CN116536418A - Plasma tFs/tRNAs marker related to lung adenocarcinoma and application thereof - Google Patents
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Abstract
The invention relates to a plasma tFs/tRNAs marker related to lung adenocarcinoma and application thereof, wherein the plasma tFs/tRNAs marker is one or a combination of tRF-1:29-Pro-AGG-1-M6, tRF-55:76-Tyr-GTA-1-M2; the nucleotide sequence of the tRF-1:29-Pro-AGG-1-M6 is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the tRF-55:76-Tyr-GTA-1-M2 is shown in SEQ ID NO: 2. The invention obtains the plasma tFs/tRNAs marker related to lung adenocarcinoma, and can make diagnosis of lung adenocarcinoma more convenient and easy to implement through development and application of the marker and the diagnostic kit, and provide assistance for finding a novel small molecular drug target with potential therapeutic value.
Description
Technical Field
The invention belongs to the field of biological diagnosis and medicine, and in particular relates to a plasma tFs/tRNAs marker related to lung adenocarcinoma and application thereof.
Background
Lung cancer has long been the most serious malignancy in the world that threatens human life, with the highest subtype being lung adenocarcinoma (LUAD) (Sung et al, 2021; zhang et al, 2020). Despite the great progress in therapy, the 5-year Overall Survival (OS) of LUAD does not exceed 15% because a significant proportion of patients are usually diagnosed when symptoms become apparent (Al-dhearsi et Al 2021). Although low-dose computed tomography (LDCT) is the highest early diagnosis technique for lung adenocarcinoma detection rate in the past, it has significant drawbacks such as high price, high false positive rate, etc. (National Lung Screening Trial Research Team et al., 2011). Traditional serum biomarkers such as carcinoembryonic antigen (CEA) and cytokeratin fragment antigen 21-1 (CYFRA 21-1) often fail to achieve early diagnosis of lung adenocarcinoma due to insufficient specificity (I and Cho, 2015). Therefore, the discovery of new early diagnosis biomarkers has great significance for overcoming lung adenocarcinoma, and provides a practical and effective help for timely discovery, early treatment and even improvement of prognosis of lung adenocarcinoma.
With the aid of high throughput sequencing technology, large amounts of non-coding RNAs (ncrnas) have been shown to play an important role in the development and progression of cancer (Zhou et al, 2011). Transfer RNA derived non-coding small RNAs (tsRNAs) are of great interest, the major members of which are tRNA derived fragments (tRNAs) and tRNA derived stress-induced RNAs (tRNAs), which are obtained by specific cleavage of a precursor or mature tRNA by a nuclease (Xu et al, 2017;Saikia and Hatzoglou,2015). The study of tRNAs/tRNAs was traced back to the 70 s of the 19 th century, when Borek E et al found that a large number of tRNAs/tRNAs in tumor tissue were derived from highly circulating tRNAs (Borek et al, 1977). Thereafter, a series of studies detected a large number of tRNAs/tRNAs in cells, tissues and fluids (Honda et al 2015;Sharma et al, 2016; godoy et al 2018), and demonstrated that the stably present tRNAs/tRNAs play important regulatory functions in gene expression, protein translation and epigenetic modification (Kuscu et al 2018; lyons et al 2020;Watanabe et al, 2011). Recent evidence suggests that tRFs/sirnas are aberrantly expressed in neurological diseases, metabolic diseases and cancers (Oberbauer et al 2018). In particular, studies have shown that tiRNA-Gln-CTG-003, tiRNA-His-GTG-001 and tRF-Ala-AGC-002 are abnormally expressed in advanced ovarian cancer tissues, and that 5' -tRF-LysCTT is significantly over-expressed in bladder cancer patients (Chen et al, 2021;Papadimitriou et al, 2020). In addition, shao Y et al showed that tRF-Leu-CAG was up-regulated in non-small cell lung cancer tumor tissues and cell lines, and its expression in non-small cell lung cancer serum was observed to be significantly correlated with tumor stage progression (Shao et al, 2017). The expression of tRF-31-79MP9P9NH57SD in serum from non-small cell lung cancer patients was found to be elevated by Li J et al and its level of expression was related to clinical staging and lymph node malignancy (Li et al 2022). Therefore, it is reasonable to believe that tRNAs/tRNAs can be candidate molecular markers for monitoring cancer.
At present, related researches on the aspects of tFs/tRNAs and lung adenocarcinoma are not reported yet, and if the plasma tFs/tRNAs which are abnormally expressed in the lung adenocarcinoma can be screened as biomarkers, and corresponding diagnostic kits are developed, the diagnosis status of the lung adenocarcinoma can be greatly promoted.
Disclosure of Invention
The invention aims to provide a plasma tFs/tRNAs marker related to lung adenocarcinoma and application of the marker in preparation of a kit for diagnosing lung adenocarcinoma. In addition, the invention also provides a specific primer of the plasma tFs/tRNAs marker related to lung adenocarcinoma and application thereof.
