CN116144772B - Application of Hsa_circ_0006117 in preparation of lung adenocarcinoma treatment drugs - Google Patents

Application of Hsa_circ_0006117 in preparation of lung adenocarcinoma treatment drugs Download PDF

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CN116144772B
CN116144772B CN202211448618.5A CN202211448618A CN116144772B CN 116144772 B CN116144772 B CN 116144772B CN 202211448618 A CN202211448618 A CN 202211448618A CN 116144772 B CN116144772 B CN 116144772B
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徐秋月
赵婷
段勇
张艳亮
龚容
赵滢
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First Affiliated Hospital of Kunming Medical University
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Abstract

The invention discloses an application of Hsa_circ_0006117 in preparing a medicine for treating lung adenocarcinoma. The relative expression level of Hsa_circ_0006117 in the plasma of a patient with the LUAD is related to the invasion phenomenon of the disease and participates in promoting the occurrence and development of the invasion phenomenon of the LUAD. Bioinformatics analysis showed that hsa_circ_0006117 promoted tumor progression in LUAD by mirnas spike action.

Description

Application of Hsa_circ_0006117 in preparation of lung adenocarcinoma treatment drugs
Technical Field
The invention belongs to the technical field of molecular biology and immunology, and particularly relates to an application of Hsa_circ_0006117 in preparation of a lung adenocarcinoma treatment drug.
Background
Lung cancer is one of the common malignant tumors that are severely threatening the health and life of humans worldwide. GLOBOCAN statistics show that in 2020, the global new cases of lung cancer account for about 11.4% of all new cancer cases, and the related cases of death account for about 18.0% of cancer cases of death. In recent years, lung adenocarcinoma (Lung Adenocarcinoma, LUAD) has become the most common histological type in Non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC), with significantly higher incidence of lung squamous carcinoma, accounting for about 40% of lung cancer. Early lung adenocarcinoma generally has no obvious symptoms, is often accompanied by invasion of nerves, pleura and blood vessels or lymphosis during diagnosis, and has poor prognosis. Although in recent years, molecular targeted therapy, immunotherapy and other therapeutic means and clinical management of lung cancer have been significantly advanced, only less than 10% of advanced patients have a survival period of more than five years.
At present, clinical tumor examination is mainly based on imaging examination and histopathological biopsy. The sensitivity of the imaging examination mainly depends on the resolution and the focus size, objectivity exists in judging the tumor property, and certain radioactive damage can be generated on the organism; histopathological biopsies are the gold standard for tumor diagnosis, but up to 25% of lung cancer biopsies fail to evaluate due to failure to obtain sufficient tissue. The conventional serological tumor markers in clinic at present often cannot meet the clinical requirements in detection sensitivity and specificity. With the rapid development of high throughput RNA sequencing technology and molecular biology, liquid biopsy is receiving increasing attention in the biological field. Liquid biopsy is a non-invasive biopsy method that has been studied by scientists and oncologists for several years to reflect tumor information by identifying and quantifying circulating-derived tumor biological material in body fluids, including circulating tumor cells (circulating tumor cell, CTCs), circulating tumor DNA (circulating tumor DNA, ctDNA), circulating tumor RNA (circulating tumor RNA, ctRNA), outer vesicles (extracellular vesicles, EV), and tumor education platelets (tumor-educated platelets, TEP), among others.
With intensive studies on the biological functions and unique molecular structures of Circular RNAs (circrnas), the results indicate that circrnas are one of ideal candidates for novel liquid biopsy markers for tumors. CircRNA is a class of RNA molecules with unique covalently closed structures formed by reverse splicing of precursor RNAs, and is widely expressed in eukaryotes and viruses. Research shows that the circRNA is mainly involved in regulating various pathophysiological processes of tumor cells such as cell stem property, cell cycle, apoptosis, autophagy, angiogenesis, replication immortalization, cytoenergetics and the like through three action mechanisms of miRNA (ribonucleic acid), interaction with proteins (protein sponge, protein scaffold and protein recruitment) and serving as a translation template, and plays an important biological role in the formation and disease progression of tumors. Studies have shown that circrnas are generally expressed tissue-specific and even cell-type-specific, their relative stability and detectability in various body fluids (such as plasma, saliva and urine) has been demonstrated, and the presence of specific circrnas in body fluids has been demonstrated by high throughput sequencing methods.
However, to date, circRNA still faces significant challenges and difficulties in research and clinical transformations in the field of tumor fluid biopsy: (1) the naming rules of the circRNA are not unified, so that the rapid development of the whole research field is greatly hindered; (2) because the abundance of the circRNA in the body fluid is relatively low, the existing RNA extraction and enrichment method is difficult to accurately detect and identify in the body fluid; (3) most of the related researches are basically focused on basic researches, heterogeneity exists among research platforms, no mature standardized workflow exists, comparability and consistency are lacking, and repeatability and traceability of clinical detection are directly affected.
Disclosure of Invention
In view of the above, the present invention provides an application of hsa_circ_0006117 in preparing a medicament for treating lung adenocarcinoma.
In order to solve the technical problems, the invention discloses an application of Hsa_circ_0006117 in preparing a medicine for treating lung adenocarcinoma.
Alternatively, hsa_circ_0006117 is differentially expressed in lung adenocarcinoma (Lung Adenocarcinoma, LUAD) tissue and paracancerous tissue, hsa_circ_0006117 being over-expressed in LUAD tissue compared to paired paracancerous tissue.
Alternatively, hsa_circ_0006117 was differentially expressed in the LUAD patient and healthy control group plasma, and hsa_circ_0006117 was significantly higher in the LUAD patient plasma than in the healthy control group.
Alternatively, the relative expression level of hsa_circ_0006117 in the plasma of a LUAD patient is correlated with the disease invasion phenomenon, and participates in promoting the occurrence and development of the LUAD invasion phenomenon.
Alternatively, plasma hsa_circ_0006117 is significantly differentially expressed pre-operatively and post-operatively; hsa_circ_0006117 may promote tumor progression of LUAD through miRNA-spike effects.
