CN106047875B - LncRNA molecules and its application in nasopharyngeal carcinoma auxiliary diagnosis - Google Patents

Abstract

The invention discloses a kind of lncRNA molecules and its applications in nasopharyngeal carcinoma auxiliary diagnosis.The lncRNA molecular names are TCONS_00003617, and nucleotide sequence is as shown in SEQ ID NO.1.Expression quantity of the TCONS_00003617 in tissues of nasopharyngeal carcinoma is apparently higher than cancer beside organism, it can be used as nasopharyngeal carcinoma molecular marker or target spot, applied to the early diagnosis of nasopharyngeal carcinoma clinic, Index for diagnosis or targeted therapy, and be conducive to further elucidate the occurrence and development mechanism of nasopharyngeal carcinoma, signal path etc., great application prospect and theory value.

Description

lncRNA molecules and their application in auxiliary diagnosis of nasopharyngeal carcinoma

technical field

The invention belongs to the fields of molecular biology and oncology, and specifically relates to an lncRNA molecule and its application in clinical diagnosis, treatment, prognosis evaluation or scientific research of nasopharyngeal carcinoma.

Background technique

Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) is one of the most common malignant tumors in southern China. It has the highest incidence rate in southern China, especially in Guangdong Province, ranking first in the world, so it is also called "Guangdong cancer". NPC is sensitive to radiation, and radiotherapy is the first choice, and its prognosis varies greatly depending on the stage of the disease. Radiotherapy alone is effective for stage I and stage II patients, and the 10-year disease-related survival rate, recurrence-free survival rate, and distant metastasis-free survival rate are all high. However, the onset of NPC is insidious, and the early symptoms lack specificity. About 75% of the patients are already in stages III and IV when they are diagnosed. The efficacy of advanced NPC is still quite disappointing, prone to recurrence and distant metastasis, and the 5-year survival rate is only 35%. Therefore, early detection and early treatment are the key to improving the survival rate of patients with nasopharyngeal carcinoma. The occurrence and development of nasopharyngeal carcinoma is a dynamic process involving multiple factors and multiple genes. Therefore, it is necessary to explore the molecular mechanism of its dynamic development and look for molecular markers related to nasopharyngeal carcinoma, so as to further clarify its pathogenesis and develop a drug that can be applied to Reagents or products for clinical early diagnosis, treatment and prognosis evaluation are of great significance to improving the diagnosis and treatment level of nasopharyngeal carcinoma and reducing the recurrence and metastasis rate.

There are a large number of transcribed long non-coding RNA (Long Noncoding RNA, IncRNA) in the human genome, which is a type of functional RNA molecule with a transcript length of more than 200nt nucleotide units. Studies have found that lncRNA is involved in many important regulatory processes such as genomic imprinting and chromatin modification, transcriptional activation, transcriptional interference, and intranuclear transport, and also plays an important regulatory role in carcinogenesis and tumor suppressor pathways. new hotspot. LncRNA has been shown to be involved in the occurrence and development of many tumors, however, there are only a few reports on nasopharyngeal carcinoma.

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide an lncRNA molecule and its application in auxiliary diagnosis of nasopharyngeal carcinoma.

Another object of the present invention is to provide the application of the lncRNA molecules mentioned above in the treatment or prognosis assessment of nasopharyngeal carcinoma.

Another object of the present invention is to provide the application of the above-mentioned lncRNA molecules in the preparation of drugs for the prevention or treatment of nasopharyngeal carcinoma.

The technical scheme that the present invention takes is:

An lncRNA molecule highly expressed in nasopharyngeal carcinoma tissue is named TCONS_00003617, and its nucleotide sequence is shown in SEQ ID NO.1.

Application of lncRNA molecule TCONS_00003617 in auxiliary diagnosis of nasopharyngeal carcinoma or evaluation of treatment/prognosis of nasopharyngeal carcinoma.

Application of lncRNA molecule TCONS_00003617 in preparation of nasopharyngeal carcinoma auxiliary diagnosis or nasopharyngeal carcinoma treatment/prognosis assessment reagent or kit.

Application of the reagent for detecting lncRNA molecule TCONS_00003617 in the preparation of nasopharyngeal carcinoma auxiliary diagnosis or nasopharyngeal carcinoma treatment/prognosis assessment reagent or kit.

