CN116875699A - Early non-small cell lung cancer lncRNA marker and detection kit thereof - Google Patents

Abstract

The present invention provides an early stage non-small cell lung cancer lncRNA marker, the gene sequence of which is shown in SEQ ID NO. 1. The present invention also provides the application of the above-mentioned lncRNA markers in preparing a kit for detecting early-stage non-small cell lung cancer. The present invention also provides a kit for detecting the above-mentioned lncRNA markers, including an upstream primer sequence for detecting lncRNA markers as shown in SEQ ID NO.2; and a downstream primer sequence for detecting lncRNA markers as shown in SEQ ID NO.3. shown; also includes primers for amplifying the internal reference gene GAPDH. The invention also provides a method of using the above-mentioned kit. The present invention finds that LncRNAAIRN is specifically increased in the plasma of patients with early-stage non-small cell lung cancer. Therefore, it can be detected and used for early diagnosis of non-small cell lung cancer through simple and stable fluorescence real-time quantitative PCR technology.

Description

Early non-small cell lung cancer lncRNA marker and detection kit thereof

Technical Field

The invention belongs to the field of biological detection, and relates to a detection kit, in particular to an early non-small cell lung cancer lncRNA marker and a detection kit thereof.

Background

Non-small cell lung cancer (NSCLC) is derived from all epithelial malignant carcinomas ranging from bronchi to terminal alveoli, and commonly includes squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and the like. The incidence rate and the death rate of lung cancer are the first in China, more than 80% of cases belong to NSCLC, and meanwhile, the diffusion speed of NSCLC is relatively slow, so that most cases are in middle and late stages when being diagnosed, the five-year survival rate is extremely low, and therefore, the early screening and diagnosis of NSCLC are extremely important.

LncRNA is a non-coding RNA with a length exceeding 200nt, and is considered to be transcription "garbage" once due to the lack of Open Reading Frames (ORFs), however, as research progresses, more and more evidence shows that LncRNA participates in various biological processes through abundant mechanisms, is indistinguishable from the occurrence and development of human diseases, can regulate gene expression at the epigenetic, cis-or trans-transcriptional and posttranscriptional levels, and participates in biological processes such as X chromosome silencing, genome imprinting, chromatin modification, transcriptional activation, transcriptional interference, nuclear transport and the like, but the number of LncRNA related to the diseases identified through experiments is only 1% less than that of the identified genes, and the biological functions of the LncRNA are still to be further mined. Studies show that lncRNA is used as a cancer or cancer suppressor gene, and the downstream target gene is regulated through interaction with microRNA, so that abnormal proliferation, metastasis, invasion, transformation, apoptosis and the like of NSCLC cells can be influenced, drug resistance and radiotherapy sensitivity of NSCLC cells can also be influenced, and lncRNA UCA1, lncRNA XIST, lncRNA PVT1, PVT1-5, lncRNA MALAT1 and the like are hopeful to become markers of treatment prognosis, however, the indexes are more related to treatment process and lack early-stage specific diagnosis markers, but based on the research results, lncRNA can be stably detected in peripheral circulation, and the finding of lncRNA markers for early diagnosis of NSCLC is highly hopeful.

At present, smoking history is a main risk factor of lung cancer, for NSCLC high-risk groups, imaging means low-dose spiral CT scanning is the only feasible screening method, mortality can be reduced to a certain extent through early discovery, but the requirements of large-scale screening are not met due to high false positives and high cost, and the effect of reducing the mortality cannot be achieved through chest radiography and sputum cell culture. In addition, the discovery of a variety of biomarkers, including proteins, lipids, non-coding RNAs, and the like, such as carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), cytokeratin 19 fragment antigen (CYFRA 21-1), can also be used as non-invasive biomarkers, however, due to non-ideal sensitivity and specificity, diagnostic efficacy of markers for early detection of NSCLC is limited. Therefore, there is a need for an economic, convenient, rapid, stable and reliable early screening marker and a detection kit thereof, which assist in improving the early diagnosis rate of NSCLC and are beneficial to reducing the death rate of NSCLC.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides an early non-small cell lung cancer lncRNA marker and a detection kit thereof, and the early non-small cell lung cancer lncRNA marker and the detection kit thereof aim to solve the technical problems in the prior art that the diagnosis effect on early non-small cell lung cancer is poor.

