CN108866187B - Long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis and application thereof - Google Patents
Long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis and application thereof Download PDFInfo
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- CN108866187B CN108866187B CN201810638515.2A CN201810638515A CN108866187B CN 108866187 B CN108866187 B CN 108866187B CN 201810638515 A CN201810638515 A CN 201810638515A CN 108866187 B CN108866187 B CN 108866187B
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Abstract
The invention relates to an lncRNA marker related to lung cancer auxiliary diagnosis, the nucleotide sequence of which is SEQ ID No.1, and a detection method and application thereof in peripheral blood of the lncRNA marker, compared with the prior art, the invention has the advantages that the inventor successfully screens lncRNA XLOC _009167 with abnormal expression in lung cancer, and separates and detects lung cancer specific lncRNA XLOC _009167 from human whole blood and shows remarkable high expression. In addition, the expression of the lncRNA XLOC _009167 is in obvious positive correlation with the pathological type of the lung cancer, and the lung cancer-treating gene has higher sensitivity and specificity for distinguishing lung cancer patients from healthy people, lung cancer patients and pneumonia patients, wherein the sensitivity can reach 78.7 percent, the specificity can reach 61.8 percent, and the gene provides a new treatment target as a target gene medicine for preparing a medicine for treating liver cancer.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis and application thereof.
Background
According to the statistics report of cancers in 2018 in the United states, the cancers are the main public health problems worldwide, wherein the incidence rate and death rate of lung cancer are the first malignant tumors in China for a long time. There are various causes of cancer death, and there is no way to completely prevent cancer, and many patients have been diagnosed with cancer at a middle or late stage, missing the gold stage of cancer treatment. Therefore, early diagnosis is a key for discovering and intervening in advance, and nowadays, clinically used means such as X-ray examination, Magnetic Resonance Imaging (MRI), Computed Tomography (CT), and bronchofiberscope examination can perform early diagnosis of lung cancer to different degrees, which improve early diagnosis rate and prognosis of lung cancer in a certain range, but still have many limitations, such as high invasiveness, low sensitivity, and low specificity.
With the continuous development of oncology and molecular biology technologies, the research of tumor markers has become the hot door in the field of cancer diagnosis. In recent years, continuous research shows that Long non-coding RNA (Long non-coding RNA) plays a crucial role in many biological processes, the term "non-coding RNA" refers to RNA that does not code protein, most of the non-coding RNA in the human genome is non-coding RNA, only 2% of transcripts generated in the human genome are coding RNA, and the rest 98% are non-coding RNA, and in the non-coding RNA, the non-coding RNA is divided into short non-coding RNA and Long non-coding RNA according to size, and the non-coding RNA is functional RNA molecules that cannot be translated into protein, and the non-coding RNA is widely involved in human physiology and pathology and is closely related to human tumors. Such as cell cycle checkpoints, gene regulation, cell resistance, etc., and also play an important role in the development and progression of cancer. Meanwhile, the detection of circulating nucleic acid in peripheral blood of lung cancer patients is gradually attracting attention, blood is the most common clinical test material in early clinical diagnosis and treatment of lung cancer, and the blood collection method is easy to accept by people due to small trauma and no pain. Therefore, screening lung cancer specific lncRNA based on human peripheral blood and using the same as a novel molecular marker have important detection and diagnosis potential.
Disclosure of Invention
The invention aims to solve the technical problem of providing a long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis aiming at the current situation of the prior art.
Another technical problem to be solved by the present invention is to provide a method for detecting the lncRNA marker, which is not directly used for diagnosis and treatment of diseases.
The invention also provides an application of the lncRNA marker in preparation of an auxiliary diagnosis kit for lung cancer.
The last technical problem to be solved by the invention is to provide an application of the lncRNA marker in preparing a medicament for treating lung cancer.
The technical scheme adopted by the invention for solving the technical problems is as follows: the long-chain non-coding RNA marker related to the auxiliary diagnosis of the lung cancer is characterized in that the nucleotide sequence of the long-chain non-coding RNA marker is Seq NO. 1.
Further, the long non-coding RNA marker is lncRNA xoc _009167, which is upregulated in specific expression in serum of lung cancer patients.
