LINC02413 as a novel colon cancer marker to predict patient prognosis.

Contents of the invention

The purpose of the present invention is to provide a biomarker long-chain non-coding RNA LINC02413 and a kit for predicting the prognosis of colon cancer, which are used to predict the prognosis and survival of colon cancer patients.

To achieve the above purpose, the present invention adopts the following technical solution: the biomarker for predicting the prognosis of colon cancer is long-chain non-coding RNA LINC02413, and the nucleic acid sequence of the long-chain non-coding RNA LINC02413 is shown in SEQ NO:1.

Application of long non-coding RNA LINC02413 biomarker in the preparation of preparations for predicting the prognosis of colon cancer.

The preparation for predicting the prognosis of colon cancer is a real-time fluorescent quantitative PCR detection kit.

The real-time fluorescent quantitative PCR detection kit for predicting the prognosis of colon cancer includes specific primers for detecting the expression of the target gene LINC02413, and the primer sequences are SEQ NO:2 and SEQ NO:3.

The real-time fluorescent quantitative PCR detection kit for predicting the prognosis of colon cancer comprises specific primers for the expression of the internal reference gene TUBA1A, and the primer sequences are SEQ NO:4 and SEQ NO:5.

The real-time fluorescent quantitative PCR detection kit for predicting the prognosis of colon cancer contains real-time fluorescent quantitative SYBR dye, RNase-free water.

The volume ratio of real-time fluorescent quantitative SYBR dye, target gene LINC02413 specific primer, internal reference gene TUBA1A specific primer, RNase-free water in the real-time fluorescent quantitative PCR detection kit for predicting the prognosis of colon cancer is 10:1:1: 6.

The beneficial effect of the present invention is that: the present invention finds that LINC02413 derived from colon cancer tissue is related to the survival rate of patients through fluorescence quantitative PCR and survival curve analysis, and the higher the content, the higher the survival rate. This method provides strong technical support for predicting the prognosis and survival of colon cancer patients, and is helpful to improve the postoperative life quality of colon cancer patients, formulate postoperative treatment plans, and improve survival rate, which has far-reaching clinical significance.

Description of drawings

Figure 1 shows the real-time fluorescent quantitative PCR analysis of the expression difference of LINC02413 in normal tissues and colon cancer.

Figure 2 is a survival curve analysis of the effect of the expression level of LINC02413 derived from colon cancer tissue on the prognosis of colon cancer patients.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Example 1

The biomarker used to predict the prognosis of colon cancer is long-chain non-coding RNA LINC02413, and the nucleic acid sequence of the long-chain non-coding RNA LINC02413 is shown in SEQ NO:1.

The preparation for predicting the prognosis of colon cancer is a real-time fluorescent quantitative PCR detection kit, a real-time fluorescent quantitative PCR detection kit, including specific primers for detecting the expression of the target gene LINC02413, and the primer sequences are shown in SEQ NO:2 and SEQNO:3, The real-time fluorescent quantitative PCR detection kit contains specific primers for the expression of the internal reference gene TUBA1A, and the primer sequences are shown in SEQ NO:4 and SEQ NO:5.

Preparation of reagents for detecting the expression of LINC02413 is used to prepare a kit for the prognosis of patients with colon cancer (for 50 reactions), and the required reagents include: SYBR Green qPCR Mix 500 μL, 3 μM target gene LINC02413-specific primer 50 μL, 3 μM internal reference gene TUBA1A 50 μL of specific primers, 300 μL of RNase-free water.

The reagents used to extract total RNA from colon cancer tumors or normal control tissues include: Trizol 120mL, RNA degradation inhibiting solvent 40mL, chloroform 80mL, isopropanol 80mL, DEPC water 10mL.

Reagents for reverse transcription of LINC02413 into cDNA using total RNA as a template, including: 100 μL of a mixture of 10× random primers and OligodT primers, 150 μL of 5× reverse transcription reaction buffer, 10 mM deoxynucleotide triphosphate bases containing Mg ²⁺ dNTP 100 μL, 200 U/μL M-MLV reverse transcriptase 50 μL.

Example 2

Detection of LINC02413 in Tissue Samples

1. Collect the colon cancer tumor tissue or normal control tissue to be tested, wash it with normal saline, put it into a cryopreservation tube filled with a solvent that inhibits RNA degradation, and put it in a -80°C refrigerator for later use.

2. Extraction of RNA from tissues

(1) Add liquid nitrogen to the mortar first, then cut the tissue into small pieces and grind it into powder in liquid nitrogen, take 100mg of tissue powder with a liquid nitrogen pre-cooled spoon and add it to the EP tube filled with 1ml of Trizol solution middle. The total volume of tissue powder should not exceed 10% of the volume of Trizol used, and it should be thoroughly mixed evenly.