The reagent for detecting the lncRNA molecule of claim 1 comprises a pair of specific primers for amplifying the lncRNA molecule of claim 1.

The specific primer pair for amplifying the lncRNA molecule described in claim 1, the sequence is as follows:

F: 5'-CCGCCAGTATCTGATGAAGG-3' (SEQ ID NO.4);

R: 5'-TGGGAGATGGGAAAGTAGCC-3' (SEQ ID NO. 5).

Application of lncRNA molecule TCONS_00003617 as a therapeutic target for nasopharyngeal carcinoma.

Application of the lncRNA molecule TCONS_00003617 in the preparation of drugs for the prevention or treatment of nasopharyngeal carcinoma.

A kit for auxiliary diagnosis or prognosis assessment of nasopharyngeal carcinoma, comprising:

(1) A pair of specific primers for amplifying the lncRNA molecule TCONS_00003617;

(2) standard DNA template;

(3) PCR reaction solution.

A method for detecting lncRNA, comprising the steps of:

(1) Extract the total RNA of the sample;

(2) Prepare sample Cdna;

(3) Quantitative amplification of lncRNA;

The lncRNA is TCONS_00003617.

The beneficial effects of the present invention are:

The present invention successfully screens out highly expressed lncRNA molecules in nasopharyngeal carcinoma tissues, which can be used as molecular markers or targets of nasopharyngeal carcinoma for early clinical diagnosis, prognosis judgment or targeted therapy of nasopharyngeal carcinoma, and is conducive to further elucidating nasopharyngeal carcinoma The mechanism of occurrence and development of cancer, signaling pathways, etc., have great application prospects and theoretical value.

Description of drawings

Figure 1 Denaturing agarose gel electrophoresis detection of total RNA in nasopharyngeal carcinoma and adjacent tissues;

Fig. 2 Microarray fluorescence scanning diagram;

Fig. 3 sample fluorescence intensity distribution diagram;

Fig. 4 chip hybridization signal intensity scatter diagram;

Fig. 5 Hierarchical cluster analysis diagram;

Figure 6 lncRNA in qRT-PCR: TCONS_00003617 corresponding melting curve and diffusion curve;

Figure 7 qRT-PCR detection of lncRNA: TCONS_00003617 expression increase multiple histogram;

Figure 8 Pie chart of the physical examination results of the non-cancer group;

Figure 9 Histogram of abnormal physical examination of patients in the non-cancer group;

The gender classification bar chart of Fig. 10 experimental group and control group;

The age classification bar chart of Fig. 11 experimental group and control group;

Figure 12 The denatured agarose gel electrophoresis diagram of total RNA in peripheral blood samples, wherein 41-49 are the electrophoresis results of RNA continuity and integrity, and 16/24/25/27 are the electrophoresis results of RNA degradation;

Figure 13 Amplification and melting curve graph of TCONS_00003617. The left figure is the amplification curve, and the right figure is the dissolution curve;

Fig. 14 Scatter plot of relative expression of TCONS_00003617 in experimental group and non-cancer group (P=0.538).

Detailed ways

The present invention will be further described below in conjunction with the examples, but not limited thereto. The molecular biology experimental techniques used in the following examples include PCR amplification, plasmid extraction, plasmid transformation, DNA fragment ligation, enzyme digestion, gel electrophoresis, etc., unless otherwise specified, generally operate according to conventional methods. For details, please refer to " Molecular Cloning Experiment Guide (Third Edition) (translated by Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitang, etc., 2002, Beijing: Science Press), or according to the conditions suggested by the manufacturer.

Example

1. Screening of lncRNA molecules related to nasopharyngeal carcinoma

1. Case collection

A total of 19 patients who were hospitalized in the Nasopharyngeal Radiotherapy Department of the First People's Hospital of Foshan City, Guangdong Province from February 2014 to August 2014 were collected. All patients did not undergo radiotherapy and chemotherapy before operation, other systemic medical history was excluded, and the functions of vital organs were within the normal range. The age ranged from 21 to 61 years, with a median age of 45 years. The clinical data of the samples are shown in Table 1-1, and 6 cases were randomly selected. Used for microarray research, and the remaining 13 cases were used for further verification. Informed consent of the patients was obtained for the collection of specimens in the experiment, and the authorization was obtained from the Ethics Committee of the First People's Hospital of Foshan City, Guangdong Province.

