CN109679957B - IncRNA L NC-004208 and application of detection reagent thereof in preparation of brain glioma prognosis reagent - Google Patents
IncRNA L NC-004208 and application of detection reagent thereof in preparation of brain glioma prognosis reagent Download PDFInfo
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Abstract
The invention discloses IncRNA L NC-004208 and application of a detection reagent thereof in preparation of a glioma prognosis reagent, wherein IncRNA L NC-004208 is abnormally highly expressed in a temozolomide-resistant glioma cell line through research and application of IncRNA sequencing, the first time, the grade, the treatment condition, the recurrence or the recurrence, other diseases, other medicines, the recurrence and death time and the like of 104 patients with glioma are inquired in detail through telephone follow-up of the patients, the survival time and the survival state are registered, the average time and the progression-free survival period of the high-expression patients are found to be obviously shorter than those of the low-expression or non-expression patients and are related to the reactivity of temozolomide, L NC-004208 is a molecular NC marker related to glioma prognosis, the IncRNA L _004208 is highly expressed, and the prognosis of the patients is poor.
Description
Technical Field
The invention belongs to the field of tumor molecular biology, and particularly relates to IncRNA L NC-004208, application of a detection reagent thereof in preparation of a brain glioma prognosis reagent, and a corresponding reagent.
Background
Brain gliomas are the most common malignancies of the central nervous system, accounting for about 80% of primary malignant brain tumors, and have an annual incidence of about 6/100,000 worldwide according to european association of neurological tumors (EANO). The treatment method of brain glioma is a worldwide problem, particularly malignant glioblastoma, and even if an optimal treatment scheme is adopted, the prognosis of a patient is still not ideal, and the average survival time is only 13.3-15 months. Temozolomide (TMZ) is a new drug with a good effect in treating brain glioma, and has been approved by FDA in the united states and recommended by national comprehensive cancer network guidelines to become the first chemotherapeutic drug for treating brain glioma. TMZ belongs to a novel imidazole oral antineoplastic agent, is easy to permeate a hemangioma barrier, and an active product MTIC thereof plays a cytotoxic role through N3, N7 and O6 guanine and O3 adenine DNA alkylation. Because of the factors inherent in tumor cells or the occurrence of acquired resistance, patients with brain glioma gradually develop resistance to TMZ treatment, and the curative effect of tumor treatment is reduced.
O6-methylguanine-DNA methyltransferase (MGMT) inhibits TMZ-mediated DNA damage by transferring an abnormal O6 methyl group on the DNA, resulting in glioma cell TMZ resistance. Recent studies have shown that, in addition to MGMT, abnormalities in various signaling pathways such as Epidermal Growth Factor Receptor (EGFR) are involved in TMZ resistance, but the specific mechanism is not yet elucidated. Therefore, the molecular mechanism of TMZ resistance is deeply discussed, and effective measures are taken for intervention, so that an important new idea is provided for improving the chemotherapy curative effect of the brain glioma.
In recent years, research finds that the expression profiles of lncRNAs in different classes of brain glioma tissues have significant difference, and the onset and prognosis of brain glioma are closely related to the expression and dysfunction of lncRNAs. Molecular pathology plays an increasingly important role in tumor research and diagnosis in the precise medical age. With the rapid progress of the research of tumorigenesis development mechanism and the development and clinical application of targeted drugs, the molecular pathological detection related to tumors is also expanded from tissue or cytology specimens to liquid biopsy; the adopted analysis means is more sensitive, multi-level and high-flux, and the intelligent method is gradually developed. Accurate molecular typing has become an indispensable link in the whole process of tumor diagnosis, treatment and management. In particular, the search and practice of therapeutic modalities that span tumorigenic sites and use genotypes as direct targets have placed new demands on molecular pathology.
Disclosure of Invention
The invention provides IncRNA L NC-004208 with a sequence shown in SEQ NO: 1. the invention provides a new strategy for diagnosing, prognosing and treating brain glioma by using IncRNA L NC-004208 as a target, and provides new experimental evidence for researching the molecular mechanism of IncRNAs in the resistance of the brain glioma TMZ.