The aim of the invention is realized by the following technical scheme:
plasma tFs/tRNAs markers associated with lung adenocarcinoma, wherein the plasma tFs/tRNAs markers are one or a combination of tRF-1:29-Pro-AGG-1-M6, tRF-55:76-Tyr-GTA-1-M2; the nucleotide sequence of the tRF-1:29-Pro-AGG-1-M6 is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the tRF-55:76-Tyr-GTA-1-M2 is shown in SEQ ID NO: 2.
The application of the plasma tFs/tRNAs marker in preparing a kit for diagnosing lung adenocarcinoma.
The invention provides a kit for diagnosing lung adenocarcinoma, which comprises primers for amplifying one or a combination of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2.
Wherein, the sequence of the primer used for amplifying the tRF-1:29-Pro-AGG-1-M6 is as follows:
F:5’-GATCGGCTCGTTGGTCTAGG-3'(SEQ ID NO：3)，
R:5’-CTTCCGATCTCGAGAATCATACC-3’(SEQ ID NO：4)。
the sequences of the primers used for amplifying the tRF-55:76-Tyr-GTA-1-M2 are as follows:
F:5’-CAGTCCGACGATCTCGAATCC-3'(SEQ ID NO：5)，
R:5’-GCTCTTCCGATCTTGGTCCTTC-3’(SEQ ID NO：6)。
the kit also comprises an internal reference primer for amplifying an internal reference U6; the sequence of the internal reference primer is as follows:
F:5’-GCTTCGGCAGCACATATACTAAAAT-3’(SEQ ID NO：7)，
R:5’-CGCTTCACGAATTTGCGTGTCAT-3’(SEQ ID NO：8)。
the invention also provides a specific primer of the plasma tFs/tRNAs marker related to lung adenocarcinoma,
the specific primers of the tRF-1:29-Pro-AGG-1-M6 are as follows:
F:5’-GATCGGCTCGTTGGTCTAGG-3'，
R:5’-CTTCCGATCTCGAGAATCATACC-3’；
the specific primers of the tRF-55:76-Tyr-GTA-1-M2 are as follows:
F:5’-CAGTCCGACGATCTCGAATCC-3'，
R:5’-GCTCTTCCGATCTTGGTCCTTC-3’。
the invention also provides application of the specific primer in preparation of lung adenocarcinoma diagnostic reagents.
Compared with the prior art, the invention has the advantages that:
the present invention is based on high throughput sequencing technology and analyzes the expression profile of plasma tFs/tRNAs in lung adenocarcinoma patients and healthy controls. TiRNA-1:34-Val-CAC-2, tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 were screened as candidate tRNAs/tRNAs and further validated by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the diagnostic efficacy of plasma tFs/tRNAs was assessed using the subject's working profile (ROC). Finally, the present invention predicts potential target genes of tFs/tRNAs and their regulatory network by using bioinformatics technology, and further discusses their major cellular biological functions and related molecular mechanisms in lung adenocarcinoma.
According to the invention, 7 tRNAs/tRNAs are screened by qRT-PCR, and the results show that the tRNAs-1:34-Val-CAC-2, tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 are expressed in lung adenocarcinoma and are matched with sequencing results. These three were selected as candidate tRFs/sirnas and used for further ROC curve analysis. Further validation of candidate tFs/tRNAs showed that tRF-55:76-Tyr-GTA-1-M2 was up-regulated in lung adenocarcinoma, while tRF-1:29-Pro-AGG-1-M6 was significantly down-regulated compared to healthy subjects. AUCs of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 reach 0.882 and 0.896, respectively, and show better diagnostic value in patients with lung adenocarcinoma. The present study also assessed their correlation between expression levels in lung adenocarcinoma and clinical pathology features. The results show that the expression of tRF-55:76-Tyr-GTA-1-M2 in lung adenocarcinoma patients is obviously increased, which suggests that the expression is positively correlated with the malignancy of lung adenocarcinoma. Whereas the expression level of tRF-1:29-Pro-AGG-1-M6 was inversely related to the clinical stage of lung adenocarcinoma, indicating that its high expression has great potential in inhibiting tumor progression. Furthermore, the expression of tRF-1:29-Pro-AGG-1-M6 was down-regulated in patients after tumor resection compared to lung adenocarcinoma preoperatively, while the expression level of tRF-55:76-Tyr-GTA-1-M2 was inversely elevated. From this it can be concluded that the expression levels of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 are closely related to the presence of tumors in lung adenocarcinoma patients before and after surgery.
The invention screens and determines that the tRF-55:76-Tyr-GTA-1-M2 and the tRF-1:29-Pro-AGG-1-M6 have higher sensitivity and specificity in diagnosing lung adenocarcinoma.