Table 1-1 collects relevant clinical parameters of 19 cases of nasopharyngeal carcinoma

2. Sample collection

1) Firstly, prepare cryopreservation tubes for tissue preservation, and use a black oily marker to mark the sample grouping, serial number, collection date and other information on the cryopreservation tubes;

2) Nasopharyngeal carcinoma and paracancerous tissue with forceps for outpatient biopsy (operated by a nasal endoscopy technician with more than 5 years of operating experience);

3) Divide the specimens of cancer and paracancerous tissues taken by forceps into 2 parts, one of which is sent for histopathological identification, and the other is placed in a prepared cryopreservation tube and tightened, and quickly put into a liquid nitrogen transport tank for cooling. After being completely frozen, transfer to a -80°C refrigerator for storage;

4) Fill in the specimen registration form, specifying the sample number, grouping, source patient name, medical record number, sample collection date and sample processing process, etc.;

5) After the pathological properties are confirmed, the specimens are taken out for further research.

3. Total RNA sample preparation, purification and quality inspection

(1) TRIZOL method to extract RNA samples;

(2) Purify the total RNA sample by using RNeasy column;

(3) RNA quantification and quality control (Nanodrop ND 1000 UV-Vis spectrophotometer and gel electrophoresis were used to determine the concentration and quality of total RNA in the sample).

The results of total RNA determination of nasopharyngeal carcinoma and adjacent tissues by Nanodrop spectrophotometer are shown in Table 1-2. The denatured agarose gel electrophoresis images of total RNA in nasopharyngeal carcinoma and paired paracancerous tissues are shown in Figure 1. The bands of 18S rRNA and 28S rRNA are clear, and the ratio of the brightness of the two bands is about 1:2, indicating that the extracted Tissue total RNA has good integrity, no degradation, no pollution, high purity and reliable quality.

Table 1-2 Determination of total RNA in nasopharyngeal carcinoma and adjacent tissues by Nanodrop spectrophotometer

*For a spectrophotometer, the O.D.A260/A280 ratio should be close to 2.0 for pure RNA (ratio between 1.8 and 2.1 is acceptable). The O.D.A260/A230 ratio should be greater than 1.8.

4. Labeling reaction

The labeling reaction used Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-0442) kit, and the operation steps were carried out according to the kit instructions.

5. Purification of labeled/amplified RNA and quality control of labeled cRNA

Labeled/amplified RNA was purified using the RNeasy Mini Kit (Qiagen p/n 74104). Take 1.5 μl of purified labeled cRNA and quantify it with NanoDrop ND-1000. The specific activity of labeled cRNA can be calculated by the following formula:

If the amount is less than 1.65 μg and the specific activity is less than 9.0 pmol Cy3 per μg cRNA, hybridization cannot be performed and cRNA is prepared again.

6. Chip hybridization

1) Add 500 μl of nuclease-free water to the 10X blocking reagent freeze-dried bottle, shake and mix;

2) Equilibrate in a water bath at 60°C. For each microarray experiment, add the following components in the order listed in the table below into a 1.5ml nuclease-free microcentrifuge tube, and mix thoroughly and gently on a vibrating mixer;

3) Incubate at 60°C for a full 30 minutes to fragment the RNA;

4) Add 55ul 2x GEx hybridization buffer HI-RPM to terminate the fragmentation reaction on the whole genome microarray chip Human LncRNA Array v3.0 (8x60K) of American Arraystar Company;

5) Carefully blow and suck to mix thoroughly, be careful not to cause air bubbles, do not mix on an oscillating mixer, mixing on an oscillating mixer will cause air bubbles;

6) Centrifuge at 13,000rpm for 1 minute at room temperature to separate the sample from the wall and cover and help reduce air bubbles. Put the sample on ice and load it to the chip as soon as possible;

7) Align the clean sealing gasket base with the mark facing upwards with the rectangular section of the hybridization chamber base and install it into the Agilent SureHyb hybridization chamber base to ensure that the sealing gasket base is completely flush with the hybridization chamber base and does not warp.

