CN107326090B - Quantitative detection method of platelet LncRNA for diagnosing non-small cell lung cancer - Google Patents
Quantitative detection method of platelet LncRNA for diagnosing non-small cell lung cancer Download PDFInfo
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Abstract
The invention discloses a quantitative detection method of platelets LncRNA for diagnosing non-small cell lung cancer, belonging to the field of tumor molecular biology. At present, the diagnosis of lung cancer mainly depends on the clinical signs of tumor tissues with wounds, radiographic images, biochemical detection, disease detection and the like, and has tissue heterogeneity. In order to solve the problem, the invention proves that the expression of platelet long-chain non-coding RNA MAGI2-AS3 and ZFAS1 of NSCLC patients is lower than that of normal people, and a kit for real-time fluorescence quantitative PCR for diagnosing non-small cell lung cancer is prepared on the basis of the expression. By combining expression quantity data obtained by real-time fluorescent quantitative PCR amplification of the MAGI2-AS3 and ZFAS1, a Logistic regression fitting data model for diagnosing the non-small cell lung cancer is established, and the model has higher diagnosis efficiency and sensitivity to the non-small cell lung cancer and has profound clinical significance and popularization.
Description
Technical Field
The invention belongs to the field of tumor molecular biology, and particularly relates to application of a reagent for detecting expression quantity of platelet long-chain non-coding RNA MAGI2-AS3 and ZFAS1 in preparation of a non-small cell lung cancer diagnosis reagent.
Background
Lung cancer is one of the most common malignant tumors in the world, and the morbidity and mortality of lung cancer are the first of various tumors, wherein 80% of lung cancer is non-small cell lung cancer (NSCLC). NSCLC is also largely classified as squamous lung carcinoma (AD), adenocarcinoma of the lung (SCC). Although non-small cell lung cancer has made great progress in diagnosis and basic research in recent decades, overall, the prognosis of non-small cell lung cancer has not improved significantly, and its 5-year survival rate is only 5% -20%. Therefore, for the treatment of lung cancer, the most important thing is to find effective early diagnosis markers, which provides a theoretical basis for resisting the invasion and metastasis of cancer cells.
LncRNA, a genetic molecule involved in cancer biology, has emerged as a new place in recent scientific research. LncRNA transcripts longer than 200nt, generally do not encode protein or have limited ability to encode protein, and usually in the form of RNA regulate the expression level of target genes at various levels (epigenetic regulation, transcriptional regulation, post-transcriptional regulation, etc.), playing an important role in the development of non-small cell lung cancer. LncRNA MALAT-1 and LncRNA CCAT2 are highly expressed in lung cancer, induce cell migration, and stimulate the growth and invasion of tumors in vivo; the expression of LncRNA MEG3 in NSCLC cancer tissue is lower than that in normal tissue, and is related to pathological stage and tumor size; furthermore, lower levels of MEG3 expression patient prognosis are relatively poor; MEG3 overexpression reduces cell proliferation of NSCLC, can induce apoptosis in vitro, and blocks tumor development in vivo; LncRNAH19 mainly plays a role in promoting the growth of lung cancer, and the interference of the expression of H19 gene can reduce the colony forming capability and the independent adherence capability of lung cancer cells. With more and more researches, LncRNA has an important meaning for the treatment and diagnosis of non-small cell lung cancer.
The diagnosis for lung cancer mainly depends on the aspects of traditional tumor tissue clinical signs, radiographic images, biochemical detection, disease detection and the like, and most methods are traumatic, bring pain to patients and have the limitations of tissue heterogeneity and the like; to reduce the limitations of obtaining tumor tissue, blood-based liquid biopsy methods for tissue sample analysis have become a popular trend in recent years, and currently mainly involve the detection of plasma free DNA, tumor platelets, exosomes and circulating tumor cells. Platelets, the second most abundant cell type in peripheral blood, are derived from circulating anucleated fragments of bone marrow megakaryocytes, and although an anucleated cell fragment, are rich in RNA, and are capable of absorbing some tumor markers released by tumors. However, in the prior art, a mature technology for accurately diagnosing tumors by using markers in platelets does not exist.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a detection method for jointly using the results of fluorescent quantitative PCR of platelets LncRNAMAGI2-AS3 and ZFAS1 for diagnosing NSCLC, and the method has higher sensitivity and specificity; further provides a non-small cell lung cancer diagnosis kit with high cost performance and easy popularization and application.