The invention obtains the plasma tFs/tRNAs marker related to lung adenocarcinoma, and the diagnosis of the lung adenocarcinoma can be more convenient and easy to implement through the development and the application of the plasma tFs/tRNAs marker and the diagnosis kit, thereby providing a foundation for clinical treatment and providing assistance for finding a novel small molecular drug target with potential treatment value.
The kit related to the plasma tFs/tRNAs marker, namely the kit for diagnosing lung adenocarcinoma, can be used for assisting early diagnosis of lung adenocarcinoma patients, is beneficial to reflecting the disease state of the lung adenocarcinoma patients, and provides better support for clinical treatment.
Drawings
FIG. 1 is a graph showing the expression of tRNAs/tRNAs in plasma of patients with lung adenocarcinoma and healthy controls;
wherein (a) the correlation coefficient heat map of all samples, the darker the color in the panel, the higher the correlation coefficient between the two samples; (B) PCA plots of tFs/tRNAs expression profile between lung adenocarcinoma patients and healthy controls; (C) The wien plot shows the number of tRFs/sirnas detected and collected in the tRFdb in this project; (D) Wien figures show tRFs/sirnas that are common and specifically expressed between early and healthy controls, late and healthy controls.
FIG. 2 is a graph of plasma tFs/tRNAs subtype analysis of lung adenocarcinoma patients and healthy controls;
wherein the distribution of the (A-C) tFs/tRNAs subtypes in healthy control group, early stage lung adenocarcinoma and late stage lung adenocarcinoma; (D-F) the number of tRFs/sirnas subtype corresponding to tRNA isoforms in healthy controls, early lung adenocarcinoma and late lung adenocarcinoma; (G-I) subtype frequency of tFs/tRNAs in healthy control group, early lung adenocarcinoma and late lung adenocarcinoma as a function of their length.
FIG. 3 is a graph showing the results of differential expression analysis of tRNAs/tRNAs in early lung adenocarcinoma, late lung adenocarcinoma and healthy controls.
Wherein (a) the hierarchical cluster heatmap shows tRFs/sirnas differentially expressed between early lung adenocarcinoma and healthy controls, late lung adenocarcinoma and healthy controls, early lung adenocarcinoma and late lung adenocarcinoma; (B) Volcanic plot of the differential expression of tRFs/sirnas between early and healthy controls, late and healthy controls; (C) Relative expression levels of 7 tRBs/tRNAs (tRNA1:34-Val-CAC-2, tRF-1:15-Ala-AGC-2-M11, tRF-1:24-Ser-AGA-1-M7, tRF-1:29-Pro-AGG-1-M6, tRF-55:76-Tyr-GTA-1-M2, tRF-59:75-Trp-CCA-1-M5 and tRF-61:77-Thr-AGT-1-M2) in healthy controls, early lung adenocarcinoma and late lung adenocarcinoma; * p <0.05, < p <0.01 and p <0.001; ns, have no meaning.
FIG. 4 is a graph of expression levels and diagnostic value of candidate plasma tRNAs/tRNAs in lung adenocarcinoma;
wherein the relative expression levels of tiRNA-1:34-Val-CAC-2, tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 in the lung adenocarcinoma patient as compared to the normal control; (D, E) diagnostic efficacy of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 in lung adenocarcinoma patients.
FIG. 5 is a graph of the expression levels and diagnostic value of candidate plasma tRNAs/tRNAs both pre-and post-lung adenocarcinoma;
wherein, the relative expression amounts of (A-C) tiRNA-1:34-Val-CAC-2, tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 in the lung adenocarcinoma patients before and after the operation; (D, E) diagnostic efficacy of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 in lung adenocarcinoma patients pre-and post-operatively.
FIG. 6 is a diagram of bioinformatics analysis of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2;
wherein, (A, B) the positions of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 in the secondary structure of the clover of the tRNA and their target targets. (C) target genes for tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2. (D) GO enrichment analysis of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2. (E) KEGG pathway analysis of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2.
FIG. 7 is an amplification curve of tRF-1:29-Pro-AGG-1-M6, tRF-55:76-Tyr-GTA-1-M2, and U6.
FIG. 8 is a melting curve of tRF-1:29-Pro-AGG-1-M6, tRF-55:76-Tyr-GTA-1-M2, and U6.
Detailed Description
The present invention is described in detail below with reference to the drawings and examples of the specification:
1. materials and methods
1.1 clinical information
All plasma samples related to the invention were collected from lung adenocarcinoma patients and healthy persons who had been diagnosed in the foggy province hospital from 1 month 2021 to 3 months 2022.
All lung adenocarcinoma patients were histopathologically confirmed to exclude hypertension, diabetes, severe liver and kidney diseases, metastases and other systemic diseases, 19 men and 28 women, ages 28-80 years, and average ages 56.02 + -10.42 years. Lung adenocarcinomas were staged according to the 2010 international cancer control consortium (UICC)/united states joint cancer committee (AJCC) tumor-lymph node-metastasis (TNM) staging system.