(2) Leave at room temperature for 5 minutes, then add 200 μL of chloroform, tightly cap the EP tube and shake vigorously for 0.5 minutes. Centrifuge at 12,000 rpm for 10 minutes.

(3) Take the upper aqueous phase into a new EP tube, add 500 μL of isopropanol, and gently invert to mix. Place at room temperature for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes.

(4) Carefully discard the supernatant, add 1ml of 75% ethanol, vortex, and centrifuge at 12000 rpm for 5 minutes at 4°C. Repeat the operation once.

(5) Discard the supernatant, and dry at room temperature or in vacuum for 5-10 minutes. Dissolve the RNA with 50 μL of DEPC-treated water.

(6) Measurement of RNA concentration: measure with a nucleic acid concentration measuring instrument, add 1 μL of RNA sample to the sample hole, and directly determine the concentration of RNA according to the reading. The OD260/OD280 ratio measured by the nucleic acid concentration analyzer, if the ratio is between 1.8-2.0, the RNA purity is considered to be very good. Finally, the RNA was stored in a -80°C refrigerator for future use.

3. Reverse transcription of LINC02413 RNA

The reverse transcription kit (D7170S) from Shanghai Beyontien Biotechnology Co., Ltd. was used. Specific steps are as follows:

(1) Genomic DNA removal: Thaw template Total RNA, 5×gDNA Eraser Buffer on ice, thaw 5×RTBuffer, 10×RT Primer Mix, DEPC-treated Water at room temperature (15-25°C), and place immediately after thawing on ice. Mix each solution well and centrifuge briefly before use to allow all liquid to settle to the bottom of the tube. For the denaturation of RNA, heat denature the RNA sample at 65°C for 5 minutes, and immediately place it in ice water to cool. Prepare the reaction mixture on ice according to the components in the table below, then dispense into each reaction tube, and finally add the RNA sample. The reaction system and conditions are shown in Table 1.

Table 1

(2) Incubate at 37°C for 2 minutes on a PCR machine or in a water bath. Quickly place on ice for later use.

(3) Reverse transcription system preparation: Prepare the reaction solution on ice, and perform reverse transcription reaction immediately after mixing gently. Prepare the mixed solution according to the reverse transcription reaction system in the table below. The reaction system and conditions are shown in Table 2.

Table 2

(4) Incubate at 42°C for 60 minutes to carry out the reverse transcription reaction, then incubate at 80°C for 10 minutes to inactivate the reverse transcriptase and put it on ice. For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance the efficiency of reverse transcription.

(5) The obtained cDNA can be used immediately or frozen at -80°C for subsequent real-time fluorescent quantitative PCR. It is advisable to avoid excessive repeated freezing and thawing of the cDNA.

4. Real-time quantitative PCR using specific primers of LINC02413

Specific primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., including specific primers for detecting the expression of LINC02413, the primer sequences are SEQ NO: 2 and SEQ NO: 3, and specific primers for the expression of the internal reference gene TUBA1A, The primer sequences are SEQ NO:4 and SEQ NO:5. Other reagents for real-time quantitative PCR use BeyoFast™ SYBR Green qPCR Mix (2×) from Shanghai Beyontien Biotechnology Co., Ltd. The specific steps are as follows:

(1) Thaw and mix the various solutions required for the PCR reaction. BeyoFast™ SYBR Green qPCR Mix is completely thawed and mixed and placed in an ice box.

(2) Set up a PCR reaction system (taking a 96-well plate as an example) on an ice bath. The reaction system and conditions are shown in Table 3.

table 3

(3) Generally, the amount of DNA template is 1-10ng cDNA as a reference amount, and good detection effect can be obtained when the final concentration of primers is 0.2-0.5μM, and the final concentration of primers can also be adjusted according to the situation. If necessary, serially dilute the template to determine the optimal amount of template to use. When the cDNA obtained from the reverse transcription PCR reaction is directly used as a template, the amount added should not exceed 10% of the total volume of the PCR reaction. The recommended reaction system for a 96-well plate is 20 μl, and the reaction system can also be scaled up or down according to actual experimental needs.

(4) Gently pipette to mix or slightly Vortex to mix, and centrifuge at room temperature for a few seconds to make the liquid accumulate at the bottom of the tube.

(5) Place the set PCR reaction tube or PCR reaction plate on the fluorescent quantitative PCR instrument to start the PCR reaction.