8) Slowly dispense 100 μl of hybridization samples into the sealed wells in a "drag while loading" manner, and slowly place the chip on the SureHyb sealing gasket base so that the "Agilent" label barcode faces down and the number barcode faces above, confirm this sandwich arrangement;

9) Cover the SureHyb hybridization chamber on the film base of the sandwich structure and slide the clips to assemble the two pieces, and manually fasten the clips to the hybridization chamber;

10) Vertically rotate the installed hybridization chamber to the sealing ring, estimate the activity of air bubbles, put the installed substrate hybridization chamber into the hybridization furnace oven, set to 65°C, set the hybridization rotator to 10rpm, 65 Hybridization at °C for 17 hours.

7. Wash the chip with Gene Expression Wash Buffer

8. Scan and extract data

1) Scanning operation steps:

① Install the chip on the chip holder;

② Put the loaded chip tray on the scanner conveyor belt;

③Confirm the scan settings for monochrome scanning:

④ Click Scan Slot m-n on the main window of the scan control, the letter m indicates the beginning slot where the first chip is located, and the letter n indicates the end slot where the last chip is located.

Figure 2 is a microarray fluorescence scanning image. Among them, 1cancer-6cancer is the fluorescence scanning image of nasopharyngeal carcinoma tissue, and 1pracancer-6pracancer is the scanning image of paracancerous tissue of nasopharyngeal carcinoma. All the hybridization points in the fluorescent scanning images are uniform, free from pollution, and the hybridization signal intensity is high, indicating that the experimental results meet the requirements.

Figure 3 is a box diagram of the fluorescence intensity distribution of the sample. It is a simple method to quickly and intuitively display the distribution of the data set. It is often used to compare the intensity distributions from all samples. The X-axis is the number of each microarray, and the Y-axis is the normalized fluorescence intensity. The fluorescence intensity of each microarray is basically the same, indicating that the experimental results meet the requirements.

Fig. 4 is a scatter diagram of chip hybridization signal intensity. A scatterplot between two compared samples or between two compared groups of samples is a visualization method for evaluating the expression changes of lncRNA genes. The X-axis and Y-axis values in the scatter plot are the normalized signal values of the samples. Green line Fold change line (default Fold change 2.0). The area outside the green line above and below the diagonal line of lncRNAs above indicates lncRNAs with a fold change of more than 2.0.

Figure 5 is a hierarchical cluster analysis diagram. Hierarchical clustering is one of the most widely used clustering methods to analyze lncRNA expression data. Cluster analysis arranges samples into groups based on their expression levels, thus allowing us to infer relationships between samples. Differences in long non-coding gene expression profiles between samples. It shows that most of the signals are abnormally expressed in nasopharyngeal carcinoma tissues.

In the end, a total of 6320 differentially expressed lncRNAs were screened by the chip, and 3 of them with up-regulated expression were selected for qRT-PCR verification. Seqname, GeneSymbol, and Fold change in Table 1-3 represent the sequence name, gene symbol, and fold change, respectively. Among them, the nucleic acid sequence of TCONS_00003617 is shown in SEQ ID NO.1. Its expression level in nasopharyngeal carcinoma tissues is significantly higher than that in paracancerous tissues, and it is very likely to become a molecular marker or target, and it can be used in the early clinical diagnosis of nasopharyngeal carcinoma It has broad application prospects in the direction of , prognosis judgment or targeted therapy.

Table 1-3 The up-regulation fold of differentially expressed LncRNA

9. Screen lncRNAs with large expression differences for qRT-PCR verification

(1) Primer design

According to the principle of primer design for real-time quantitative fluorescent PCR, primers for real-time quantitative fluorescent PCR were designed using Primer5.0 software. Among them, the upstream of LncRNA real-time quantitative fluorescent PCR primers is specific, and the downstream is random universal primers, and the upstream and downstream of LncRNA real-time quantitative PCR fluorescent primers are both specific, which need to be designed with software. The primers used in this study were all synthesized by Shanghai Kangcheng Biotechnology Co., Ltd., and GAPDH was the internal reference calibration gene.