Compared with the prior art, the invention can obtain the following technical effects:
1) Hsa_circ_0006117 has potential clinical significance as a novel diagnostic marker for LUAD.
2) The relative expression level of the plasma hsa_circ_0006117 of the LUAD patient is related to the invasion phenomenon of the disease, and the relative expression level is obviously and differently expressed before and after the operation, and the relative expression level participates in the occurrence and development process of promoting the invasion phenomenon of the LUAD.
3) Bioinformatics analysis showed that hsa_circ_0006117 promoted tumor progression of LUAD by miRNA spike action.
Of course, it is not necessary for any of the products embodying the invention to achieve all of the technical effects described above at the same time.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a graph showing the number of circRNAs differentially expressed between lung cancer and paracancerous tissues at different times in accordance with the present invention;
FIG. 2 is a schematic representation of the structure of candidate circRNAs of the present invention;
FIG. 3 is a basic formation of the circRNA of the present invention;
FIG. 4 shows the gel electrophoresis results of cDNA and gDNA amplification products of Hsa_circ_0006117, hsa_circ_0007418 and hsa_circ_0000288 of the present invention;
FIG. 5 shows the comparison result of Hsa_circ_0006117circRNA chip analysis result and circbase;
FIG. 6 shows the comparison result of Hsa_circ_0000288circRNA chip analysis result and circbase;
FIG. 7 shows the comparison result of Hsa_circ_0007418circRNA chip analysis result and circbase;
FIG. 8 shows the expression differences of hsa_circ_0006117 and hsa_circ_0000288 before and after RNase digestion according to the present invention;
FIG. 9 is a standard curve of beta-actin (A), hsa_circ_0006117 (B) and housekeeping gene hsa_circ_0000288 (C) according to the present invention;
FIG. 10 is a graph showing the differential expression of hsa_circ_0006117 and hsa_circ_0000288 in LUAD tissue and paracancerous tissue by QRT-PCR of the present invention;
FIG. 11 is a graph showing the differential expression of hsa_circ_0006117 in plasma samples of the LUAD group and healthy control group by QRT-PCR;
FIG. 12 is a ROC curve of the invention evaluating the diagnostic value of plasma hsa_circ_0006117 for LUAD;
FIG. 13 is a graph showing the relationship between the relative expression level of hsa_circ_0006117 in plasma and the LUAD invasion phenomenon according to the present invention;
FIG. 14 is a graph of a predicted course of lung adenocarcinoma patient nomograms according to the present invention;
FIG. 15 is a calibration curve of a nomographic invasion prediction model of a patient with lung adenocarcinoma according to the invention;
FIG. 16 is a graph of the predictive power of the DCA curve evaluation lung adenocarcinoma patient nomogram invasion predictive model of the present invention;
FIG. 17 is a graph of the ROC curve of the present invention evaluating the predictive power of a nomographic invasion predictive model for patients with lung adenocarcinoma;
FIG. 18 is a comparison of relative expression levels of hsa_circ_0006117 in post-operative plasma of a LUAD patient of the invention;
FIG. 19 is a diagram showing the network of miRNA-mRNA interactions of the bioinformatics analysis of the present invention predicting hsa_circ_ 0006117;
FIG. 20 shows the GO analysis results of the Hsa_circ_0006117 target gene of the present invention;
FIG. 21 shows the GO analysis results of the Hsa_circ_0006117 target gene of the present invention;
FIG. 22 shows the results of a KEGG analysis of the Hsa_circ_0006117 target gene of the present invention.
Detailed Description
The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how to apply the technical means to solve the technical problems and achieve the technical effects of the present invention can be fully understood and implemented.
Example 1 sample preparation
1.1 collection and processing of tissue samples
Collecting fresh tumor tissue specimens from the operation excision of lung cancer patients in the thoracic surgery of the hospital and the thoracic surgery of the aged from 9 months in 2020 to 11 months in 2021, and obtaining the tissue beside the cancer, which is more than or equal to 3cm away from the tumor tissue. Inclusion and exclusion criteria were as follows:
inclusion criteria: (1) the diagnosis of the disease is LUAD; (2) no treatment is received before the operation, including operation, radiotherapy, chemotherapy, immunotherapy, etc.; (3) patient clinical information (e.g., sex, age, smoking history, drinking history, date of surgery, histological type of tumor, size, serological tumor marker detection data, presence or absence of lymph node metastasis and invasion phenomena, etc.) was collected intact.
Exclusion criteria: (1) combining other malignant tumors; (2) suffering from immune system diseases (including systemic lupus erythematosus, rheumatoid arthritis, hyperthyroidism, etc.); (3) suffering from chronic wasting diseases (including tuberculosis, diabetes, etc.); (4) with infectious diseases.
Specimen processing: (1) preparation: adding at least 1.5mL RNAstore sample preservation solution (at least 1mL RNAstore is needed for every 100mg tissue sample) into the cryopreservation tube before collecting the sample; (2) tissue cutting: cutting the tissue sample into small fragments with the thickness of any side not exceeding 0.5cm as far as possible, placing the small fragments into RNAstore preservation solution, and placing the small fragments in a refrigerator at 4 ℃ for permeation overnight; (3) preserving at low temperature: when the sample needs to be stored for a long time, the sample is transferred to a refrigerator at the temperature of-80 ℃ for storage until the sample is used.
1.2 collection and processing of plasma samples
EDTA anticoagulated plasma samples collected from patients with chest surgery (including aged chest surgery) before and after surgical treatment of the LUAD and healthy subjects in this hospital from 9 in 2020 to 11 in 2021 were respectively the LUAD group and the healthy control group.
The inclusion criteria for each group were: (1) LUAD group: with the LUAD tissue specimen group; (2) healthy control group: no obvious abnormality in chest or lung CT results; the detection results of various conventional laboratories are not obviously abnormal.
The exclusion criteria for each group were the same as for the LUAD tissue sample group.
Specimen processing: (1) low speed centrifugation: centrifuging at 4deg.C for 10min at 3000r/min, transferring the plasma layer into 1.5mL sterile EP tube; (2) split charging and preserving: and (3) subpackaging and storing the centrifuged plasma sample in sterile freezing tubes according to the sample amount, wherein each tube is at least required to be preserved for 300L, marks are made on the tube wall, and the blood plasma sample is immediately stored in a refrigerator at-80 ℃ until being used.