The present invention provides a method for detecting a long non-coding RNA marker associated with the auxiliary diagnosis of lung cancer, which is not directly used for diagnosis and treatment of diseases, to solve the second technical problem, and is characterized by comprising the steps of:
1) collecting blood, and extracting total RNA in plasma;
2) reverse transcribing the total RNA into cDNA;
3) carrying out fluorescent quantitative PCR detection on the cDNA solution in the step 2) by using primers for specifically amplifying the upstream and downstream of the lncRNA XLOC _ 009167.
Further, the specific amplification upstream and downstream primers of lncRNA XLOC _009167 are as follows:
P1:5’-gaactccagctgcatccactt-3’；
P2:5’-gacctgttgcttctgcttgg-3’。
the invention provides an application of the long-chain non-coding RNA marker in preparing a lung cancer auxiliary diagnosis kit for solving a third technical problem, which is characterized in that: the kit specifically comprises the following reagents: the concentration of the primer pair P1 and P2 is 1mM, 0.5ml, 2ml of TaqDNA polymerase II and 1ml of redistilled water.
The invention provides an application of the long-chain non-coding RNA marker in preparing a lung cancer medicament for solving a fourth technical problem, which is characterized in that: the medicine consists of nucleic acid containing Seq NO.1 shown in a sequence table in limited amount and a pharmaceutically acceptable carrier or auxiliary material.
Compared with the prior art, the invention has the following advantages: the present inventors successfully screened lncRNA XLOC _009167 for abnormal expression in lung cancer, and isolated and detected lung cancer-specific lncRNA XLOC _009167 from human whole blood and showed significantly high expression. In addition, the expression of the lncRNA XLOC _009167 is in obvious positive correlation with the pathological type of the lung cancer, and the molecular marker has higher sensitivity and specificity for distinguishing lung cancer patients from healthy people, lung cancer patients and pneumonia patients, wherein the sensitivity can be up to 78.7%, and the specificity can be up to 61.8%.
Drawings
FIG. 1 is a chip analysis of lncRNA in lung cancer tissue and corresponding paracancerous normal tissue (the right side is the name of lncRNA with abnormal expression and significant difference; arrow indicates lncRNA XLOC-009167);
FIG. 2 is a diagram showing the results of agarose gel electrophoresis for detecting IncRNA XLOC-009167 (in which the left side is the size of DNA standard; M represents a DNA marker, and lanes 1-4 are PCR products);
FIG. 3 is a graph showing the results of fluorescence quantitative PCR validation of the relative expression of lncRNA XLOC _009167 in lung cancer tissues and paracancerous normal tissues;
FIG. 4 is a graph showing the results that the expression of LncRNA XLOC _009167 in the blood of lung cancer patients is significantly higher than that of normal persons;
FIG. 5 is a graph showing the results of the ROC curve detection analysis of the diagnostic value of lncRNA XLOC _009167 for lung cancer patients and normal persons;
FIG. 6 is a graph of the results for lncRNA XLOC _009167 expression significantly higher in TNM II-IV stage tumors than TNM I stage tumors; .
FIG. 7 is a graph showing the results of ROC curve analysis of the diagnostic value of lncRNA XLOC _009167 for diagnosing stages I and II-IV of lung cancer;
FIG. 8 is a graph of the results of lncRNA XLOC _009167 significantly higher expression in lung cancer patients' blood than in pneumonia patients;
FIG. 9 is a graph of the results of the ROC curve detection assay lncRNA XLOC _009167 for diagnostic value in diagnosing lung cancer patients and pneumonia patients.
Detailed Description
The present invention is further illustrated by the following figures, sequence listing and examples.
Example 1
Chip analysis of lung cancer differential lncRNA: after approval by the institutional medical ethics committee, the patient's cancer and paracancerous tissues and blood samples were collected. Total RNA was extracted from 4 pairs of lung cancer tissues and tissues adjacent to the cancer, and RNA quality was evaluated. The Arraystar human LncRNA chip V3.0 chip was selected, and RNA labeling and chip hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). The chip results are shown in fig. 1, and a plurality of lncrnas with significant expression upregulation are obtained by analyzing lung cancer tissues and cancer-adjacent tissues through the chip, wherein the first 4 lncrnas with higher expression upregulation have related research reports, so an lncRNA XLOC _009167 with relatively higher upregulation fold is selected, as shown by an arrow in fig. 1.
Example 2
Lung cancer characteristic lncRNA xoc _009167 validation: the fluorescent quantitative RT-PCR validation was performed in particular in an expanded sample (n ═ 61) where PCR fragments of lncRNA XLOC _009167 were found to be about 708bp long as shown in fig. 2, the detailed sequences obtained by sequencing are shown in the sequence listing, and statistically analyzed to be expressed, lncRNA XLOC _009167 was significantly up-regulated (P <0.05) in lung cancer tissues (as shown in fig. 3).