The detailed sequences of the primers are shown in Table 1-4:

Table 1-4 Real-time quantitative fluorescent PCR primers

(2) Synthetic cDNA

(3) qRT-PCR detection

①Preparation of serially diluted DNA templates for drawing standard curves

For each gene and housekeeping gene that needs to be measured, select a cDNA template that expresses the gene for PCR (reaction system as shown in Table 1-5) reaction: flick the bottom of the tube to mix the solution, centrifuge briefly at 5000rpm, set the PCR reaction: 95 °C, 10 min; 40 PCR cycles (95 °C, 10 sec; 60 °C, 60 sec (collection of fluorescence)).

Table 1-5 PCR reaction system

The PCR product and 100bp DNA Ladder were electrophoresed on 2% agarose gel, stained with ethidium bromide, and detected whether the PCR product was a single specific amplification band.

Perform a 10-fold gradient dilution of the PCR product: set the concentration of the PCR product to 1, and dilute it to 1×10 ^-1 , 1×10 ^-2 , 1×10 ^-3 , 1×10 ^-4 , 1×10 ^-5 , 1×10 ^-6 , 1×10 ^-7 , 1×10 ^-8 , 1×10 ^-9 , these are several gradient concentrations of DNA.

②qRT-PCR reaction

All cDNA samples were configured in Realtime PCR reaction system. The system configuration is shown in Table 1-6, and the solution is mixed by flicking the bottom of the tube, and briefly centrifuged at 5000rpm. Loading:

a. Add 8ul of the mixture to each well of the 384-PCR plate;

b. Then add the corresponding 2μl cDNA;

c. Carefully stick on the Sealing Film, and briefly centrifuge to mix;

d. Place the prepared PCR plate on ice before setting up the PCR program.

Table 1-6 Realtime PCR reaction system

Place the above 384-PCR plate on the Realtime PCR instrument for PCR reaction, all the indicators are carried out according to the following procedures: 95°C, 10min; 40 PCR cycles (95°C, 10 seconds; 60°C, 60 seconds (collect fluorescence) ).

In order to establish the melting curve of the PCR product, after the amplification reaction is completed, press (95°C, 10 seconds; 60°C, 60 seconds; 95°C, 15 seconds); and slowly heat from 60°C to 99°C (automatically performed by the instrument - Ramp Rate is 0.05°C/sec).

(4) qRT-PCR results and calculations

The target gene and housekeeping gene of each sample were subjected to qRT-PCR reaction. According to the drawn serially diluted DNA standard curve, the concentration results of the target gene and housekeeping gene of each sample are directly generated by the machine. The concentration of the target gene in each sample is divided by the concentration of its housekeeping gene, which is the corrected relative content of the gene in this sample. Statistical software SPSS 17.0 was used for data analysis. 2- ^△△ CT is used to represent the relative expression change fold of lncRNA in each sample. The t-test of two independent samples was used when the data conformed to the normal distribution, and the rank sum test of two independent samples was used when the data did not conform to the normal distribution. P<0.05 was considered statistically significant.

qRT-PCR results are shown in Figure 6 and Figure 7. It can be seen from Figure 6 that the reaction process of the whole experiment process is good, there is no non-specific amplification product, and the qRT-PCR result is reliable. Compared with adjacent tissues, TCONS_00003617 was up-regulated in NPC tissues (P<0.05). The results showed that the qRT-PCR results were consistent with the chip detection results.

The histogram of differentially expressed lncRNA verified by Real-time PCR shows that, using GAPDH as a reference, the expression of TCONS_00003617 in cancer tissue was upregulated compared with that in the paracancerous group, which was consistent with the chip results.

Example 2

In order to further verify that lncRNA: TCONS_00003617 can be used as a molecular marker or target of nasopharyngeal carcinoma, the inventor further expanded the number of samples and conducted further experiments.

1. Case collection

From December 2012 to August 2015, the peripheral blood samples of nasopharyngeal cancer patients hospitalized in the Department of Head and Neck Radiotherapy, Foshan First People's Hospital were collected as the sample bank of the experimental group, while the non-cancer peripheral blood sample banks of the control group were collected from From September 2015 to November 2015, patients in the Health Care Department of Foshan First People's Hospital and inpatients in the Department of Otorhinolaryngology. The specimen collection in the trial obtained the informed consent of the patients and was authorized by the Ethics Committee of the First People's Hospital of Foshan City, Guangdong Province. All patients with nasopharyngeal carcinoma underwent physical examination, head and neck MRI (magnetic resonance imaging, MRI), whole body bone scanning, abdominal ultrasound, and chest X-ray before treatment.