The invention provides an early non-small cell lung cancer lncRNA marker, the gene sequence of which is shown as SEQ ID NO. 1.

(shown in SEQ ID NO. 1).

The invention also provides application of the lncRNA marker in preparation of a kit for detecting early non-small cell lung cancer.

The invention also provides a kit for detecting the lncRNA marker, which comprises the following upstream primer sequences for detecting the lncRNA marker: 5'-aggagcgggactgaaata-3' (SEQ ID NO. 2); the sequence of the downstream primer for detecting the lncRNA marker is as follows: 5'-gactgcactgcgacgaga-3' (SEQ ID NO. 3); the primer also comprises an internal reference gene GAPDH amplification primer, wherein the upstream primer sequence of the internal reference gene GAPDH is as follows: 5'-gctctctgctcctcctgt-3' (SEQ ID NO. 4); the sequence of the downstream primer of the reference gene GAPDH is as follows: 5'-gactccgaccttcaccttcc-3' (SEQ ID NO. 5).

Further, cDNA, TB Green Premix Ex Taq II, ROX Reference Dye II and ultrapure water are included.

Further, the kit also comprises a total RNA extraction Reagent group, wherein the total RNA extraction Reagent group comprises Trizol Reagent solution, chloroform, isopropanol, ethanol and ultrapure water.

Further, the kit also comprises an RNA reverse transcription reagent group, wherein the RNA reverse transcription reagent group comprises a random primer, dNTP mixed solution, 5× Reverse Transcriptase M-MLV Buffer, DTT solution, an RNAse inhibitor and M-MLV reverse transcriptase.

The invention also provides a use method of the kit, which comprises the following steps:

1) Collecting 3-5mL of peripheral venous blood sample of an object to be detected by adopting an EDTA anticoagulation tube, storing the collected blood sample in a 4 ℃ environment, centrifuging at 3500rpm for 5min at 4 ℃, collecting upper plasma in an EP tube without RNase, adding 750 mu L of Trizol Reagent solution into every 250 mu L, uniformly mixing, and storing at-80 ℃ for use;

2) Taking out the EP tube filled with the plasma sample, vibrating and uniformly mixing at room temperature, standing at room temperature, and incubating for 5min;

3) Adding 0.3mL of chloroform into the EP tube in the step 2), covering the EP tube cover, oscillating for 30s, and standing at room temperature for 2min to obtain a mixed extract;

4) Centrifuging the mixed extract of step 3) at 12000rpm at 4deg.C for 15min, and collecting supernatant into new RNase-free EP tube;

5) Adding isopropyl alcohol with the same volume into the supernatant obtained in the step 4), shaking and mixing uniformly, and standing at the temperature of minus 20 ℃ for more than 30 min.

6) After the operation according to step 5), taking out and centrifuging at 12000rpm for 15min at 4 ℃, reserving bottom sediment, and discarding supernatant;

7) Adding 1mL of 75% ethanol after the operation of the step 6), washing the precipitate, centrifuging at 7500rpm at 4 ℃ for 5min, and discarding the supernatant to obtain RNA precipitate;

8) Standing and airing the RNA precipitate obtained in the step 7) at room temperature for less than 10 minutes, and then adding 10 mu L of ultrapure water for dissolving to obtain a total RNA solution;