Healthy controls exclude lung disease, tumors and other systemic diseases, 9 men and 12 women, aged 26-77 years, average 47.71 + -12.48 years.
Plasma samples of 4 early lung adenocarcinomas, 4 late lung adenocarcinomas and 4 healthy controls were randomly drawn from all subjects for sequencing analysis, the remaining samples being left for subsequent study.
Plasma samples from 47 lung adenocarcinoma patients were selected for quantitative real-time polymerase chain reaction (qRT-PCR) analysis.
Plasma samples from 12 lung adenocarcinoma patients were selected for evaluation of the expression levels and potential value of tRFs/sirnas before and after surgical excision of lung adenocarcinoma.
Relevant clinical data for all participants was collected and recorded in detail.
The study received written informed consent from all participants and was approved by the ethical committee of the fowless province, hospital (K2021-03-054).
2. Extraction and pretreatment of plasma RNA
First, total RNA was extracted from plasma using TRIzol LS reagent. Then, the concentration and purity of each RNA sample were evaluated using a Nanodrop ND-1000 spectrophotometer. Next, the absorbance of all RNA samples at 260 and 280nm wavelength was measured and the OD260/OD280 ratio was calculated (ratio required to be 1.8-2.1). RNA integrity was then checked using agarose gel electrophoresis. In addition, total RNA was pre-treated to remove some RNA modifications that interfere with the construction of small RNA sequencing libraries. The process is as follows: 3' -aminoacyl (charged) deacylation is 3' -OH for 3' -linker ligation; 3' -cP (2 ',3' -cyclic phosphate) is 3' -OH for 3' -linker ligation; 5' -OH (hydroxy) phosphorylates to 5' -P for 5' -linker ligation; m1A and m3C are demethylated to achieve efficient reverse transcription.
3. Library preparation and tRBS/tRNAs sequencing
The pretreated total RNA was used to prepare a sequencing library. First, total RNA from each sample was ligated to 3 'and 5' small RNA adaptors in sequence. cDNA was then synthesized and amplified using the specific RT primers and amplification primers of Illumina. Subsequently, 134-160 bp PCR amplified fragments were extracted and purified from the PAGE gel. Finally, the completed library was quantified by an Agilent 2100 Bioanalyzer. These libraries were denatured into single stranded DNA molecules, captured on Illumina flow cells, amplified in situ as sequencing clusters, and sequenced for 50 cycles on Illumina NextSeq 500 system according to the manufacturer's instructions.
Data analysis of tRBS/tRNAs sequencing
Image analysis and base calls were performed using Solexa pipeline v 1.8.8 (Off-Line Base Caller software, v 1.8). Sequencing quality was checked by FASTQC. The raw data file in FASTQC format was generated by Illumina sequencer. To check sequencing quality, a mass score map is drawn for each sample. The quality score Q is logarithmically related to the base call error probability (P). After checking the sequencing quality with Illumina, the sequencing reads were trimmed for 5', 3' -adapter, the useless reads were removed (length <14nt or length >40 nt) and recorded in FASTA format. The FASTA format trimmed reads allowed only 1 mismatch with the mature tRNA sequence, then the unmapped reads were aligned using the bowtie software, allowing only 1 mismatch with the precursor tRNA sequence. The remaining reads were aligned, allowing only 1 mismatch with the mirnas reference sequence of mirdieep 2. The abundance of tRFs/sirnas was assessed using their sequencing counts and normalized to parts per million of Counts (CPM) Ji Douqu. Based on the alignment statistical analysis (alignment rate, read length, fragment sequence bias), we determined whether the results were available for subsequent data analysis. If possible, expression profiles and differentially expressed tRNAs/tRNAs and miRNAs were calculated. Fold change of 1.5 or more and p value of 0.05 or less were used to screen for differentially expressed tFs/tRNAs. The Principal Component Analysis (PCA), correlation analysis, pie charts, wen charts, hierarchical clustering, scatter charts and volcanic charts of the differentially expressed tRNAs/tRNAs were statistically calculated and plotted in R or perl language.
qrt-PCR analysis
The rtStar was used separately according to the manufacturer's instructions TM tRF&tiRNA Pretreatment Kit and rtStar TM First-Strand cDNA Synthesis Kit was subjected to RNA pretreatment and cDNA synthesis. The synthesized cDNA was analyzed by qRT-PCR on a LightCycler480 real-time quantitative PCR system (Roche, switzerland) according to the protocol of the manufacturer 2X PCR Master Mix Kit. All reactions were performed in triplicate, and the relative expression level of tFs/tRNAs was 2 -ΔΔCt And 2 -ΔCt The method performs calculation by taking U6 as an internal reference. Among them, specific primers for amplifying sequences are shown in Table 1.
5.1, preparing a real time PCR reaction system for all cDNA samples. The system configuration is as follows:
the solution was mixed at the bottom of the flick tube and centrifuged briefly at 5000 rpm.