The study was passed by the ethical committee of the university of Kunming medical science (approved ethical number: 8216080364) and enrolled patients had filled in an informed consent.
1.3 statistical analysis
Statistical analysis and mapping was performed using open source statistical software and GraphPad rrrism (version 8.0.1). The metering data are described as arithmetic mean ± standard deviation. The relative expression levels of the target circrnas between the cancerous and paracancerous tissues, preoperative and postoperative groups were analyzed using paired T-test (normal distribution) or Wilcoxon signed rank test (non-normal distribution), and two independent samples were used between the remaining control and experimental groups, either T-test (normal distribution) or Mann-Whitney U-test (non-normal distribution). ROC curves were constructed to evaluate the potential clinical application value of the circRNA of interest as a LUAD liquid biopsy diagnostic marker. The correlation between the plasma relative expression level of the target circRNA and the clinical factors was analyzed by a dot-two-column correlation analysis method. The discrimination capability of the relative expression level of the plasma circRNA on the pathological characteristics of the tumor is further evaluated through the establishment of a tumor prediction model. * Represents P < 0.05, P < 0.01, P < 0.001.
General clinical data for subjects are shown below:
the study included 246 EDTA anticoagulated plasma specimens (including 186 LUAD patients, 60 healthy controls), and general clinical data are shown in Table 1. The LUAD group patients in the plasma samples were aged 54±10 years and the healthy group was aged 45±12 years. In the LUAD group of patients, 41 cases (22.0%) of smoking history, 15 cases (8.1%) of drinking history, 32 cases (17.2%) of tumor size not less than 3cm, 9 cases (4.8%) of lymph node metastasis, 42 cases (22.6%) of pleura, airway, vessel, blood vessel or nerve invasion occurred.
TABLE 1 general clinical data for plasma samples
Note that: * Clinical information loss was shown, with 13 LUAD patients missing tumor size information and 55 missing EGFR level information.
Example 2 screening and identification of candidate circRNAs
2.1 screening of candidate circRNAs
2.1.1 preliminary screening of candidate CircRNAs in combination with microarray analysis results and Circbase database
And (3) screening candidate circRNAs according to conditions that the difference multiple FC is more than or equal to 2, the Raw density is more than or equal to 200 and the P value is smaller and better by analyzing and verifying 26 circRNA chips of lung cancer tissues and adjacent normal tissues. Basic information such as the Gene length (Genomic length,200 bp-2000 bp range), parent Gene (Parenal Gene), chromosome position, gene sequence, etc. of candidate circRNAs is queried through a Circbase database.
2.1.2 analysis of potential Functions of candidate circRNAs by the CSCD database
Tumor specific circular RNA database (Cancer-specific circRNA database, CSCD) a database containing a variety of tumor cells and normal cell specific circRNAs was constructed by pooling RNA sequencing data of 87 Cancer cell samples and 141 normal cell samples of 19 Cancer types. In this database, the structural pattern map of candidate circRNAs is queried by their chromosomal location information, including information about miRNA response elements (MiRNA Response Elements, MREs), protein binding sites and protein coding regions (Open Reading Frame, ORFs), for predicting potential biological functions of the candidate circRNAs.
The analysis result of the circRNA chip shows that 537 of the circRNAs are totally expressed in the lung cancer tissues from stage I to stage III and the paracancerous tissues, and the circRNA chip comprises 143 overexpressed circRNAs and 394 overexpressed circRNAs (shown in figure 1). Combining Circbase, CSCD and other databases and related researches in the past, primarily screening 4 candidate circRNAs with up-regulated expression and related tumor, wherein the candidate circRNAs are respectively: hsa_circ_0006117, hsa_circ_0007418, hsa_circ_0000288 and hsa_circ_0082564, the basic information of which is shown in Table 2.
The structure pattern of 4 candidate circRNAs was queried by CSCD database, and the remaining three circRNAs except hsa_circ_0082564 were queried as shown in fig. 2. In the structural pattern diagram, red small triangles represent binding sites for circRNAs to mirnas, blue portions represent protein binding sites for circRNAs, and green portions represent open reading frames (i.e., protein coding regions) for circRNAs. The results show that: each of the 3 candidate circRNAs has a number of miRNA binding sites and protein binding sites; in addition, hsa_circ_0006117 also has more protein coding regions and has certain translation potential. Namely hsa_circ_0006117, hsa_circ_0007418, hsa_circ_0000288 and hsa_circ_0082564 may function as both mirnas in a single direction and in a biological process of disease through interactions with proteins; in addition, hsa_circ_0006117 has a certain translation potential. The circumcbank and circumactiometer databases showed that there were a number of potential downstream miRNAs for each of the 4 candidate circumrnas.
In summary, 4 candidate circRNAs (hsa_circ_ 0006117, hsa_circ_0000288, hsa_circ_0007418, and hsa_circ_ 0082564) may be used as candidate circRNAs.
TABLE 2 basic information on candidate circRNAs
2.2 identification of candidate circRNAs
Unlike the classical forward splice formation mode of linear RNA molecules, circRNA is spliced in reverse (downstream 3 'end linked to upstream 5' end base sequence) from RNA precursor (pre-mRNA) to form a unique covalent closed loop structure, as shown in FIG. 3. That is, the remaining region of the circRNA is identical to the linear RNA except that the reverse splice site is different from the linear RNA. Thus, a key step in the detection of circRNA is the design of the circRNA-specific primers (reverse design across splice junctions). Primers were designed by the circ interactome database and synthesized by the company Shanghai, inc. of Biotechnology. To ensure that the cyclic form of the target circRNAs can be specifically detected, we performed a circularity validation of candidate circRNAs.