9) Measuring concentration of 1 μL of the RNA solution obtained in the step 8) by using an ultra-micro ultraviolet spectrophotometer, respectively measuring absorbance at wavelengths of 260nm and 280nm, wherein the absorbance of A260/A280 of a sample with good quality is 1.8-2.0, and calculating the concentration of RNA obtained by calculation to obtain the concentration of RNA for reverse transcription reaction

Total RNA volume required; the rest sample is placed at-80 ℃ for preservation or direct total RNA reverse transcription reaction; 10 Setting total RNA reverse transcription reaction system with total volume of 20 muL, its components, component content and reaction bar

The pieces are as follows:

11 Real-time fluorescent quantitative PCR amplification is carried out on the product obtained by reverse transcription, and the reagent and component content of a real-time fluorescent quantitative PCR amplification reaction system are prepared, wherein the total volume of the reagent and the component content is 15 mu L, and the reagent and the component content are as follows:

the amplification reaction conditions were: 3min at 95 ℃;95℃15s,54℃30s,72℃25s,40 cycles.

12 Observing a real-time fluorescent quantitative PCR amplification curve and a dissolution curve, setting a base line and a threshold value to obtain a Ct value, and obtaining a relative quantitative formula according to fluorescent quantitative PCR: 2 ^-△△Ct And calculating the expression level of LncRNA AIRN in the plasma of the patient to be tested, and further analyzing the expression specificity of the lncRNA molecule in early NSCLC.

The invention combines transcriptome bioinformatics analysis technology, and utilizes early NSCLC clinical research resources (plasma samples) to find that LncRNA AIRN is specifically increased in early NSCLC plasma. The lncRNA transcript belongs to a nucleic acid molecule, can be secreted and dissociated in extracellular fluid, can be stably expressed in human plasma, and can be independently or exist in exosomes, and the detection of the nucleic acid molecule can be realized by a simple and stable fluorescent real-time quantitative PCR technology; in general, proteins or lipid molecules are regulated and controlled after transcription, mature molecules secreted outside cells are influenced by biochemical and physical factors such as various enzymes, pH and the like, and the protein or lipid molecules are used as traditional markers, so that the results are unstable, the specificity and the sensitivity are insufficient, and the detection technical threshold and the detection cost are high.

Compared with the prior art, the invention has the technical effects of being positive and obvious. Compared with low-dose spiral CT scanning, the method is simple to operate, does not depend on professional technicians, only needs to operate according to the steps of the instruction book, and has a stable result; the sample data is simple to collect, only a small amount of venous blood is needed, the requirement on the sample preservation condition is low, and the marker can be freely stored in the blood plasma for a long time and is stable; the method can be used for NSCLC early screening, is cheaper, quick, stable and efficient, is more suitable for wide screening, and has higher clinical transformation value.

Drawings

FIG. 1 is an agarose gel electrophoresis of the detection product of the LncRNA AIRN of the biomarker of the present invention.

FIG. 2 shows the recognition binding sites for the upstream and downstream primers and the biomarker LncRNA AIRN in the present invention.

FIG. 3 shows the expression levels of LncRNA AIRN in plasma of early stage NSCLC patients in the test examples of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

The invention discloses a kit for early detection of NSCLC, which specifically comprises the following components:

(1) Primer design

Primer design of fluorescence real-time quantitative PCR is carried out by utilizing Primer Premier 5 software, in the design process, tm value is controlled between 55 ℃ and 63 ℃, the base length of the Primer is 15-25, GC content is 40% -65%, bases are randomly distributed, no complementary sequence exists between two primers, the 5 'end and middle delta G value of the Primer are relatively higher, and the delta G value of the 3' end is lower; in addition, the size of the target fragment amplified by the upstream and downstream primers is controlled, the total fragment length of LncRNA AIRN is 4374bp, and the product is not suitable to be overlong in the fluorescent real-time quantitative PCR detection process, so that the binding site is selected in a conservation region during the primer design, the product is controlled to be 100-300bp, and the formation of the secondary structure of the primer is reduced to the greatest extent by adjusting the base pairing starting point, thereby improving the detection specificity and the reaction rate.