Example 3
The method for extracting the total RNA of the whole blood comprises the following steps: taking 0.25ml of whole blood containing RNA protective solution, adding 0.75ml of TRIzol LS reagent (Invitrogen company, USA), reversing the mixture up and down, mixing the mixture evenly, and standing the mixture for 15 min; ② adding 0.2ml chloroform, oscillating vigorously for 15 seconds, standing for 10min at room temperature; ③ centrifugation is carried out for 15 minutes at 12000 Xg and 4 ℃; transferring the upper aqueous phase to another EP tube of diethyl pyrocarbonate (DEPC), adding isopropanol with the same volume, and standing for 10 minutes at room temperature; centrifuging at 12000 Xg for 10min at 4 deg.C, and removing supernatant; sixthly, washing the RNA sediment by 1ml of 75 percent ethanol, centrifuging for 5 minutes at the temperature of 4 ℃ at 7500 Xg, and removing the supernatant; and drying at room temperature for about 5-10 minutes. Mu.l RNase-free water was added, and the mixture was pipetted several times and left at 56 ℃ for 10 minutes to dissolve the RNA. The concentration of the obtained RNA was 400-2000 ng/ml.
Example 4
The method for reverse transcription into cDNA comprises the following steps: : the following reagents were first added to the DEPC-treated PCR tubes: random primer 2. mu.l, dNTP mix 1. mu.l, RNA template 2. mu.l, DEPC water 8. mu.l, mix well and put into metal bath 65 ℃ for 5min, then ice bath 2min, add the following reagents: mu.l of reverse transcriptase (200U/. mu.l), 4. mu.l of 5 Xtimes reverse transcription reaction buffer, 0.5. mu.l of RNase inhibitor (40U/. mu.l), 3.5. mu.l of RNase-free water, and 20. mu.l in total volume. The reverse transcription procedure comprises 10min at 37 deg.C, 50min at 70 deg.C, 20min at 50 deg.C, and storing at 4 deg.C, and transferring to-80 deg.C refrigerator for long-term storage.
Example 5
Fluorescent quantitative PCR detection of lncRNA XLOC _ 009167: using the cDNA of example 4 as a template, PCR primers for amplification of lncRNA XLOC _009167, i.e., the forward primer: 5'-gaactccagctgcatccactt-3', respectively; a downstream primer: 5'-gacctgttgcttctgcttgg-3'). The PCR reaction system is as follows: quantitative SYBR assay Taq enzyme II, 10. mu.l, 1. mu.l upstream primer, 1. mu.l downstream primer, 1. mu.l cDNA template, ddH2O7. mu.l, 20. mu.l in total. Detecting on a fluorescence quantitative PCR instrument MX3500, the reaction program is 95 ℃ for 2min, 45 cycles are carried out at 95 ℃ for 30s, 55 ℃ for 1min and 72 ℃ for 1min, and finally, the Ct value is recorded after the sample is stored at 4 ℃.
Example 6
Analysis of the relationship between lncRNA XLOC _009167 expression and patient pathology: the statistical results are shown in Table 1 in combination with clinical data and various pathological factor analyses, from which it can be seen that lncRNA XLOC _009167 expression has no significant association with age, sex, Body Mass Index (BMI), tumor size, tumor metastasis and smoking history (P > 0.05). However, the relative expression of lncRNA XLOC _009167 was significantly correlated with tumor stage. In particular, the relative expression level of lncRNA XLOC _009167 in TNM II-IV stage tumors was significantly higher than in TNM I stage tumors (P < 0.05). The relationship between the relative expression levels of lncRNA XLOC _009167 and TNM staging suggests that lncRNA XLOC _009167 may be valuable for the diagnosis of early stage lung cancer.
TABLE 1 relationship between lncRNA XLOC _009167 expression and clinical and pathological factors
BMI, body mass index; P/N, positive/negative; #, Pearson' s2test
Example 7
The lung cancer patients were differentiated from healthy populations using blood lncRNA xoc 009167: blood samples were obtained from example 1 and analyzed for lncRNA XLOC _009167 expression levels in patient and normal human blood according to the methods described in examples 3-5. As can be seen from the results in fig. 4, lncRNA XLOC _009167 was significantly higher expressed in patients than in normal humans (P < 0.0001). This indicates that lncRNA XLOC _009167 is detectable in the patient's blood and is higher than normal.