2.2.1 candidate circRNAs can only be amplified by reverse designed primers
Because of the unique reverse splice formation of circRNA, PCR amplification can be performed with both normally designed primers (convergent primers) and reverse designed primers (divergent primers) across the splice junction. However, the convergent primer amplifies the circular form of the desired circRNA and also amplifies the homologous linear gene, while the divergent primer amplifies only the circular form of the desired gene. That is, both cDNA reverse transcribed from circRNA and directly extracted gDNA can be PCR amplified by convergent primers, whereas gDNA can only be amplified by cDNA but not directly by divergent primers.
2.2.1.1LUAD extraction of tissue gDNA:
the invention uses the tissue genome DNA extraction kit (centrifugal column) of the TianGen to extract the tissue gDNA. Before the experiment, a proper amount of absolute ethyl alcohol is added into the buffer solution GD and the rinsing solution PW in advance according to the instruction of the kit. The specific experimental steps are as follows:
(1) sample processing: weighing 10mg (not more than 15 mg) of tissue sample, crushing into cell suspension by using a homogenizer, centrifuging for 1min at 10,000r/min, and discarding the supernatant;
(2) 200 mu L of buffer GA is added, vortex vibration is carried out to thoroughly suspend the buffer GA;
(3) adding 20 mu L of proteinase K into the suspension, shaking and mixing uniformly, placing in a water bath shaker at 56 ℃ for 40min-1h to completely dissolve tissues, and centrifuging briefly;
(4) adding 200 mu L buffer solution GB into the solution, fully reversing and uniformly mixing, standing for 10min in a 70 ℃ water bath oscillator to clear white precipitate, and centrifuging briefly;
(5) adding 200 mu L absolute ethyl alcohol into the solution, fully oscillating and uniformly mixing for 15s, and briefly centrifuging;
(6) placing the adsorption column CB3 into a collecting pipe in advance, transferring the sample (including sediment) obtained in the previous step into the adsorption column CB3, centrifuging for 30s at 12,000r/min, discarding the waste liquid, and placing the adsorption column CB3 into the collecting pipe;
(7) adding 500 mu L buffer GD into an adsorption column CB3, centrifuging for 30s at 12,000r/min, discarding waste liquid, and placing the adsorption column CB3 into a collecting pipe;
(8) adding 600 mu L of rinsing liquid PW into the adsorption column CB3, centrifuging for 30s at 12,000r/min, discarding the waste liquid, and placing the adsorption column CB3 into a collecting pipe;
(9) repeating the operation step (8);
and (3) centrifuging for 2min at 12,000r/min, and pouring out the waste liquid. Opening a CB3 cover of the adsorption column, drying at room temperature for about 5min, and thoroughly airing residual rinsing liquid in the adsorption material;
transferring the adsorption column CB3 into a new 1.5mL Ep tube, suspending and dripping 80 mu L of elution buffer TE into the middle part of the adsorption film, standing for 2min at room temperature, centrifuging for 2min at 12,000r/min, and collecting the obtained DNA solution into a centrifuge tube;
and (3) DNA preservation: directly carrying out qRT-PCR reaction or storing at-80 ℃.
2.2.1.2LUAD preparation of tissue cDNA:
(1) RNA extraction:
the invention uses RNeasy Mini Kit (Qiagen) to extract total RNA of tissue specimens. The preparation of reagents is needed before the experiment, including the treatment of lysate (beta-mercaptoethanol is added according to 20 mu L/mL, and the mixture can be preserved for 1 month at room temperature after beta-mercaptoethanol is added) and the dilution of buffer RPE (96% -100% absolute ethanol is added according to the instruction of the kit). The specific extraction steps are as follows:
(1) sample cutting treatment: weighing the samples with an electronic balance, wherein each sample is 25mg (not more than 30 mg), and shearing the samples into small fragments as much as possible;
(2) sample homogenization treatment: 500 mu L of lysate RLT is added to every 25mg of tissue, 2 to 3 soaked magnetic beads are placed into the tissue, and the tissue is thoroughly homogenized in an electric tissue grinder (65 Hz,120s or so). After homogenization is completed, the mixture is centrifuged for 2min at 12,000 r/min;
(3) carefully transferring the supernatant into a new 1.5mL sterile Ep tube, adding 70% ethanol with the volume of 1 time, blowing, sucking and mixing uniformly;
(4) transferring the uniformly mixed sample (including precipitate) into an Rneasy Mini adsorption column immediately, lightly covering the upper cover, centrifuging for 30s at 12,000r/min (passing through the column twice when the sample volume exceeds 700 mu L), and discarding effluent;
(5) adding 700 μl of buffer RW1 to Rneasy Mini adsorption column, lightly covering the upper cover, centrifuging at 12,000r/min for 30s, and discarding the effluent;
(6) adding 500 mu L of buffer RPE to an Rneasy Mini adsorption column, lightly covering the upper cover, centrifuging for 30s at 12,000r/min, and discarding effluent;
(7) adding 500 μl buffer RPE into Rneasy Mini adsorption column, lightly covering, centrifuging at 12,000r/min for 2min, and discarding effluent;
(8) placing the Rneasy Mini adsorption column in a new 1.5mL enzyme-free sterile Ep tube, opening the cover of the adsorption column, and drying at room temperature for 2-5min;
(9) RNA elution: dropwise adding 30 mu L of RNase-Free ddH into an adsorption column in a suspending manner 2 0, covering the adsorption column cover, standing at room temperature for 2min, and centrifuging at 10,000r/min for 1min to elute RNA;
and (3) RNA preservation: blowing and sucking the mixed RNA solution by a pipette, and directly performing reverse transcription reaction or temporarily storing at-80 ℃;
determination of purity and concentration of RNA: RNase-Free ddH was used before detection 2 0 washing the sample adding holes twice. By RNase-Free ddH 2 0 is a blank control, 1. Mu.L of the sample was added to the well (no air bubbles were generated) and the quality of RNA extraction was judged by A260/A280, A260/A230 and the continuous wavelength absorption peaks. A260/A280 values are generally in the range of 1.8-2.1, otherwise DNA or protein contamination may occur.