(2) The fluorescent quantitative PCR reagent set comprises an amplification primer of an LncRNAAIRN biomarker, an amplification primer of an internal reference gene GAPDH, cDNA, TB Green Premix Ex Taq II, ROX Reference Dye II and ultrapure water, wherein the sequences of the amplification primer of the biomarker and the amplification primer of the internal reference gene GAPDH are as follows:

upstream primer of biomarker: 5'-AGGAGCGGGACTGAAATA-3';

downstream primer of biomarker: 5'-GACTGCACTGCGACGAGA-3';

the upstream primer of the reference gene: 5'-GCTCTCTGCTCCTCCTGT-3';

primer downstream of reference gene: 5'-GACTCCGACCTTCACCTTCC-3';

wherein the amplification product of LncRNAAIRN is 207bp, and the agarose electrophoresis band position is shown in figure 1; the binding sites of the upstream and downstream primers to LncRNA air were predicted using NCBI BLAST website, and the results are shown in fig. 2.

(3) Total RNA extraction reagent set: including Trizol Reagent solution, chloroform, isopropanol, ethanol, and ultrapure water.

(4) RNA reverse transcription reagent group: random primers, dNTP mix, 5X Reverse Transcriptase M-MLV Buffer, DTT solution, RNase inhibitor and M-MLV reverse transcriptase.

Example 2

The method for early detection of NSCLC was performed using the kit provided in example 1.

1. Test subjects

As an experimental group, 14 plasma samples from NSCLC early stage patients belonging to the pulmonary hospital of Shanghai, university, were randomly selected, and patients were screened for high risk by low dose helical CT scanning and were highly suspected to be NSCLC imagewise, i.e. early stage NSCLC patients.

14 cases of plasma samples from healthy people belonging to Shanghai city pulmonary hospital at the university of Tongji are randomly selected as a control group, and the healthy people are similar in age, have no history of coronary heart disease, have no surgery, have normal blood fat, blood sugar and blood pressure, have no familial genetic diseases, and have no liver, kidney and heart dysfunction.

The study was approved by the ethics committee of the medical science of the department of pneumology, shanghai, university, and informed consent was obtained for all subjects.

2. Test method

And (3) collecting 3-5mL of peripheral venous blood sample of the object to be detected by adopting an EDTA anticoagulation tube, storing the collected blood sample in a 4 ℃ environment, centrifuging at 4 ℃ and 3500rpm for 5min, collecting upper plasma in an EP tube without RNase, adding 750 mu L of Trizol Reagent solution into every 250 mu L, uniformly mixing, and storing at-80 ℃ for use.

While blood was collected, clinical data such as gender, age, medical history, history of smoking and drinking, clinical stage of NSCLC, imaging characteristics, etc. of each subject were collected.

The above plasma samples were removed prior to the assay and slowly thawed on ice, and then the expression level of the biomarker LncRNA air in the samples was detected using the kit for early detection of NSCLC provided in example 1, and the marker specificity of the LncRNA molecules in early NSCLC was analyzed.

2.1 extraction of Total RNA from plasma samples

(1) Taking out the EP tube filled with the plasma sample, vibrating and uniformly mixing at room temperature, standing at room temperature, and incubating for 5min;

(2) Adding 0.3mL of chloroform into the EP tube in the step 1), covering the EP tube cover, oscillating for 30s, and standing at room temperature for 2min to obtain a mixed extract;

(3) Centrifuging the mixed extract of step 2) at 12000rpm at 4deg.C for 15min, and collecting supernatant into new RNase-free EP tube;

(4) Adding isopropyl alcohol with equal volume into the supernatant obtained in the step 3), shaking and mixing uniformly, and standing at-20 ℃ for more than 30 min.