5.2, sample addition
a. 8ul of the mixture was added to each well corresponding to 384-PCR plates.
b. Then, 2. Mu.l of the corresponding cDNA was added.
c. Carefully stick the Sealing Film and briefly mix by centrifugation.
d. The prepared PCR plate was placed on ice before setting up the PCR program.
5.3, placing the 384-PCR plate on a real time PCR instrument for PCR reaction.
All indexes are carried out according to the following procedures: 95 ℃ for 10min;40 PCR cycles (95 ℃,10 seconds; 60 ℃,60 seconds (fluorescence collected)). To establish a melting curve of the PCR product, after the amplification reaction is completed, the PCR product is subjected to the amplification reaction (95 ℃,10 seconds; 60 ℃,60 seconds; 95 ℃ 15 seconds); and slowly heated from 60 ℃ to 95 ℃ (instrument automated-Ramp Rate 0.075 ℃/sec).
5.4 results and calculations
The target gene and housekeeping gene (U6) of each sample were subjected to the real time PCR reaction, respectively. According to the drawn gradient diluted DNA standard curve, the concentration results of the genes of each sample order and housekeeping genes are directly generated by a machine. The concentration of the gene of interest in each sample is divided by the concentration of its housekeeping gene, i.e., the corrected relative content of this gene for that sample.
As shown in FIG. 7, in the normal control blood sample and the lung adenocarcinoma blood sample, the melting curves of tRF-1-29-Pro-AGG-1-M6 (FIG. 7 a), tRF-55-76-Tyr-GTA-1-M2 (FIG. 7 b) and internal reference U6 ((FIG. 7 c) were amplified efficiently, consistent with the results of gene sequencing FIG. 8 shows that the melting curves of tRF-1-29-Pro-AGG-1-M6 (FIG. 8 a), tRF-55-76-Tyr-GTA-1-M2 (FIG. 8 b) and internal reference U6 (FIG. 8 c) were all good, and the melting curves showed that the specificity of the primers corresponding to the single specific peak.
TABLE 1 primer sequences for qRT-PCR
Wherein F represents the forward (upstream primer); r represents the reverse direction (downstream primer)
6. Electrochemiluminescence method for determining CEA, NSE and SCC expression in serum
The expression levels of serum CEA, NSE and SCC were quantified on a Cobas E602 machine (Roche Diagnostics, switzerland) using the original kit according to the manufacturer's instructions. The critical values for CEA, NSE and SCC are 5ng/mL, 16.3ng/mL and 2.7ng/mL, respectively.
Bioinformatics analysis of tRBS/tRNAs
The exact location of each tRF in the secondary structure of the derived tRNA was determined according to the GtRNAdb database (http:// GtRNAdb. Ucsc. Edu /). The potential target genes for tRNAs/tRNAs were then mined by the TargetScan (http:// www.targetscan.org/vert_72 /) and Miranda (http:// www.microrna.org/microra /) databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis was performed on tRFs/sirnas using KOBAS 3.0 software.
8. Statistical analysis
Statistical analysis was performed using SPSS Statistics 24.0 (SPSS, chicago, IL) and GraphPad Prism 8.0 software (GraphPad Software, la Jolla, calif.). Unpaired t-test was used to evaluate the differences between all lung adenocarcinoma patients and healthy control groups. Paired t-test was used to assess the pre-and post-operative differences in lung adenocarcinoma patients. Diagnostic value is determined by calculating the Area Under Curve (AUC) from a subject operating characteristic (ROC) curve. All measured data are expressed as mean and Standard Error of Mean (SEM). The Johnson index was used to calculate the optimal cut-off value for tFs/tRNAs. p <0.05 is considered statistically significant.
9. Results
9.1 expression profiling of tFs/tRNAs in plasma
As a core criterion for measuring the rationality and reliability of sample selection, the closer the value of the sample correlation coefficient is to 1, the higher the similarity between any two samples. The study shows that the selected 12 plasma samples are suitable for the sequencing analysis by calculating the sample correlation coefficient, and the result is accurate and reliable (figure 1A). The results of PCA showed significant differences in the expression profile of tFs/tRNAs between lung adenocarcinoma patients and healthy controls (FIG. 1B). As shown in FIG. 1C, a total of 506 tRNA derivatives, including 431 novel tRNA derivatives that were not annotated in the tR fdb database, were identified by sequencing analysis of tRs/tRNAs. Furthermore, as shown in FIG. 1D, there are tRNAs/tRNAs that are co-and specifically expressed between early lung adenocarcinoma and healthy control group, late lung adenocarcinoma and healthy control group, early and late lung adenocarcinoma.