(2) Reverse transcription reaction
The reverse transcription Kit used in this experiment was FastKing RT Kit (with gDNase). All reagents were thawed at room temperature before the experiment, and after dissolution, each solution was vortexed and mixed well and centrifuged briefly for use. The specific experimental steps are as follows:
1) gDNA removal reaction: this step was performed on ice to prepare a reaction mixture as shown in Table 3. Centrifuging briefly after sample addition is completed, and incubating at 42 ℃ for 3min;
TABLE 3gDNA removal reaction System
2) Reverse transcription reaction: the reverse transcription reaction system was prepared as shown in Table 4. In order to ensure the accuracy of sample addition, at least the reagent components are mixed and prepared into the same EP tube according to the quantity of the reaction hole number +1, and are thoroughly mixed and added into each reaction hole respectively.
TABLE 4 reverse transcription reaction system
3) And adding the prepared reverse transcription reaction system into the gDNA removal reaction system, and briefly centrifuging. The reaction conditions are as follows: 45℃for 45min and 95℃for 5min. After the reverse transcription is completed, the obtained cDNA is put on ice for subsequent experiments or stored at-20 ℃ for use.
(3) QRT-PCR reactions
In the invention, a Tiangen SuperReal PreMix Plus (SYBR Green) kit is used for qRT-PCR reaction, and the kit is required to be stored at the temperature of minus 20 ℃ in a dark place. Before the invention, all reagents were dissolved at room temperature and thoroughly mixed and used after a short centrifugation. The specific experimental steps are as follows:
1) Primer dilution:
centrifuging EP with primer dry powder at low temperature and high speed for 30s with a centrifuge at high speed of 10,000r/min, and adding RNase-Free ddH with corresponding volume according to instruction 2 0, blowing and sucking, and diluting to a concentration of 100 mM/. Mu.L. In order to be capable of long-term storage and avoiding loss, the sample is packaged and stored at-20 ℃ according to experimental requirements, and is diluted to be 10 mM/mu L when in use.
2) QRT-PCR reaction:
the reaction system was formulated as in table 6, this step being operated on ice. In order to ensure the accuracy of sample addition, at least according to the amount of the sample addition hole number +1, other reaction components except cDNA in a reaction system are mixed and arranged in the same Ep tube, thoroughly mixed and centrifuged for use. In order to ensure the amplification efficiency, the amount of cDNA template used in the reaction system is preferably not more than 100ng. After three repeated tests, the tissue cDNA concentration is mostly in the range of 1000-1300 ng/. Mu.L, so that the tissue cDNA needs to be diluted 20 times and then the subsequent experiments are carried out. After the sample addition is completed, the eight-joint tube is covered tightly, the eight-joint tube is lightly and elastically mixed and all bubbles are removed, all the liquid is collected to the bottom of the tube by short centrifugation, and then qRT-PCR reaction (the bubbles in the reaction liquid are removed as completely as possible during the amplification) is carried out according to the table 5 (reaction system) and the table 6 (amplification program). Three replicates were run for each sample. The experiment uses a two-step amplification procedure.
TABLE 5QRT-PCR reaction System
TABLE 6 two-step QRT-PCR procedure
(4) 2% agarose gel electrophoresis
After the QRT-PCR reaction is completed, agarose gel electrophoresis is performed on the amplified products of gDNA and cDNA to determine if the single band is clear and single, while comparing the amplicon sizes to be consistent with expectations. The specific operation steps are as follows:
(1) dilution of TAE electrophoresis buffer: diluting the 50×tae running buffer to 1×tae running buffer (50×tae buffer: deionized water=1:49);
(2) 2% agarose gel was prepared: 2g agarose powder was dissolved in 100. Mu.L of 1 XTAE electrophoresis buffer and heated to transparency in a microwave oven; when the temperature is cooled to be lower than that of scalding hands, adding 6 mu L of nucleic acid dye, gently shaking the mixture, pouring the gel into an electrophoresis mould prepared in advance, and completely solidifying the agarose gel after about 25 minutes;
(3) sample adding electrophoresis: mixing 5 μL of amplification product with 1 μL of 6×loading Buffer, adding sample, and electrophoresis at 120V for 20-30min;
(4) after electrophoresis, the gel is taken out, exposed on a gel imager, photographed and stored.
2.2.2Sanger sequencing
To ensure that the designed primer sequences accurately amplify the circular form of the circRNA, sanger sequencing was performed on cDNA amplicons specifically amplified and of the desired size as described above. The sequencing results were then aligned with the circRNA gene sequences in the circbase database to determine if the amplified product was the circRNA of interest.
2.2.3RNase R digestion experiments
Due to the special closed loop structure of circRNA, the lack of a 5 'cap structure and a 3' A tail, is highly resistant to exoribonucleases (RNase R). RNase R is able to digest most linear RNA, whereas circular circRNA is generally not digested. To further eliminate the possibility of detecting linear RNA, 3 tissue specimens were randomly selected and the total RNA extracted was split into two fractions, one fraction was digested with RNase R enzyme and the other fraction was not subjected to any treatment, followed by simultaneous RT-qPCR reactions. The RNase R enzyme digestion assay procedure was as follows:
(1) Preparing an RNase R enzyme digestion reaction system: see table 7.
TABLE 7RNase R enzyme digestion reaction system
Note that: * The total amount of RNA in the system is not more than 5mg. To make the test comparable, the volume of RNA used in this system was 8L as in the reverse transcription reaction system described above.
(2) Placing the reaction system at 37 ℃ for incubation for 15min;
(3) After digestion is completed, the temperature is kept at 70 ℃ for 10min to inactivate enzymes;
(4) Reverse transcription and qRT-PCR reactions were performed as described in tables 3, 4 and 6 above, respectively.
qRT-PCR validation (primer specificity validation) was performed on the three candidate circRNAs with potential biological functions. Tissue RNA was extracted as described above in 2.2.1.2, reverse transcription reactions were performed according to tables 3 and 4, and qRT-PCR reactions were performed according to Table 6. Through multiple primer designs, amplification curves of candidate circRNAs hsa_circ_0006117, hsa_circ_0000288 and hsa_circ_0007418 are S-shaped, a dissolution curve is a single peak, specific amplification can be realized, and primer sequences are shown in table 8.