(5) After the operation according to the step 4), taking out and centrifuging at 12000rpm for 15min at 4 ℃, reserving bottom sediment, and discarding supernatant;

(6) Adding 1mL of 75% ethanol (enzyme-inactivating treatment, preparing in situ and pre-cooling on ice) after the operation (5) is finished, lightly washing the precipitate, centrifuging at 7500rpm for 5min at 4 ℃, and discarding the supernatant to obtain RNA precipitate;

(7) Standing and airing the RNA precipitate obtained in the step 6) at room temperature for less than 10 minutes, and then adding 10 mu L of ultrapure water for dissolving to obtain a total RNA solution;

(8) Measuring the concentration of 1 mu L of the RNA solution obtained in the step 7) by using an ultra-micro ultraviolet spectrophotometer, respectively measuring the absorbance at the wavelengths of 260nm and 280nm, wherein the absorbance of A260/A280 of a sample with good quality is 1.8-2.0, and calculating the total RNA volume required by the reverse transcription reaction according to the calculated RNA concentration; the remaining samples were stored at-80℃or were subjected to total RNA reverse transcription directly.

2.2 Total RNA reverse transcription

The total RNA reverse transcription reaction system with a total volume of 20. Mu.L was set, and the components, the component contents and the reaction conditions thereof are shown in Table 1:

TABLE 1 Total RNA reverse transcription reaction System and reaction conditions

2.3 real-time fluorescent quantitative PCR amplification

And (3) carrying out real-time fluorescence quantitative PCR amplification by taking the cDNA product obtained by the reverse transcription of 2.2 as a template. The reagent and component contents of the real-time fluorescent quantitative PCR amplification reaction system were prepared with a total volume of 15. Mu.L, as shown in Table 2:

TABLE 2 real-time fluorescent quantitative PCR reaction system

The amplification reaction conditions were: 3min at 95 ℃;95℃15s,54℃30s,72℃25s,40 cycles.

3. Data analysis

Each test data was statistically analyzed using SPSS25.0 statistical software and matched comparison of control (i.e., healthy) and experimental (i.e., early NSCLC patient) groups using rank-sum test, considered statistically significant when P < 0.05.

And (3) observing a real-time fluorescence quantitative PCR amplification curve and a dissolution curve, and setting a base line and a threshold value to obtain a Ct value.

Experimental data; the experimental data are compared and quantified according to a fluorescence quantitative PCR relative quantitative formula: 2 ^-△△Ct The expression level of the biomarker in the plasma of healthy people in the control group and in the early NSCLC patients in the experimental group was calculated respectively, each sample was repeated three times by using the kit procedure, and the average value was obtained as the final value of the sample.

4. Analysis of results

According to the relative quantitative formula of fluorescence quantitative PCR: 2 ^-△△Ct The expression level of lncrnaiavrn in healthy human plasma and in the plasma of patients with early stage NSCLC was calculated.

The results are shown in figure 3, where LncRNAAIRN expression levels in plasma of patients with early stage NSCLC were 4.55-fold higher than average in plasma of healthy humans, with P <0.0001.

The detection result is consistent with the conclusion of the primary screening result of the patient imaging.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

Claims (7)

1. An early non-small cell lung cancer lncRNA marker is characterized in that the gene sequence is shown as SEQ ID NO. 1.

2. The use of the lncRNA marker of claim 1 in the preparation of a kit for detecting early non-small cell lung cancer.

3. A kit for detecting the lncRNA marker of claim 1, characterized in that: the sequence of the upstream primer for detecting the lncRNA marker is as follows: 5'-AGGAGCGGGACTGAAATA-3';

the sequence of the downstream primer for detecting the lncRNA marker is as follows: 5'-GACTGCACTGCGACGAGA-3'; the primer also comprises an internal reference gene GAPDH amplification primer, wherein the upstream primer sequence of the internal reference gene GAPDH is as follows: 5'-GCTCTCTGCTCCTCCTGT-3'; the sequence of the downstream primer of the reference gene GAPDH is as follows: 5'-GACTCCGACCTTCACCTTCC-3'.