9.2 plasma tFs/tRNAs subtype analysis
The pie chart of subtype tRFs/sirnas distribution shows that the number of tRFs/sirnas per subtype varies in early lung adenocarcinoma, late lung adenocarcinoma and healthy control group (fig. 2A, 2B, 2C). The amounts of tRF-1, tRF-3a, tRF-3b and tRF-5a were increased in early and late stage lung adenocarcinoma compared to the normal control group. Furthermore, tRF-1 and tRF-5b were significantly elevated in advanced lung adenocarcinoma compared to early lung adenocarcinoma. As can be seen from parts D, E and F of FIG. 2, arg-TCT, leu-TAA, phe-GAA and Ser-CGA contained in lung adenocarcinoma are four tRNA isomers that are not present in healthy controls. Furthermore, although tRNA isoforms have identical anticodons, the types and numbers of tRs and the subtypes of tRNAs are not identical in healthy control, early and late lung adenocarcinomas. The frequencies of the tRNAs/tRNAs subtypes in tRNAs of different sequence lengths are also different. As shown in fig. 2G, 2H and 2I, the present study further found that there was also a significant difference in the frequency of tRFs/sirnas subtypes with the same sequence length in the control, early and late lung adenocarcinoma.
9.3 analysis of differential expression of tRNAs/tRNAs
Unsupervised hierarchical cluster heatmaps showed significant changes in tRFs/sirnas expression between any two of the healthy control group, early lung adenocarcinoma group, and late lung adenocarcinoma group (fig. 3A). As shown in FIG. 3, panel B, there were 40 up-regulated and 34 down-regulated tFs/tRNAs between the early lung adenocarcinoma patient and healthy controls; among tRNAs/tRNAs differentially expressed between advanced lung adenocarcinoma and healthy controls, 24 were up-regulated and 25 were down-regulated; there were 23 increased expression of tFs/tRNAs and 16 decreased expression of tFs/tRNAs in patients with advanced lung adenocarcinoma compared to early lung adenocarcinoma. According to CPM results, 7 tRNAs/tRNAs showing higher homogeneity and heterogeneity among the different groups (tRNA1:34-Val-CAC-2, tRNA1:15-Ala-AGC-2-M11, tRNA1:24-Ser-AGA-1-M7, tRNA1:29-Pro-AGG-1-M6, tRNA55:76-Tyr-GTA-1-M2, tRNA59:75-Trp-CCA-1-M5 and tRNA61:77-Thr-AGT-1-M2) were selected and their expression levels were evaluated by qRT-PCR. The results showed that tiRNA-1:34-Val-CAC-2, tRF-1:24-Ser-AGA-1-M7, tRF-1:29-Pro-AGG-1-M6, tRF-55:76-Tyr-GTA-1-M2 and tRF-61:77-Thr-AGT-1-M2 were differentially expressed in lung adenocarcinoma (FIG. 3C). Finally, the project selects 3 tFs/tRNAs with most significant differential expression (tRNA1:34-Val-CAC-2, tRNA1:29-Pro-AGG-1-M6 and tRNA55:76-Tyr-GTA-1-M2) as candidate tRNAs/tRNAs for subsequent study according to the relative expression amount of each tFs/tRNAs.
9.4 expression levels and diagnostic efficacy of candidate plasma tRs/tRNAs in lung adenocarcinoma
The specific expression levels of tiRNA-1:34-Val-CAC-2, tRF-1:29-Pro-AGG-1-M and tRF-55:76-Tyr-GTA-1-M2 in the plasma of patients with lung adenocarcinoma were further analyzed by qRT-PCR. Compared with the normal control group, the expression level of tRF-1:29-Pro-AGG-1-M6 in lung adenocarcinoma is significantly reduced, while the expression level of tRF-55:76-Tyr-GTA-1-M2 is significantly up-regulated. (FIGS. 4B, 4C). However, as shown in FIG. 4A, there was no significant difference in expression of plasma tiRNA-1:34-Val-CAC-2 in lung adenocarcinoma compared to healthy controls. The diagnostic value of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 in lung adenocarcinoma was further analyzed on this basis. Wherein the AUCs of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 were 0.882 (95% CI=0.794-0.970) and 0.896 (95% CI=0.821-0.970), respectively. Furthermore, the optimal cut-off values for tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 were 0.9575 (sensitivity 85.7%, specificity 76.6%) and 2.277 (sensitivity 78.7%, specificity 85.7%). It follows that tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 have great potential in the diagnosis of lung adenocarcinoma.
9.5 correlation of expression levels of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 with clinical pathological characteristics
The correlation of the expression levels of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 with the clinical pathology was further evaluated. As shown in Table 2, the expression of tRF-1:29-Pro-AGG-1-M6 was related to TNM stage, N stage, and CEA expression level, while there was no apparent correlation with age, sex, T stage, M stage, diameter, and NSE and SCC expression levels. Furthermore, the expression of tRF-55:76-Tyr-GTA-1-M2 in lung adenocarcinoma was significantly correlated with TNM stage, T stage, N stage, M stage, diameter and expression levels of CEA and SCC, but was independent of age, sex and expression levels of NSE.