The technical scheme for realizing the aim is as follows:
the invention provides an application of a reagent for detecting the expression quantity of long-chain non-coding RNA MAGI2-AS3 and ZFAS1 of platelets in preparing a reagent for diagnosing non-small cell lung cancer, which is characterized in that the reagent for detecting the expression quantity of long-chain non-coding RNA MAGI2-AS3 and ZFAS1 is a real-time fluorescent quantitative PCR detection reagent; the real-time fluorescent quantitative PCR detection reagent comprises a detection primer which is designed and synthesized according to the nucleotide sequences of long-chain non-coding RNA MAGI2-AS3 and ZFAS1 and is specifically used for real-time fluorescent quantitative PCR, wherein the specific primer is shown AS SEQ ID NO.1-4 in a sequence table;
the detection method of the platelet long non-coding RNA comprises the steps of sample RNA extraction, sample cDNA preparation and long non-coding RNA amplification, and specifically comprises the following steps:
1) extraction of platelet RNA: extracting platelet RNA according to the steps required by the blood total RNA rapid extraction kit instruction of BioTeKe company;
2) preparation of cDNA: PrimeScript using TaKaRa kitTMRT reagent kit, reverse transcription of the extracted platelet RNA to synthesize cDNA;
3) amplification of long non-coding RNA: performing fluorescent quantitative PCR amplification by using an Ultra SYBR Mixed kit of CW BIO and taking the cDNA reverse transcribed in the step 2) as a template according to the operation steps specified by the instruction;
obtaining the expression multiple 2 of the target Lnc RNA expression quantity in a sample to be detected relative to the change of an internal reference gene GAPDH through real-time fluorescent quantitative PCR amplification-ΔCtSubstituting into formula Y (12.330) (MAGI2-AS3) +11.876 (ZFAS1) -3.675, wherein (MAGI2-AS3) and (ZFAS1) are the expression levels of MAGI2-AS3 and ZFAS1 in platelets 2-ΔCtWherein, the Ct value of the target gene-the Ct value of the reference gene GAPDH is the Ct value of each gene detected by real-time fluorescence quantitative PCR; when the Y value is less than 0.370, the non-small cell lung cancer is judged to be positive, otherwise, the non-small cell lung cancer is judged to be negative.
In the second aspect of the invention, a real-time quantitative PCR detection kit for non-small cell lung cancer diagnosis is provided, which comprises a specific detection primer for real-time fluorescent quantitative PCR designed according to the nucleotide sequence of long-chain non-coding RNA MAGI2-AS3 and ZFAS1, wherein the specific primer is shown AS SEQ ID NO.1-4 in a sequence table.
The detection preparation can be used for detecting the expression levels of long-chain non-coding RNA MAGI2-AS3 and ZFAS1 in platelets, and a Logistic regression fitting data model for diagnosing the non-small cell lung cancer is established by combining the quantitative expression results of the platelets MAGI2-AS3 and ZFAS1, has higher diagnosis efficiency and sensitivity to the non-small cell lung cancer, and has profound clinical significance and popularization.