Table 2-1 Overview of clinical data of patients with nasopharyngeal carcinoma

However, in the analysis of clinical data of 102 patients with nasopharyngeal carcinoma, it was found that 8 (7.8%) patients gave up treatment or refused follow-up after being diagnosed or adjuvant chemotherapy, 2 (2.0%) patients had a history of genetic diseases, and 9 ( 8.8%) patients had a history of hepatitis B virus infection, 14 (13.7%) had a recent infection history, and 18 (17.6%) patients had a history of hypertension or diabetes or coronary heart disease. According to the inclusion criteria of patients with nasopharyngeal carcinoma in the experimental group: age 18-70 years old, nasopharyngeal histopathologically confirmed nasopharyngeal carcinoma, no surgery or radiotherapy and chemotherapy before enrollment, excluding other systemic history, all vital organ functions are normal Range, KPS score (Karnofsky score, KPS) ≥ 80, and finally 58 (56.9%) met the inclusion criteria, excluding the influence of other systemic diseases. Therefore, 58 patients with nasopharyngeal carcinoma who met the inclusion criteria were selected for subsequent experimental analysis.

All 58 patients with nasopharyngeal carcinoma received radiotherapy, among which 55 (94.8%) received intensity-modulated radical radiotherapy, and 3 (5.1%) patients received highly palliative radiotherapy. The body position requirements and target delineation of radiotherapy follow the standardized operation of radiotherapy. Intensity-modulated radiation dose: primary tumor (GTVnx) 68-72Gy, cervical involved lymph node area (GTVnx) 68-70Gy, clinical target volume 1 (CTV-1) 60Gy, clinical target volume 2 (CTV-2) 54Gy, total The dose is divided into 30 times, once a day, 5 days a week. At the same time, patients with nasopharyngeal carcinoma also receive chemotherapy. The concurrent chemotherapy mainly uses cisplatin alone, and the induction or adjuvant chemotherapy mainly uses PF (cisplatin + 5-fluorouracil) or TPF (docetaxel + cisplatin + 5-fluorouracil). -fluorouracil) combined with chemotherapy. Of the 58 patients with nasopharyngeal carcinoma enrolled, 3 (5.2%) received palliative chemoradiotherapy, 4 (6.9%) received induction or adjuvant chemotherapy + radiotherapy, and 7 (12.1%) received only concurrent chemoradiotherapy , 44 (75.9%) cases received adjuvant chemotherapy + concurrent chemoradiotherapy.

In the peripheral blood specimen bank of non-cancer patients, a total of 78 patient specimens were included in the bank, including 41 males and 37 females, with an age range of 20-68 years old and a median age of 41 years old. (The clinical data are shown in Table 2-2).

Table 2-2 Clinical data of patients in the non-cancer group

Among the 78 non-cancer patients, thalassemia was found in 2 (2.6%) cases, coronary heart disease was confirmed in 3 (3.8%) cases, abnormal tumor indicators were found in 4 (5.1%) cases, blood was found in 8 (10.3%) cases. Abnormal tolerance or diagnosed diabetes, 9 (11.5%) cases were diagnosed with hypertension by physical examination, 11 (14.1%) cases were diagnosed with hepatitis B virus carrier, 18 (23.1%) cases had a history of infection within 1 week before blood drawing. Criteria: exclude any history of tumor disease, aged 18-70 years, all vital organ functions are within the normal range, exclude other medical history, and finally 29 (37.2%) patients have no obvious abnormalities in physical examination and meet the inclusion criteria, and follow-up trials (Figure 8, Figure 9).

The Chi-square test was used to analyze the differences in gender and age groups between the 58 cases in the experimental group and the 29 cases in the control group that met the inclusion criteria, and the differences were not statistically significant (Figure 10, Figure 11).