TABLE 2 correlation of expression levels of tRF-1-29-Pro-AGG-1-M6 and tRF-55-76-Tyr-GTA-1-M2 with clinical pathological characteristics of patients with lung adenocarcinoma
9.6 expression and potential value of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 in lung adenocarcinoma preoperatively and postoperatively
The value of 3 candidate tRFs/sirnas in monitoring lung adenocarcinoma treatment was assessed by analyzing their expression in 12 pairs of pre-and post-operative plasma samples of lung adenocarcinoma. Although there was no difference in expression of tiRNA-1:34-Val-CAC-2 before and after lung adenocarcinoma surgery, there was a significant down-and up-regulation of expression of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2, respectively, after lung adenocarcinoma surgery compared to pre-surgery in lung adenocarcinoma patients. Further ROC analysis demonstrated that expression levels of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 before and after lung adenocarcinoma surgery could be effective to help distinguish between the patient's treatment states, with AUCs of 0.899 (95% ci=0.770-1.000) and 0.896 (95% ci=0.745-1.000), respectively. The above results demonstrate that tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 can be used as valuable plasma markers for judging the surgical efficacy of patients with lung adenocarcinoma.
9.7 prediction and functional analysis of potential target genes of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2
In addition to exhibiting positions of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 on the clover secondary structure of their respective tRNA, FIGS. 6A and 6B also show their respective target targets. the regulatory network diagrams of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 show that one tsRNA is capable of corresponding to multiple mRNAs (FIG. 6C). GO functional analysis shows that the target genes of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 are widely distributed in cytoplasm, nucleus and cytoplasm and play important roles in nucleus. Furthermore, both target genes can also play a role in cell growth and development by promoting biological processes such as protein binding and the same protein binding (fig. 6D). KEGG pathway enrichment analysis showed that the target genes for tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 were mainly enriched in cancer-associated signaling pathways, including metabolic pathways, pyrimidine metabolism, MAPK signaling pathways, calcium signaling pathways, and HIF-1 signaling pathways, etc. (FIG. 6E).
10. Analysis of the invention
Lung adenocarcinoma is the most common subset of lung cancers among cancer patients today. The key cause of poor prognosis of lung adenocarcinoma is the inability of many patients to obtain timely and effective diagnosis and treatment. TsRNA is an emerging biomarker that has been discovered by more and more researchers for its stable presence in the circulation of body fluids and is actively used for diagnosis, treatment and monitoring of diseases (Xu et al, 2017; zhang et al, 2021). Recent studies have shown that the key members tRFs and sirnas of tsrnas are biomarkers of great potential that can affect tumor development and progression by acting on protein translation and gene expression in tumor cells (Ivanov et al, 2011;Haussecker et al, 2010; zhu et al, 2020). The invention analyzes the expression profile of tFs/tRNAs in the plasma of lung adenocarcinoma patients based on a high-throughput sequencing technology. It was unexpectedly found that 431 new tRFs/sirnas were not annotated in the tRFdb database and that a more thorough study of them would help to explore their value in lung adenocarcinoma. Furthermore, there were 350 and 344 differentially expressed tRFs/sirnas in early and late lung adenocarcinoma, respectively, compared to healthy controls. Identification of complementary subtypes of tFs/tRNAs revealed that lung adenocarcinoma patients had abnormally elevated levels of expression of tRF-1, tRF-3a, tRF-3b, and tRF-5 a. Further analysis found that the subtypes tRF-1, tRF-3a, tRF-3b and tRF-5a, which correspond to the tRNA isoforms Arg-TCT, leu-TAA, phe-GAA and Ser-CGA, respectively, were not expressed in the healthy control group, but were abnormally expressed in lung adenocarcinoma. Furthermore, studies have indicated that tRNAs/tRNAs are differentially expressed in tissue samples of lung adenocarcinoma. From another point of view, the study reveals that the tRNAs/tRNAs are abnormally expressed in the plasma samples of patients with lung adenocarcinoma, which also provides support for exploring the tRNAs/tRNAs as potential biomarkers of lung adenocarcinoma.
According to the invention, 7 tRNAs/tRNAs are screened by qRT-PCR, and the results show that the tRNAs-1:34-Val-CAC-2, tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 are expressed in lung adenocarcinoma and are matched with sequencing results. These three were selected as candidate tRFs/sirnas and used for further ROC curve analysis. Further validation of candidate tFs/tRNAs showed that tRF-55:76-Tyr-GTA-1-M2 was up-regulated in lung adenocarcinoma, while tRF-1:29-Pro-AGG-1-M6 was significantly down-regulated compared to healthy subjects. In contrast, AUCs of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 reached 0.882 and 0.896, respectively, showing better diagnostic value in patients with lung adenocarcinoma. More encouraging, the present invention also evaluates their correlation between expression levels in lung adenocarcinoma and clinical pathology. The results show that the expression of tRF-55:76-Tyr-GTA-1-M2 in lung adenocarcinoma patients is obviously increased, which suggests that the expression is positively correlated with the malignancy of lung adenocarcinoma. Whereas the expression level of tRF-1:29-Pro-AGG-1-M6 was inversely related to the clinical stage of lung adenocarcinoma, indicating that its high expression has great potential in inhibiting tumor progression. Furthermore, the expression of tRF-1:29-Pro-AGG-1-M6 was down-regulated in patients after tumor resection compared to lung adenocarcinoma preoperatively, while the expression level of tRF-55:76-Tyr-GTA-1-M2 was inversely elevated. From this it can be concluded that the expression levels of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2 are closely related to the presence of tumors in lung adenocarcinoma patients before and after surgery. The invention screens and determines that the tRF-55:76-Tyr-GTA-1-M2 and the tRF-1:29-Pro-AGG-1-M6 have higher sensitivity and specificity in diagnosing lung adenocarcinoma.