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FIG. 1 is a work flow diagram of the present invention;
FIG. 2 shows the expression levels of the platelets MAGI2-AS3 and ZFAS1 in the present invention
AD-lung adenocarcinoma; SCC-squamous cell carcinoma; control-healthy Control group; relative expression 2-ΔCtPresentation, comparison between sets by non-parametric test<0.05，**P<0.01***p<0.001,****p<0.0001
A: expression of target gene MAGI2-AS3 in non-small cell lung cancer platelets
B: the expression condition of the target gene ZFAS1 in non-small cell lung cancer platelets;
FIG. 3 diagnostic ROC graph of platelet target genes MAGI2-AS3 and ZFAS1 in the invention
Wherein the Sensitivity-Sensitivity, specificity-specificity is
A: ROC curve of MAGI2-AS3 in platelets in lung adenocarcinoma and lung squamous carcinoma
B: ROC curve of ZFAS1 in lung adenocarcinoma and lung squamous carcinoma in blood platelet
C: ROC curves of the combined diagnosis of lung adenocarcinoma and lung squamous carcinoma of MAGI2-AS3 and ZFAS1 in platelets;
FIG. 4 is a graph showing the relationship between the platelet target gene and the clinical pathological parameters in the present invention
A: analytical graph of relationship between NSCLC platelets ZFAS1 and TNM (tumor stage)
B: analytical graph of relationship between NSCLC platelets MAGI2-AS3 and TNM (tumor stage)
C: analysis of relationship between NSCLC platelets MAGI2-AS3 and N (regional lymph node involvement)
D: graph of relationship between NSCLC platelets MAGI2-AS3 and M (distant metastasis);
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
[ example 1 ]
The platelet of 101 NSCLC (68 adenocarcinoma +33 squamous carcinomas) tumor patients and 60 healthy control groups are adopted to carry out detection and model establishment on MAGI2-AS3 and ZFAS1, the working flow is shown in figure 1,
the specific detection method comprises the following steps:
1. platelet collection and preservation
Venous blood of a subject is collected in a 4ml anticoagulation tube, and platelets are centrifugally separated within 12h under a room temperature environment. Centrifuging at room temperature of 120g for 20min, separating into two layers, sucking the upper layer of platelet-rich plasma, transferring to a new EP tube (part of plasma is left when sucking, and the plasma is not sucked completely), centrifuging at 360g for 20min, obtaining the precipitate as platelets, washing twice with PBS, adding 300. mu.l DEPC water, and extracting RNA.
2. Platelet RNA extraction (blood total RNA rapid extraction kit, BioTeKe)
1) Adding 900 mul of lysis buffer RLS into the platelet suspension, and blowing and cracking;
2) centrifuging at 12000rpm for 10min at 4 deg.C, collecting supernatant, and introducing into new enzyme-free EP tube;
3) adding 180 μ l chloroform, covering the EP tube, shaking for 15s, and incubating at room temperature for 10 min;
4) centrifuging at 12000rpm for 10min at 4 deg.C, and collecting 550 μ l upper water phase;
5) adding equal volume of 70% ethanol, mixing, and transferring to adsorption column RA;
6) centrifuging at 1000rpm for 3min, and discarding the waste liquid;
7) adding 500 μ l deproteinized solution RE, centrifuging at 12000rpm for 3min, and discarding the waste liquid;
8) adding 700 mul of the rinse solution RE, centrifuging at 12000rpm for 3min, and discarding the waste liquid;
9) adding 500 mul of RE removed rinsing liquid for secondary rinsing, centrifuging at 12000rpm for 3min, and discarding the waste liquid;
10) centrifuging at 12000rpm for 4min, and throwing off residual liquid;
11) placing the adsorption column RA into a new enzyme-free EP tube, adding RNase free water in 56 deg.C water bath
Centrifuging at 12000rpm for 3min in 30 μ l to collect platelet RNA;
12) RNA concentration and purity were determined and stored at-80 ℃.
cDNA Synthesis (PrimeScript)TMRT reagent kit，Takara)
1) Removal of genomic DNA:
mixing, centrifuging, placing in PCR instrument at 42 deg.C for 2min, and storing at 4 deg.C.
2) Reverse transcription:
mixing, centrifuging, and storing in PCR instrument at 37 deg.C for 15min, 85 deg.C for 5s, and 4 deg.C.
Screening of LncRNA: three GEO databases GSE19188, GSE30219 and GSE27262 are used for screening long-chain non-coding RNA MAGI2-AS3 and ZFAS1 which have obvious expression difference with normal tissues in lung adenocarcinoma and lung squamous carcinoma.
5. Real-time fluorescent quantitative PCR:
using the Ultra SYBR mix from CW BIO, the procedure was as specified in the kit instructions: 20 μ l of the system contained 10 μ l SYBR mix, 1.6 μ l primer, 2 μ l cDNA, 6.4 μ l ddH2O, reaction sequence: pre-denaturation at 95 ℃ for 5min, (30 sec at 95 ℃, 30sec at 63.3 ℃, 30sec at 72 ℃) for 42 cycles, according to 2-ΔCtThe method of (4) calculates the expression levels of two target genes in the platelets. The primers used are shown in Table 1 below.