The above three candidate circRNAs were verified for circularity by gDNA versus cDNA amplification, cDNA product sequencing and RNase R enzyme digestion experiments. First, qRT-PCR amplification was performed on directly extracted gDNA and cDNA reverse transcribed from RNA using divergent primer pairs, and the products were subjected to agarose gel electrophoresis (as shown in FIG. 4), and the results showed that: the cDNA amplification products of hsa_circ_0006117, hsa_circ_0007418 and hsa_circ_0000288 were single and bright in band, consistent in size with the expectation, while the gDNA amplification products were essentially absent. It was demonstrated that the divergent primers hsa_circ_0006117, hsa_circ_0007418 and hsa_circ_0000288 could achieve specific amplification of circRNA.
To ensure accurate amplification of the circular form of the circRNA, the cDNA products described above were subjected to Sanger sequencing. By comparing the Sanger sequencing results with the gene sequences of candidate circRNAs in the circbase database, the results show that: the hsa_circ_0006117 and hsa_circ_0000288 sequencing results were perfectly matched to the circRNA reverse junction sequences in the circbase database (see FIGS. 5 and 6, respectively, where the arrows represent the splice sites of the circRNA), whereas the hsa_circ_0007418 primer did not successfully amplify the fragment of interest (FIG. 7).
RNase R enzyme digestion experiments were performed on the two successfully sequenced candidate circRNAs (hsa_circ_ 0006117 and hsa_circ_ 0007418). The results show that: there was no significant change in the relative expression levels of beta-actin normalization in hsa_circ_0006117 and hsa_circ_0000288 in the RNase R digested group compared to the untreated group (as shown in FIG. 8).
In summary, the hsa_circ_0006117 and hsa_circ_0000288 divergent primers designed in the invention can achieve specific amplification of circular form of the target circRNA without amplifying homologous linear RNA.
TABLE 8 primer sequences of candidate circRNAs
EXAMPLE 3 relative expression levels of candidate circRNAs in LUAD tissue
The invention adopts qRT-PCR method to detect the relative expression level of candidate circRNAs in the LUAD tissue and the adjacent tissue.
3.1QRT-PCR reactions
Tissue RNA extraction, reverse transcription and qRT-PCR reactions were performed as described in 2.2.1 above. Three replicates were run for each sample.
3.2 determination of the results of the QRT-PCR reaction
In this study, 2 was used -ΔΔCt The relative expression level of the target gene in the sample is calculated by the method, and the differential expression times between groups are determined. 2 -ΔΔCt The algorithm compares the results of the experimental sample with a calibrator (e.g., untreated or wild-type sample) or a normalizer (e.g., reference gene). Using this method, ct values of the target genes in the sample group and the control group were calibrated based on Ct of the reference gene from the same sample by calculating 2 -ΔΔCt To determine fold differences in expression. An important precondition for using the calculation method is that the amplification efficiency of the target gene and the internal reference gene is close to 100% (90% -110%).
3.2.1 verification of amplification efficiency
One example of LUAD tissue with a lower Ct value (reference) was selected, freshly extracted RNA was diluted in a 10-fold gradient with a multiple ratio, 4 gradients total, and qRT-PCR reactions were performed according to tables 6 and 7, with three multiplex assays per concentration for each gene. And drawing a standard curve by taking the logarithm of the template concentration with 10 as a base as an abscissa and the corresponding average Ct value as an ordinate, so as to judge the amplification efficiency of candidate circRNAs hsa_circ_0006117, hsa_circ_0000288 and housekeeping gene beta-actin.
3.2.2 result calculation
After the result of the target gene was normalized with a normalizer (reference gene β -actin), the relative expression level and fold of differential expression of the target gene were determined by the following formula. The calculation formula is as follows:
delta Ct (experimental group) =ct (experimental group Target gene ) -Ct (experimental group Reference gene )
Delta Ct (control) =ct (control Target gene ) -Ct (control group) Reference gene )
ΔΔΔΔ ct=Δ Ct (experimental group) delta Ct (control group)
2 -ΔΔCt =multiple of difference
3.2.3 results
3.2.3.1 amplification efficiency of two candidate circRNAs and housekeeping Gene beta-actin
The average Ct values corresponding to the series of gradient concentrations are collated in Table 9. Drawing a standard curve (as shown in figure 9) with the logarithm of template concentration based on 10 as abscissa and the corresponding average Ct value as ordinate, wherein the amplification efficiencies of beta-actin, hsa_circ_0006117 and hsa_circ_0000288 are all between 90% and 110%, and R is 2 More than 0.99, has good correlation and meets the requirement of effective amplification. Meanwhile, the amplification efficiency of the two target circRNAs is basically consistent with that of the reference gene beta-actin. The results show that beta-actin can be selected as the reference gene for this experiment and 2 can be used -ΔΔCt The method calculates the relative expression level of the target gene among the sample groups and determines the differential expression times among the groups.
TABLE 9 average Ct values corresponding to gradient concentrations of reference gene beta-actin and two candidate circRNAs
3.2.3.2 verification of differential expression of two candidate circRNAs in lung adenocarcinoma tissue
The relative expression levels of candidate circRNAs (hsa_circ_ 0006117 and hsa_circ_ 0000288) in the LUAD tissue and its paracancerous tissues were examined using qRT-PCR. By 2 -ΔΔCt Algorithm and Wilcoxon signed rank test data analysis was performed as shown in fig. 10. The results show that: the relative expression level of hsa_circ_0006117 in the LUAD tissue was up-regulated compared to the paired paracancerous tissue, the difference being statistically significant (p=0.02, P < 0.05); while there was no significant difference in the relative expression levels of hsa_circ_0007418, the difference was not statistically significant (p=0.063). The result is consistent with the analysis result of the circRNA chip, and hsa_circ_0006117 is selected as the target gene of the invention.