The second purpose of the invention is to provide an application of the reagent for detecting the expression level of IncRNA L NC-004208 in preparation of glioma prognosis or TMZ sensitivity detection preparations.
Furthermore, the reagent for detecting the expression level of IncRNA L NC-004208 is used for detecting brain glioma tissues.
Further, the reagent for detecting the expression level of IncRNA L NC-004208 comprises a real-time fluorescent quantitative detection reagent.
The primers for real-time fluorescent quantitative detection of the expression level of IncRNA L NC-004208 comprise two pairs (primers designed according to different positions can be used together or one pair can be selected)
L NC-004208 (1) -Forward primer 5'-GCCAATCCAAACCGAAAGCC-3', see SEQ NO 2;
l NC-004208 (1) -reverse primer 5'-AGACTCAGACCCCAGGCTAC-3', see SEQ NO: 3;
l NC-004208 (2) -Forward primer 5'-TTTGCTACATCCCAGCTCCA-3', see SEQ NO 4;
l NC-004208 (2) -reverse primer 5'-TCCCCCTTTCCCTTGTAGGT-3', see SEQ NO: 5.
The third purpose of the invention is to provide a reagent for prognosis or detection of TMZ sensitivity of brain glioma, wherein the IncRNA L NC-004208 has a sequence shown in SEQ NO. 1.
The brain glioma prognosis or TMZ sensitivity detection reagent is a reagent for detecting the expression level of IncRNA L NC-004208.
The reagent for detecting the expression level of the IncRNA L NC-004208 comprises a real-time fluorescent quantitative detection reagent.
The primers for real-time fluorescent quantitative detection of the expression of lnc RNA L NC-004208 comprise two pairs or one pair of the following:
l NC-004208 (1) -Forward primer 5'-GCCAATCCAAACCGAAAGCC-3'
L NC-004208 (1) -reverse primer 5'-AGACTCAGACCCCAGGCTAC-3'
L NC-004208 (2) -Forward primer 5'-TTTGCTACATCCCAGCTCCA-3'
L NC-004208 (2) -reverse primer 5'-TCCCCCTTTCCCTTGTAGGT-3'.
The reagent also contains specific primers of an internal reference gene β -actin:
forward primer 5'-CATGTACGTTGCTATCCAGGC-3', shown in SEQ NO 6;
reverse primer 5'-CTCCTTAATGTCACGCACGAT-3', see SEQ NO: 7.
The reagent also comprises:
(1) extracting total RNA from brain glioma tissue with a reagent comprising a stable solution, a reagent, chloroform, isopropanol, and non-enzymatic water;
(2) a reagent for reverse transcription of RNA L NC-004208 into cDNA by using total RNA as a template, which comprises a reverse transcription buffer solution, deoxynucleotide triphosphate, an RNase inhibitor, MM L V reverse transcriptase and a random primer;
(3) and reagents for real-time quantitative PCR of the cDNA comprise real-time fluorescent quantitative SYBR dye and enzyme-free water.
The invention discovers that lncRNA L NC _004208 shows abnormally high expression in a temozolomide resistant glioma cell line by researching and utilizing lncRNA sequencing, inquires about the first-onset time, grade, treatment condition, relapse, other diseases, other medicines, relapse, death time and the like of 104 cases of glioma patients in detail by telephone follow-up visit on the patients, and registers the survival time and the state of the patients, finds that the average survival time and the progression-free survival period of the high-expression patients are obviously shorter than those of the low-expression or non-expression patients and are related to the reactivity of temozolomide by carrying out survival analysis on L NC _004208 expression and the survival time and the state of the patients in brain glioma tissues, and indicates that L NC _004208 is a molecular NC marker related to the prognosis of the glioma, the lncRNA L _004208 expression is high, and the prognosis of the patients is poor.