TABLE 1 primer sequences
| Name of Gene | Gene sequences | Upstream primer | Downstream primer |
| MAGI2-AS3 | NR_038343 | GAGCAGAAATAGCGGGACCT | TCTCTTGGATGCAAACGGCA |
| ZFAS1 | NR_003604 | ACGTGCAGACATCTACAACCT | TACTTCCAACACCCGCAT |
| GAPDH | NM_002046 | GGTCTCCTCTGACTTCAACA | GTGAGGGTCTCTCTCTTCCT |
6. Analysis of results
1) Expression levels of MAGI2-AS3 and ZFAS 1:
by using 2-ΔCtQuantitative data analysis of two target genes shows that delta Ct is the Ct value of the target gene-Ct of the reference gene, the Ct value of the target gene is the Ct value of the target genes MAGI2-AS3 and ZFAS1 detected by a real-time fluorescence quantitative technology, the result is shown in figure 2, the expression levels of the platelets of the patients with lung adenocarcinoma and lung squamous carcinoma of the MAGI2-AS3 and ZFAS1 are lower than those of the healthy control group, and the difference is significant.
2) ROC diagnostic efficacy analysis:
ROC analysis of the diagnostic value of target genes MAGI2-AS3 and ZFAS1 in platelets in non-small cell lung cancer respectively; the results are shown in figure 3 and table 2, the diagnosis efficacy of MAGI2-AS3 in the platelet is better than that of ZFAS1, but when two LncRNAs are diagnosed in a combined way, the diagnosis efficacy is obviously higher than that of the LncRNA alone, and the sensitivity and the specificity reach more than 80 percent, which indicates that the expression levels of MAGI2-AS3 and ZFAS1 of the combined platelet can better diagnose NSCLC.
TABLE 2 sensitivity, specificity of various diagnostics
3) Relationship analysis of LncRNA and clinical and pathological parameters:
analysis of MAGI2-AS3, ZFAS1 with nonparametric tests on the basis of the type of pathology, sex, age, TNM stage, T (in the case of the primary tumor foci), N (regional lymph node involvement), M (distant metastasis)) FIG. 4 shows the results of analysis of differences in expression, using 2-ΔCtShowing Relative expression, TNM showing non-small cell lung cancer stage, N showing regional lymph node affected condition, and M showing distant metastasis condition; the results show that ZFAS1 is only negatively correlated with the TNM stage of non-small cell lung cancer, and the larger the stage is, the lower the expression of ZFAS1 is; while MAGI2-AS3 is negatively associated with N, M in addition to the stage of non-small cell lung cancer, i.e., the more severe the lymph node is affected and distant metastasis occurs, the lower the expression of MAGI2-AS 3.
Building a Logistic regression fitting data model:
y12.330 (MAGI2-AS3) +11.876 (ZFAS1) -3.675; wherein (MAGI2-AS3), (ZFAS1) is the expression level 2 of MAGI2-AS3 and ZFAS1 in platelets-ΔCtAnd positive (NSCLC patient) when the Y value is less than 0.370, and negative (non-NSCLC patient) in reverse.
Sequence listing
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Claims (1)
1. The application of a reagent for jointly detecting the expression quantity of the long-chain non-coding RNA MAGI2-AS3 and ZFAS1 of the platelet in preparing a reagent for distinguishing healthy people from lung adenocarcinoma or lung squamous carcinoma patients is characterized in that the reagent for jointly detecting the expression quantity of the long-chain non-coding RNA MAGI2-AS3 and ZFAS1 of the platelet is a real-time fluorescent quantitative PCR detection reagent; the real-time fluorescent quantitative PCR detection reagent comprises a specific detection primer which is designed and synthesized according to the nucleotide sequences of long-chain non-coding RNA MAGI2-AS3 and ZFAS1, wherein the specific detection primer is shown AS SEQ ID NO 1-4 in a sequence table.
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