EXAMPLE 4 relative expression levels of candidate circRNAs in plasma samples
4.1 extraction of total plasma RNA
Trizol is a commonly used total RNA extraction reagent, contains substances such as guanidine isothiocyanate, and can rapidly break cells, inhibit nuclease released by the cells and maintain the integrity of RNA. The invention adopts Trizol method to extract total RNA of plasma specimen, and the specific operation steps are as follows:
(1) sample treatment: taking out the collected plasma sample from the refrigerator at-80 ℃, naturally thawing at room temperature, transferring 280 mu L to a new sterile Ep tube after thawing, and centrifuging at 4 ℃ for 10min at 12,000r/min to eliminate cell sediment;
(2) adding a lysate: transferring 250 μl of plasma sample into another new sterile EP tube, adding 500 μl of lysis solution RNAiso (2 times the volume of the sample), mixing by vortex shaking, and standing at room temperature for 10min;
(3) extracting RNA by adding chloroform: adding 200 mu L of chloroform into the solution, mixing the solution for about 10 times in an upside down way, standing for 5min, centrifuging at 4 ℃ for 15min at 12,000r/min, and transferring the colorless upper layer solution into a new enzyme-free sterile EP tube;
(4) precipitating RNA by adding isopropanol: adding equal volume of isopropanol and 40 mu L of nucleic acid binding enhancer, thoroughly mixing, and standing at room temperature for 10min (since the RNA content in the blood plasma sample is low, adding nucleic acid binding enhancer to improve RNA extraction efficiency);
(5) and (3) centrifuging: centrifuging at 4deg.C for 10min at 12,000r/min, and discarding supernatant;
(6) washing the RNA precipitate: adding 75% ethanol equivalent to RNAiso Plus, reversing upside down, centrifuging at 8000r/min for 10min, and discarding supernatant;
(7) and (3) drying: opening the tube cover, and standing at room temperature for 5-10min;
(8) dissolving RNA precipitate: add 30. Mu.L of Rnase-Free ddH 2 O is dissolved and precipitated;
(9) and (3) RNA preservation: since RNA is extremely degradable, reverse transcription was performed directly after RNA extraction was completed in this study.
Quality control of total RNA: and the quality control of the tissue RNA is the same as that of the tissue RNA.
4.2RNA reverse transcription reaction and QRT-PCR reaction
The reverse transcription reactions were carried out as described in tables 1.1 and 1.2 above, with the following reaction conditions: 45℃for 45min and 95℃for 5min, and the reaction system and amplification procedure for qRT-PCR reactions are shown in tables 1.4 and 1.5, respectively. In this amplification system, the DNA was diluted 10-fold and used (the amount of cDNA template used in this reaction system was not more than 100ng, and the concentration of cDNA reverse transcribed from plasma RNA was approximately in the range of 700-900 ng/. Mu.L by three repeated tests).
4.3 result determination
Use 2 -ΔΔCt The method calculates the relative expression level of the target gene between the LUAD group and the healthy group and determines the differential expression fold between the groups. The calculation formula is 3.2.2.
4.4 results
Verification of differential expression of hsa_circ_0006117 in plasma of 4.4.1LUAD patients
The relative expression levels of hsa_circ_0006117 in plasma samples of 186 LUAD patients and 60 healthy controls were examined using qRT-PCR. By 2 -ΔΔCt The algorithm and Mann-Whitney U test were analyzed as shown in FIG. 11. The results show that: relative expression levels of hsa_circ_0006117 were significantly up-regulated in LUAD patients compared to healthy controls (P < 0.001).
ROC curves were plotted against the relative expression levels of hsa_circ_0006117 in plasma, and their potential value as LUAD liquid biopsy diagnostic markers was evaluated, as shown in FIG. 12. The results show that: AUC of plasma hsa_circ_0006117 in LUAD diagnosis ROC The value was 0.73, and the sensitivity and specificity were 53% and 86%, respectively (P < 0.0001).
Currently, lung cancer serum tumor markers recommended by the national academy of clinical biochemistry and the european tumor marker expert group are: carcinoembryonic antigen (Carcinoembryonic antigen, CEA), neuron-specific enolase (neuron specific enolase, NSE), soluble fragment antigen 21-1 of cytokeratin 19 (cyto-keratan 19fragment antigen 21-1, cyfra 21-1), gastrin-releasing peptide precursor (progastrin releasing peptide, proGRP), and squamous cell carcinoma antigen (Squamous cell carcinoma antigen, SCC-Ag). By using the above-mentioned 5 clinical common tumor markersThe object data were collected and sorted with less SCC-Ag data and ROC curve analysis was performed on the remaining 4 tumor markers. The results show that: CEA, NSE, CYFRA21-1 and AUC of ProGRP in LUAD diagnosis ROC The values were 0.60, 0.87, 0.61 and 0.72, respectively. Wherein NSE and ProGRP have relatively good clinical application value. AUC when plasma hsa_circ_0006117 is diagnosed in combination with NSE and ProGRP ROC The value was increased to 0.83, and the sensitivity and specificity were 76%, 84% (P < 0.0001), respectively.
4.4.2 calculating the maximum approximate dengue index by the formula (about dengue index = sensitivity- (1-specificity)), the corresponding relative expression level of plasma hsa_circ_0006117 is the cut-off value. The LUAD group was divided into a high expression group (n=99) and a low expression group (n=87) bounded by the cut-off value (cut-off=2.73) (see table 10). The correlation between hsa_circ_0006117 relative expression levels and 8 clinical pathology features was analyzed using a dot-two-column correlation analysis, as shown in table 3.2. The results show that: the relative expression levels of hsa_circ_0006117 in the plasma of patients in the LUAD group were related to tumor invasion phenomena (including pleural, vascular, airway or nerve invasion) (p=0.036, P < 0.05), but no statistical significance was associated with sex, age, smoking history, drinking history, tumor size, lymph node metastasis and EGFR levels.
In order to more clearly understand the relationship between the relative expression levels of plasma hsa_circ_0006117 and tumor invasion, the LUAD patients were divided into aggrension and Non-aggrension groups, and the relative expression levels of hsa_circ_0006117 between the two groups are shown in fig. 13. The results show that: plasma hsa_circ_0006117 was expressed higher (P < 0.05) in the Aggression group LUAD than in the Non-Aggression group.
TABLE 10 correlation of relative expression levels of hsa_circ_0006117 in plasma of LUAD patients with clinical pathology characteristics
Note that: * Indicating that the clinical information was missing, which is consistent with table 2.1.