Drawings
FIG. 1 is a graph of TMZ sensitive/resistant cell expression profiles analyzed by transcriptome sequencing and lncRNA sequencing techniques;
FIG. 2 is a graph of the screening for lncRNA differentially expressed in glioma TMZ resistant cells;
FIG. 3 is a graph of the screening and validation of lncRNA differentially expressed in glioma TMZ resistant cells;
FIG. 4 is a correlation of patient survival with lncRNA expression;
FIG. 5 is a graph of the correlation of patient temozolomide reactivity with lncRNA expression.
Detailed Description
The invention is further illustrated by the following detailed description, but is not to be construed as being limited thereto.
Example 1 analysis of TMZ sensitive/resistant cell expression profiles by transcriptome sequencing and IncRNA sequencing techniques
By using transcriptome sequencing technology and lncRNA sequencing technology (Illumina HiSeq)TM2000) And selecting a glioma T98G/U-118MG cell line and a TMZ resistant T98G-R/U-118MG-R cell line for expression profiling. Before screening, merging transcripts obtained by splicing samples by using Cuffmerge software, and removing transcripts with uncertain chain directions to obtain complete transcriptome information of the sequencing. The screening process was divided into the following 5 steps (FIG. 1A):
step1 screening for the exon number of transcripts: filtering a large number of single exon transcripts with low expression level and low reliability in the splicing result of the transcriptome, and selecting the transcript with the exon number of 2 (if no special requirement exists, the plant can keep the transcript with the exon number of 1);
step2 transcript Length screening: selecting transcripts with a transcript length >200 bp;
step3 transcript known annotation screening: screening out transcripts overlapping with the exon region of the database annotation by Cuffmatch software, and taking lncRNA overlapping with the exon region of the current spliced transcript in the database as the database annotation lncRNA to be included in subsequent analysis;
step4 screening for transcript expression level: calculating the expression amount of each transcript by using Cuffquant, and selecting a transcript with FPKM ═ 0.5 (the screening threshold is 2 for the single exon transcript);
step5 encoding potential screening: classified as CNCI, CPC, PFAM, phylogenf (when the species is non-mammalian, no phylogenf analysis was performed).
And analyzed by the following software (fig. 1B):
CNCI: by CNCI (Coding-Non-Coding Index) software. CNCI can effectively distinguish between coding and non-coding sequences based on the frequency spectrum of adjacent trinucleotides. CNCI can effectively predict incomplete transcripts and antisense transcript pairs;
performing B L AST comparison on the transcript and a known protein database, and evaluating the Coding Potential of the transcript by the CPC through a classifier of a support vector machine according to the biological sequence characteristics of each Coding frame of the transcript;
PFAM: performed by pfamscan software. The Pfam-A database records the high-quality structural domains of most known proteins, and the Pfam-B database more comprehensively covers the structural domain family and is a supplement to Pfam-A; the protein sequences on each coding frame of the transcript and Pfam-A and Pfam-B databases are subjected to homological search of hmmscan, the sequences of the two databases, namely the transcript with a certain structural domain, can be aligned, and the transcript is considered to have coding capacity, and the transcript which is not obtained is most likely to be non-coding transcript;
phylogens (this analysis was performed for only a fraction of mammals): by means of the PholoCSF software. The Codon replacement frequency (CSF) refers to the frequency of occurrence of a certain Codon replacement in a multiple sequence alignment, and the ratio of the Codon replacement frequency of a coding region to that of a non-coding region is a method for effectively distinguishing whether a sequence can code a protein or not; the phylogenetic tree can be combined with phylogenetic trees to score the coding potential of transcripts by their codon substitution frequency for coding and non-coding regions on the basis of genomic alignments of multiple species of transcripts. phylogencs use whole genome sequence alignment files across multiple species to define whether a segment of a genomic region has coding potential. Through literature query, the phyloyCSF threshold values are different among different species, so that a certain number of known coding and lncRNA genes of the research species are randomly selected to perform threshold value analysis, and then candidate transcript analysis results are screened. This analysis is limited to mammals.
The final analysis resulted in 5166 lncRNA being identified (FIGS. 1C-F). Differential expression analysis was performed using cuffdiff software to screen 94 differentially expressed lncRNA (FIG. 2).