To further validate that lncRNA XLOX _009167 used to differentiate lung cancer patients from healthy controls corresponded to an AUC value of 0.7398 (95% CI 0.6493-0.8303, sensitivity 78.7%, specificity 61.8%, fig. 5). Significant upregulation of lncRNA XLOC _009167 in whole blood of lung cancer patients indicates that lncRNA XLOC _00967 has value as a novel diagnostic marker. Furthermore, to investigate whether there was a correlation between the expression of lncRNA XLOC _009167 in different TNM stages, we also evaluated the expression of lncRNA XLOC _009167 in different TNM stages of lung cancer patients. Interestingly, the results show that lncRNA XLOC _009167 upregulates expression in TNM II-IV patients (P <0.05, fig. 6). AUC value 0.6600 (95% CI 0.5193-0.8007, sensitivity 50.0%, specificity 88.0%, fig. 7). These findings provide the basis for lncRNA XLOC _009167 as a novel diagnostic marker to detect lung cancer at an early stage.
Example 8
To explore the ability of lncRNA XLOC _009167 to discriminate lung cancer patients from pneumonia patients, the relative expression of lncRNA XLOC _009167 in 30 pneumonia patients was examined. The results show that lncRNA XLOC _009167 is expressed at significantly higher levels in lung cancer patients than in pneumonia patients (P <0.01, fig. 8). The corresponding AUC was 0.7005 (95% CI 0.5771-0.8240, sensitivity 90.1%, specificity 50.0%, fig. 9). These results indicate that lncRNA XLOC _009167 can be used to differentiate lung cancer patients from pneumonia patients (see table 2).
TABLE 2 diagnostic value of lncRNA XLOC _009167 for the diagnosis of pneumonia and lung cancer
Sequence listing
<110> Ningbo university
<120> long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 708
<212> RNA
<213> Artificial sequence (Homo sapiens)
<400> 1
gtgaggagcg cctgggccgg gcagcgaccc cgtctgggag gataatgaac tccagctgca 60
tccacttgct gcaaaggacg tgatttcatt ctttatggaa gagccccacc cctacacccc 120
atggtccgct gcgtccgact ggtggaagca ggatctgtgg tgagatacct cagcacaagc 180
atatgcaggc ctgtggtaga cgccggaagt agggcccttt gtctccaaga atgggcagac 240
tctcaacagg tgaaagaaaa acagtactcc aggtaggggc caagcagaag caacaggtca 300
aagaaaggcc tggaagtgga aaagccctgg gttcagtttg ctaagagccc aggagactag 360
ctggatacaa ggccggaaga agggtggggc tctgatgcac cagcttctcc aatcttcctg 420
gctccaggga ggagatgcat ttgaacaatc ctgaaaaact tttggatccc aagtctgact 480
caacattgtg acttgcttac tccgtgtcac tcttctagat gcttttgaaa tatattatca 540
taacaatgag taagttctat ttccattctc agaaaagaaa actgattcaa gattgtgtaa 600
cttgctgctt ctggttggtg agatttaaaa aaaaaaaaaa gattatgtaa cttgttcaaa 660
gtcatatgga ttacaaacta tagcgcaggg acttaatttc agtctggc 708
Claims (1)
1. The application of a primer of a long-chain non-coding RNA marker in preparing a kit for distinguishing lung cancer patients from pneumonia patients is characterized in that: the nucleotide sequence of the long-chain non-coding RNA marker is Seq NO.1, the specific expression of the long-chain non-coding RNA marker lncRNA XLOC _009167 in the serum of a lung cancer patient is up-regulated, and the specific amplification upstream and downstream primers of the lncRNA XLOC _009167 are as follows:
P1: 5’-gaactccagctgcatccactt-3’；
P2: 5’-gacctgttgcttctgcttgg -3’。
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| CN107488722A (en) * | 2017-09-18 | 2017-12-19 | 复旦大学附属中山医院 | Purposes of the LncRNA in the reagent for preparing diagnosis adenocarcinoma of lung |
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| CN107488722A (en) * | 2017-09-18 | 2017-12-19 | 复旦大学附属中山医院 | Purposes of the LncRNA in the reagent for preparing diagnosis adenocarcinoma of lung |
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