(6) PCR reaction program: pre-denature the template before the real-time fluorescent quantitative PCR reaction, usually set at 95° C. for 2 minutes. The following PCR program is used. This program is based on ABI 7900HT fluorescent quantitative PCR instrument as an example: a. Pre-denaturation: 95°C for 2min; b. Denaturation: 95°C for 15sec; c. Annealing/extension: 60°C for 15-30sec; d. Repeat steps b and c for a total of 40 cycles; e. Melting curve analysis (optional): 95°C 15sec, 60°C 15sec, 95°C 15sec; f. Use the software provided by the fluorescent quantitative PCR instrument to analyze the results. The three-step method only needs to add a step of 72°C for 30 sec after annealing/extension, and then repeat steps b, c and this additional step for a total of 40 cycles.

5. Data analysis of LINC02413 expression level

The experimental data included 60 cases of colon cancer patients and their normal controls. The results of real-time quantitative PCR were analyzed by relative quantitative method, that is, 2^ ^-△△Ct method. The details are as follows: First, sort out the Ct values of all genes in an experiment, and then subtract the Ct value of the internal reference gene TUBA1A from the Ct value of the target gene LINC02413 in each group of tumor samples, and the obtained number is △Ct; The formula is: △Ct=Ct (target gene LINC02413)-Ct (internal reference gene TUBA1A); then, calculate the △Ct of each target gene LINC02413 in each group of tumor samples. Subtract the △Ct of the normal control tissue group from the △Ct of the tumor tissue sample in this experiment, and take the inverse of all the results at the same time. The result of this step is -△△Ct. Finally, perform a power of 2 operation on -△△Ct, that is, 2^ ^-△△Ct to get the fold change of the expression amount. Statistical analysis was performed using a nonparametric t-test in triplicate. Results As shown in Figure 1, the expression level of LINC02413 in colon cancer tumor tissue was higher than that in normal control tissue, and the difference was statistically significant ( p <0.05).

Through the follow-up statistical data of 60 colon cancer patients included in the above experiment, including the time of first onset, treatment, recurrence and death time, etc., the follow-up time is at least 12 months. In the selected patients with colon cancer, the expression value of the fluorescent real-time quantitative PCR analysis was selected as a reference standard, and the obtained results were compared with the corresponding normal tissues. The results are shown in FIG. 2 . Patients with higher expression of LINC02413 in tumor tissue than normal control tissue were defined as LINC02413 high expression group, and the rest were low expression group. Through Kaplan-Meier survival analysis, the survival time of patients with high expression of LINC02413 was significantly shorter than that of patients with low expression of LINC02413, and the prognosis was worse, the difference was statistically significant ( p <0.05). Therefore, LINC02413 can be used as a specific molecular marker for the prognosis of colon cancer patients.

The above description is a preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, the present invention is not limited by the above embodiments, and some changes can also be made without departing from the overall concept of the present invention. and improvements, these should also be considered as protection scope of the present invention.

SEQUENCE LISTING

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Claims (7)

1. The biomarker used to predict the prognosis of colon cancer is long-chain non-coding RNA LINC02413, and the nucleic acid sequence of the long-chain non-coding RNA LINC02413 is shown in SEQ NO:1. 2. The application of the biomarker as claimed in claim 1 in the preparation of preparations for predicting the prognosis of colon cancer. 3. The application according to claim 2, characterized in that: the preparation for predicting the prognosis of colon cancer is a real-time fluorescent quantitative PCR detection kit. 4. A real-time fluorescent quantitative PCR detection kit for predicting the prognosis of colon cancer, characterized in that: it contains specific primers for detecting the expression of the target gene LINC02413, and the primer sequences are SEQ NO:2 and SEQ NO:3. 5. the real-time fluorescent quantitative PCR detection kit for predicting colon cancer prognosis as claimed in claim 4, is characterized in that: described real-time fluorescent quantitative PCR detection kit comprises the specific primer that internal reference gene TUBA1A expresses, and primer sequence is SEQ NO:4 and SEQ NO:5. 6. The real-time fluorescent quantitative PCR detection kit for predicting the prognosis of colon cancer as claimed in claim 5, characterized in that: said kit comprises real-time fluorescent quantitative SYBR dye and RNase-free water. 7. the real-time fluorescent quantitative PCR detection kit for predicting colon cancer prognosis as claimed in claim 6, is characterized in that: in described kit, real-time fluorescent quantitative SYBR dye, target gene LINC02413 specific primer, internal reference gene TUBA1A The volume ratio of specific primers to RNase-free water is 10:1:1:6.