4.4.3 to further determine the relationship between plasma hsa_circ_0006117 and tumor invasion, we assessed the prediction and discrimination ability of plasma hsa_circ_0006117 to tumor invasion by a tumor invasion prediction model. First, from 11 variables (age, sex, smoking history, drinking history, tumor size, lymph node metastasis, 4 common swelling markers (CEA, NSE, CYFRA-1, and ProGRP) and plasma hsa_circ_0006117 relative expression levels), sex+Tumor.size+CEA+hsa_circ_0006117 4 variables were screened as fitting factors by Lasso cox regression stepwise analysis to construct a lung adenocarcinoma patient nomographic invasion prediction model (see FIG. 14). Then, discrimination and calibration ability of the alignment chart are evaluated through a calibration curve, and the evaluation result shows that: the model predicts a tumor invasion that is relatively consistent with reality (see fig. 15).
The predictive ability of the alignment prediction model was evaluated by DCA curve and ROC curve. DCA curve analysis results show that: the prediction model has a certain prompt effect on the existence of tumor invasion phenomenon (as shown in figure 16). The ROC curve analysis results show that: the relative expression level of Sex+Tumor.size+CEA+plasma hsa_circ_0006117 is 4 factors, and the gene has certain application value in tumor invasion prediction and AUC ROC =0.71 (see fig. 17).
While the differential expression of plasma hsa_circ_0006117 between LUAD patients and healthy controls has been demonstrated by the above studies and may be related to the occurrence of tumor invasion, it is not known whether it is involved in the progression of LUAD. For this, we collected plasma samples of 29 LUAD patients pre-and post-operatively (3 d or more post-operatively) and examined the relative expression levels of hsa_circ_0006117 by qRT-PCR. The results show that: the relative expression levels of hsa_circ_0006117 in the plasma of the LUAD patient were significantly down-regulated after surgery (3 d. Gtoreq.) compared to pre-surgery (P < 0.001, FIG. 18).
Example 5CircRNA-miRNA-mRNA network prediction
First, the downstream miRNAs of the target circRNAs were queried separately by the circinter and CircBank databases, and the first 5 potential downstream miRNAs of the target circRNAs were predicted by comprehensive analysis. The circinter database provides bioinformatic analysis of miRNA and RBP binding sites on circRNA, and additional analysis of miRNA and RBP sites on the ligation and ligation flanking sequences. The CircBank database is an integrated human circRNA database containing basic information such as the miRNA binding site of the circRNA, conservation of the circRNA, m6A modification of the circRNA, mutation of the circRNA, and protein coding potential of the circRNA.
The target genes for candidate miRNAs were then predicted comprehensively by the TargetScan and Miranda databases. The TargetScan and Miranda databases are commonly used databases of predicted human miRNA target genes. The TargetScan database predicts target genes mainly by searching for conserved 8mer and 7mer sites that match the miRNA seed region. The Miranda database mainly collects the experimentally verified miRNA targets.
Finally, a network map of circRNA-miRNA-mRNA was drawn.
The circ inter and CircBank databases show that hsa_circ_0006117 has 20, 35 miRNA binding sites, respectively, with the top 5 possible downstream miRNAs with highest overall predictive scores: hsa-miR-1250-5P, hsa-miR-578, hsa-miR-636, hsa-miR-582-3p and hsa-miR-671-5p. The target genes of the 5 miRNAs are comprehensively predicted through the TargetScan and Miranda databases, and finally 4 miRNAs (hsa-miR-1250-5P, hsa-miR-152-5p, hsa-miR-578 and hsa-miR-636) and 594 target mRNAs are obtained. The network relationship diagram of the circRNA-miRNA-mRNA is drawn (as shown in figure 19), and the result shows that: hsa_circ_0006117 plays a role of miRNA by combining with hsa-miR-1250-5P, hsa-miR-671-5P, hsa-miR-636 or hsa-miR-578 and acts on TMEM, LRRC, SOX, ZNF and other disease progress related genes, so that the disease progress of the LUAD is promoted.
Example 6 functional enrichment analysis of target genes
To predict the potential biological function of hsa_circ_0006117 in LUAD, we performed GO and KEGG functional enrichment analysis on the predicted target genes of the CircRNA-miRNA-mRNA network. The first 20 items which are obviously enriched are selected from MF, BP and CC respectively, the GO name (terms) is taken as the abscissa, and the number of genes is differentiatedAnd on the ordinate, a two-level item frequency analysis chart and a bubble chart are adopted to display GO analysis results. And (3) carrying out KEGG pathway analysis on the circRNA target genes, and screening KEGG pathways of Top 20 according to the P value from small to large and the difference gene enrichment number under the pathway of more than or equal to 5, so as to know the cell signal pathways possibly involved in the circRNA. The GO analysis results show that hsa_circ_0006117 target gene is mainly involved in both Cell Composition (CC) and Molecular Function (MF). Wherein CC is localized in cell membrane and cytoplasm, MF is mainly binding protein and metal ion, and participates in ATP (Adenosine triphosphate ), cell nucleus, golgi apparatus, ca 2+ Regulation of binding, cell adhesion, nucleotide binding, and the like (fig. 20 and 21). Of the first 20 signal pathways enriched in KEGG pathway analysis results, mainly tumor cell metabolism-related pathways such as insulin secretion, insulin resistance and miRNAs action were enriched (fig. 22).
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims. And is enclosed inside.

Claims (1)

1. Use of an agent for detecting hsa_circ_0006117 for the manufacture of a product for predicting the risk of developing a tumor invasion in a patient with lung adenocarcinoma, characterized in that the agent is F CCAGATAACCAGTTCACGGATG, R GGAATCCATGCTTATCTGAAGG; the product is used for predicting the invasion risk of lung adenocarcinoma tumor by detecting the content of hsa_circ_0006117 in the plasma of a patient through qPCR, wherein the expression level of hsa_circ_0006117 in the plasma of the lung adenocarcinoma tumor invasion patient after the operation treatment is significantly reduced relative to the expression level in the patient before the operation treatment.
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