Example 2 screening and validation of lncRNA differentially expressed in glioma TMZ resistant cells
(1) Total RNA extraction from cells (TRIzol method)
Cells were seeded in 6-well plates at 37 ℃ with 5% CO2And (5) incubation in an incubator. After the cells were fully adherent, 1000. mu.l TRIzol was added to each well. After standing to completely dissolve, the mixture was transferred to a 1.5ml EP tube. 0.2ml of chloroform was added thereto, followed by vigorous shaking for 15 seconds and standing at room temperature for 3 min. Centrifugation was carried out at 12000rpm for 15min at 4 ℃ and the supernatant was transferred to a new EP tube. 0.5ml of isopropyl alcohol was added thereto, and the mixture was left at room temperature for 10 min. After centrifugation at 12000rpm for 10min at 4 ℃ a gelatinous precipitate appeared at the bottom of the EP tube. The supernatant was discarded, and the RNA pellet was washed with 1ml 75% ethanol and centrifuged at 7500rpm at 4 ℃ for 5min, and the supernatant was discarded again. After air drying for 5min, 25. mu.l of DEPC water was added and the mixture was thoroughly dissolved by pipetting several times with a pipette tip. Storing at-80 deg.C.
(2) Reverse transcription to synthesize cDNA
① mu.g of RNA was taken and the following reaction components were added to an EP tube
After the addition was complete, the mixture was gently shaken and incubated at 65 ℃ for 5 minutes and then on ice for 1 minute.
② the following substances (cDNA synthesis mixture) were added to an EP tube in order
Mixing, centrifuging and collecting. Reverse transcription program of PCR instrument: at 25 deg.C for 10min and 50 deg.C for 50 min. The reaction was terminated at 85 ℃ for 5min and cooled on ice. The synthesized cDNA was stored at-20 ℃.
(3)Real time-PCR
The PCR primer sequences were as follows:
l NC-004208 (1) -Forward primer 5'-GCCAATCCAAACCGAAAGCC-3'
L NC-004208 (1) -reverse primer 5'-AGACTCAGACCCCAGGCTAC-3'
L NC-004208 (2) -Forward primer 5'-TTTGCTACATCCCAGCTCCA-3'
L NC-004208 (2) -reverse primer 5'-TCCCCCTTTCCCTTGTAGGT-3'.
The expression level of the lncRNA differentially expressed is further verified by Real-Time PCR, and the lncRNA X L OC _024808 is proved to be remarkably highly expressed in a T98G-R, U-118MG-R cell line, and the result is shown in figure 3.
Example 3 correlation of patient survival with IncRNA expression
Survival analysis of L NC _004208 expression in glioma tissue and patient survival and status found that patients with high expression had a progression-free survival (FIG. 4A) and an average survival time that was significantly shorter than those with low or no expression (FIG. 4B).
Example 4 correlation of responsiveness of temozolomide to lncRNA expression in patients
Establishment of human brain glioma tissue sample library
Basic information collection: the basic condition of the patient, such as age, sex, age at onset, etc., personal and family medical history, previous treatment condition, and Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) result are collected preoperatively. Follow-up surveys are performed 6 months after surgery and include postoperative radiation and chemotherapy, tumor size and location (CT, MRI), and patient Progression-free survival (PFS), Overall Survival (OS), etc.
Tumor tissue sample collection: glioma patients to be treated by surgical resection are selected and signed with an informed consent. Tissue samples must be taken under sterile conditions as soon as possible after tissue isolation. Respectively cutting the obtained tissues into a plurality of pieces, putting a part of samples into an aseptic cryopreservation tube with attached patient information, immediately putting the samples into a portable liquid nitrogen bottle, and then transferring the samples into a liquid nitrogen tank of a laboratory for storage, wherein the samples are used for extracting RNA, DNA and total protein of the tissues; fixing another part of specimen in 10% formaldehyde, embedding in paraffin at selected time, and making into paraffin section.