2. Sample collection

All peripheral blood samples were drawn from the patient on an empty stomach, with an amount of about 2ml, stored in a sterile test tube containing EDTA anticoagulant, the sample number was indicated on the tube, and pre-stored in a 4°C refrigerator. Test within hours. Informed consent of patients was obtained for specimen collection in the trial and authorization was obtained from the Ethics Committee of the First People's Hospital of Foshan City, Guangdong Province.

3. Total RNA sample preparation, purification and quality inspection

1) TRIZOL method to extract RNA samples;

2) Purify the total RNA sample with RNeasy column;

3) RNA quantification and quality control (Use Nanodrop ND 1000 UV-Vis spectrophotometer and gel electrophoresis to check the total RNA concentration and quality of the sample).

Table 3 shows the results of the total RNA determination of 87 samples from the nasopharyngeal carcinoma group and the non-cancer group by the Nanodrop spectrophotometer. In total RNA denaturing agarose gel electrophoresis, the bands of 28S and 18S ribosomal RNA are very bright and dense, and the density of the upper band is about twice that of the lower band, indicating that the quality and purity of the total RNA is better. If DNA contamination occurs during RNA preparation, it will appear above the 28S ribosomal RNA band, that is, a higher molecular weight diffuse migration material or band. RNA degradation manifests as a smear of ribosomal RNA bands. Judging from the results of denatured agarose gel electrophoresis detection of peripheral blood samples of 29 cases of control group and 58 cases of nasopharyngeal carcinoma experimental group, some samples have serious problems of RNA degradation (Figure 12). In order not to affect the results of subsequent experiments, our research group selected the total RNA of peripheral blood samples from 17 non-cancer patients and 51 nasopharyngeal cancer patients for follow-up experiments.

Table 3-3 Spectrophotometer detects the total RNA extracted from 87 samples

*For a spectrophotometer, the O.D.A260/A280 ratio should be close to 2.0 for pure RNA (ratio between 1.8 and 2.1 is acceptable). The O.D.A260/A230 ratio should be greater than 1.8.

4. qRT-PCR

1) Primer design

Table 2-4 qRT-PCR primer sequences and characteristics

2) The remaining steps are consistent with the qRT-PCR used in the first part of the screening of nasopharyngeal carcinoma-related lncRNA molecules.

3) qRT-PCR results

qRT-PCR results With the housekeeping gene β-actin as the internal reference, the TCONS_00003617 gene was verified by qRT-PCR in the peripheral blood samples of nasopharyngeal carcinoma patients, and the peripheral blood samples of non-cancer patients were used as the control. The dissolution and amplification curves obtained from the experiment (Fig. 13), it can be seen from the figure that there is no specific amplification product in the experimental process, and the experimental result is reliable.

Using the Mann-Whitney U test, it was found that there was no significant difference in the expression level of TCONS_00003617 between nasopharyngeal carcinoma patients and non-cancer patients ( FIG. 14 , P=0.538).

The above experiments show that the expression of lncRNA: TCONS_00003617 in nasopharyngeal carcinoma tissues is significantly higher than that in para-cancerous tissues, and it is very likely to become a molecular marker or target in the clinical early diagnosis, prognosis judgment or targeted therapy of nasopharyngeal carcinoma. has broad application prospects.

<110> Foshan First People's Hospital

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Claims (4)

1. for detect the reagents of lncRNA molecules prepare nasopharyngeal carcinoma auxiliary diagnosis or treatment of nasopharyngeal carcinoma/prognosis evaluation reagent or Application in kit, the nucleotide sequence of the lncRNA molecules is as shown in SEQ ID NO.1.

2. application according to claim 1, which is characterized in that described to require the 1 lncRNA molecules for test right Reagent include specific primer pair for expanding lncRNA molecules described in claim 1.

3. application according to claim 2, which is characterized in that described for expanding lncRNA molecules described in claim 1 Specific primer pair, sequence is as follows：

F：5'-CCGCCAGTATCTGATGAAGG-3'(SEQ ID NO.4)；

R：5'-TGGGAGATGGGAAAGTAGCC-3'(SEQ ID NO.5).

4. a kind of kit being used for nasopharyngeal carcinoma auxiliary diagnosis or prognosis evaluation, including：

(1) it is used to expand the specific primer pair of lncRNA molecules described in claim 1；

(2) standard DNA template；

(3) PCR reaction solution.