4. A kit according to claim 3, wherein: cDNA, TB Green Premix Ex Taq II, ROX Reference Dye II and ultrapure water are also included.

5. A kit according to claim 3, wherein: the kit also comprises a total RNA extraction Reagent set, wherein the total RNA extraction Reagent set comprises Trizol Reagent solution, chloroform, isopropanol, ethanol and ultrapure water.

6. A kit according to claim 3, wherein: the kit also comprises an RNA reverse transcription reagent group, wherein the RNA reverse transcription reagent group comprises a random primer, dNTP mixed solution, 5× Reverse Transcriptase M-MLV Buffer, DTT solution, an RNase inhibitor and M-MLV reverse transcriptase.

7. A method of using the kit of claim 3, comprising the steps of:

1) Collecting 3-5mL of peripheral venous blood sample of an object to be detected by adopting an EDTA anticoagulation tube, storing the collected blood sample in a 4 ℃ environment, centrifuging at 3500rpm for 5min at 4 ℃, collecting upper plasma in an EP tube without RNase, adding 750 mu L of Trizol Reagent solution into every 250 mu L, uniformly mixing, and storing at-80 ℃ for use;

2) Taking out the EP tube filled with the plasma sample, vibrating and uniformly mixing at room temperature, standing at room temperature, and incubating for 5min;

3) Adding 0.3mL of chloroform into the EP tube in the step 2), covering the EP tube cover, oscillating for 30s, and standing at room temperature for 2min to obtain a mixed extract;

4) Centrifuging the mixed extract of step 3) at 12000rpm at 4deg.C for 15min, and collecting supernatant into new RNase-free EP tube;

5) Adding isopropyl alcohol with the same volume into the supernatant obtained in the step 4), shaking and mixing uniformly, and standing at the temperature of minus 20 ℃ for more than 30 min.

6) After the operation according to step 5), taking out and centrifuging at 12000rpm for 15min at 4 ℃, reserving bottom sediment, and discarding supernatant;

7) Adding 1mL of ethanol with the volume percentage concentration of 75% after the operation of the step 6), washing the precipitate, centrifuging at 7500rpm for 5min at 4 ℃, and discarding the supernatant to obtain RNA precipitate;

8) Standing and airing the RNA precipitate obtained in the step 7) at room temperature for less than 10 minutes, and then adding 10 mu L of ultrapure water for dissolving to obtain a total RNA solution;

9) Measuring the concentration of 1 mu L of the RNA solution obtained in the step 8) by using an ultra-micro ultraviolet spectrophotometer, respectively measuring the absorbance at the wavelengths of 260nm and 280nm, wherein the absorbance of A260/A280 of a sample with good quality is 1.8-2.0, and calculating the total RNA volume required by the reverse transcription reaction according to the calculated RNA concentration; the rest sample is placed at-80 ℃ for preservation or direct total RNA reverse transcription reaction;

10 A total RNA reverse transcription reaction system with a total volume of 20. Mu.L was set, and the components, component contents and reaction conditions thereof were as follows:

11 Real-time fluorescent quantitative PCR amplification is carried out on the product obtained by reverse transcription, and the reagent and component content of a real-time fluorescent quantitative PCR amplification reaction system are prepared, wherein the total volume of the reagent and the component content is 15 mu L, and the reagent and the component content are as follows:

the amplification reaction conditions were: 3min at 95 ℃;95℃15s,54℃30s,72℃25s,40 cycles.

12 Observing a real-time fluorescent quantitative PCR amplification curve and a dissolution curve, setting a base line and a threshold value to obtain a Ct value, and obtaining a relative quantitative formula according to fluorescent quantitative PCR: 2 ^-△△Ct And calculating the expression level of LncRNA AIRN in the plasma of the patient to be tested, and further analyzing the expression specificity of the lncRNA molecule in early NSCLC.