TMZ sensitivity/resistance was defined according to WHO solid tumor recent evaluation criteria: complete Remission (CR), Partial Remission (PR), remission (MR) are defined as TMZ sensitivity; disease Stability (SD), Progression (PD) was defined as TMZ resistance. Wherein, CR means that the tumor focus is completely disappeared and is maintained for at least more than 4 weeks; PR means that the volume of the tumor focus is reduced by more than or equal to 50 percent and is maintained for more than 4 weeks; MR means a 25-50% reduction in the volume of the tumor lesion; SD means that the volume of the tumor focus is increased or reduced by less than 25 percent, and no new focus appears; PD means that the tumor focus increases by more than or equal to 25% in volume or a new focus appears.
Analysis of brain glioma tissue samples
The subject group and the Hunan Yay hospital neurosurgery of the university of China, collected 104 samples of brain glioma, the collected samples were divided into two groups, TMZ sensitive and TMZ drug resistant according to the recent evaluation standard of WHO solid tumor, the RNA expression level of lncRNA L NC-004208 is detected in the brain glioma tissue sample by Real-Time PCR, and whether the lncRNA L NC-004208 expression level has correlation with the brain glioma TMZ drug resistance is analyzed.
The subject group successfully collected the clinical medical record data and follow-up data of 104 patients WHO had undergone TMZ chemotherapy and concurrent glioma excision, and the evaluation criteria of the chemotherapy efficacy were divided into TMZ chemotherapy-resistant patients and TMZ chemotherapy-sensitive patients according to the WHO tumor treatment response criteria and CA125 criteria of 2000. As a result, temozolomide was found to be less responsive in patients with high lncRNA expression than in patients with low expression. The sensitivity of the analyzed model was 56.81%, the specificity 63.63%, see FIG. 5.
Sequence listing
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Claims (10)
1. IncRNA L NC-004208, the sequence of which is shown in SEQ NO 1.
2. The IncRNA L NC-004208 expression quantity detection reagent is applied to preparation of glioma prognosis or TMZ sensitivity detection reagents, and the IncRNA L NC-004208 sequence is shown as SEQ NO: 1.
3. The use of claim 2, wherein the agent for detecting the expression level of IncRNA L NC-004208 is used for detecting glioma tissues.
4. The use of claim 2, wherein the reagent for detecting the expression level of IncRNA L NC-004208 comprises a real-time fluorescent quantitative detection reagent.
5. The use of claim 4, wherein the primers for amplifying lncRNA L NC _004208 comprise one or two pairs of:
6. the use of claim 4, wherein the control β -actin forward primer in the detection reagent is 5'-CATGTACGTTGCTATCCAGGC-3' and the control reverse primer is 5'-CTCCTTAATGTCACGCACGAT-3'.
7. A reagent for brain glioma prognosis or TMZ sensitivity detection is characterized in that the sequence of IncRNA L NC 004208 is shown as SEQ NO:1, and is a reagent for detecting the expression quantity of IncRNA L NC-004208.
8. The reagent for prognosis of brain glioma or detection of TMZ sensitivity of claim 7, wherein the reagent for detecting the expression level of IncRNA L NC-004208 comprises a real-time fluorescence quantitative detection reagent.
9. The reagent for prognosis of brain glioma or detection of TMZ sensitivity according to claim 8, wherein the primers for amplifying lncRNA L NC _004208 comprise one or two pairs of:
l NC-004208 (1) -Forward primer 5'-GCCAATCCAAACCGAAAGCC-3'
L NC-004208 (1) -reverse primer 5'-AGACTCAGACCCCAGGCTAC-3'
L NC-004208 (2) -Forward primer 5'-TTTGCTACATCCCAGCTCCA-3'
L NC-004208 (2) -reverse primer 5'-TCCCCCTTTCCCTTGTAGGT-3'.
10. The reagent for the prognosis of brain glioma or the detection of TMZ sensitivity according to claim 8, wherein the β -actin forward primer used for the control in the detection reagent is 5'-CATGTACGTTGCTATCCAGGC-3', and the reverse primer is 5'-CTCCTTAATGTCACGCACGAT-3'.
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| CN110205320B (en) * | 2019-06-18 | 2023-06-23 | 中国医科大学附属盛京医院 | lncRNA molecule linc00998 and application thereof in glioma treatment/prognosis evaluation |
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