The present invention explores downstream regulatory mechanisms of tRF-55:76-Tyr-GTA-1-M2 and tRF-1:29-Pro-AGG-1-M6 to find that they are involved in a variety of key biological signaling pathways, such as metabolic pathways, pyrimidine metabolism, calcium signaling pathways, MAPK signaling pathways and HIF-1 signaling pathways. The results indicate that tRF-55:76-Tyr-GTA-1-M2 and tRF-1:29-Pro-AGG-1-M6 will affect the occurrence and progression of lung adenocarcinoma through these tumor-associated signaling pathways, providing directions for us to further explore their mechanisms.
In summary, the study of the present invention reveals the expression profile of tFs/tRNAs in plasma of patients with lung adenocarcinoma and determines that tRF-55:76-Tyr-GTA-1-M2 and tRF-1:29-Pro-AGG-1-M6 can be used as novel biomarkers for diagnosing lung adenocarcinoma.
Claims (9)
1. A plasma tRFs/sirnas marker associated with lung adenocarcinoma, characterized in that: the plasma tFs/tRNAs markers are one or a combination of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2; the nucleotide sequence of the tRF-1:29-Pro-AGG-1-M6 is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the tRF-55:76-Tyr-GTA-1-M2 is shown in SEQ ID NO: 2.
2. Use of plasma tRFs/sirnas markers for the preparation of a kit for diagnosing lung adenocarcinoma, characterized in that: the plasma tFs/tRNAs markers are one or a combination of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2; the nucleotide sequence of the tRF-1:29-Pro-AGG-1-M6 is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the tRF-55:76-Tyr-GTA-1-M2 is shown in SEQ ID NO: 2.
3. The use according to claim 2, wherein: the primer sequences used for amplifying the tRF-1:29-Pro-AGG-1-M6 are:
F:5’-GATCGGCTCGTTGGTCTAGG-3'，
R:5’-CTTCCGATCTCGAGAATCATACC-3’；
the primer sequences used for amplifying the tRF-55:76-Tyr-GTA-1-M2 are as follows:
F:5’-CAGTCCGACGATCTCGAATCC-3'，
R:5’-GCTCTTCCGATCTTGGTCCTTC-3’。
4. a kit for diagnosing lung adenocarcinoma, characterized in that: it comprises primers for amplifying one or a combination of the tRF-1:29-Pro-AGG-1-M6, tRF-55:76-Tyr-GTA-1-M2.
5. The kit of claim 4, wherein: the sequences of the primers used for amplifying the tRF-1:29-Pro-AGG-1-M6 are:
F:5’-GATCGGCTCGTTGGTCTAGG-3'，
R:5’-CTTCCGATCTCGAGAATCATACC-3’。
6. the kit of claim 4, wherein: the sequences of the primers used for amplifying the tRF-55:76-Tyr-GTA-1-M2 are as follows:
F:5’-CAGTCCGACGATCTCGAATCC-3'，
R:5’-GCTCTTCCGATCTTGGTCCTTC-3’。
7. the kit of claim 4, wherein: it also includes an internal reference primer for amplifying the internal reference U6; the sequence of the internal reference primer is as follows:
F:5’-GCTTCGGCAGCACATATACTAAAAT-3’，
R:5’-CGCTTCACGAATTTGCGTGTCAT-3’。
8. a specific primer for plasma tRFs/sirnas markers associated with lung adenocarcinoma, characterized in that: the plasma tFs/tRNAs markers are one or a combination of tRF-1:29-Pro-AGG-1-M6 and tRF-55:76-Tyr-GTA-1-M2;
the specific primers of the tRF-1:29-Pro-AGG-1-M6 are as follows:
F:5’-GATCGGCTCGTTGGTCTAGG-3'，
R:5’-CTTCCGATCTCGAGAATCATACC-3’；
the specific primers of the tRF-55:76-Tyr-GTA-1-M2 are as follows:
F:5’-CAGTCCGACGATCTCGAATCC-3'，
R:5’-GCTCTTCCGATCTTGGTCCTTC-3’。
9. the use of the specific primer according to claim 8 for the preparation of a lung adenocarcinoma diagnostic reagent.
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