CN117402967A - Lung cancer marker, primer, kit and application - Google Patents
Lung cancer marker, primer, kit and application Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to a lung cancer marker, a primer, a kit and application. The lung cancer screening or diagnosing marker disclosed by the invention is a serum miRNA marker. The marker comprises: at least one of hsa-miR-10527-5p, hsa-miR-2117, hsa-miR-4457, hsa-miR-6775-5p and hsa-miR-9986, and the corresponding coding nucleotide sequence of each microRNA is shown in SEQ ID NO. 1-SEQ ID NO. 5. The application of the blood microRNA as a lung cancer screening or diagnosis marker provided by the invention has a noninvasive and efficient effect in detection, and has a wide application prospect in preparing a lung cancer screening or diagnosis kit.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a lung cancer marker, a primer, a kit and application.
Background
Lung cancer is currently the most common cancer worldwide and is also the cause of death from the first major cancer. The number of lung cancer patients is 220 ten thousand and the number of lung cancer death cases is 180 ten thousand in 2020 worldwide, accounting for about 1/5 (18.4%) of cancer death. Thus, lung cancer is a serious threat to human health.
Early lung cancer is usually asymptomatic, and early screening and accurate diagnosis thereof are important factors in improving prognosis of patients. However, the existing lung cancer screening and diagnosing means have a plurality of limitations and disadvantages. For example, the traditional lung cancer related tumor markers cannot be used for lung cancer screening and auxiliary diagnosis due to low sensitivity and specificity, and can only be used for postoperative recurrence monitoring; chest X-ray examination is less sensitive, and more lung cancer patients (especially early lung cancer) are missed; chest CT tests are highly sensitive and specific, but the nodules found by the tests can cause a lot of unnecessary panic and overdiagnosis, overdreatment, and are expensive, so they are not suitable for early screening of lung cancer in large-scale population. Therefore, a simple, convenient, noninvasive and high-efficiency lung cancer screening and diagnosing method is urgently needed in clinic.
Micrornas (mirnas) are a class of non-coding single-stranded RNA molecules of about 19-25 nucleotides in length, encoded by endogenous genes, which are widely found in eukaryotic cells. miRNA genes account for about 1% of the entire genome, which regulate gene expression by decreasing the stability of target mRNA or inhibiting translational efficiency, thereby participating in a variety of biological processes including cell proliferation, differentiation, apoptosis, development, and stress response. The miRNA in the blood often forms a complex with proteins and the like, so that the miRNA has good RNase degradation resistance, and the expression level of the miRNA in the blood is not affected after freezing, thawing or long-term storage at room temperature. In addition, the expression level of miRNA in blood is very similar for people with different sexes and ages.
With the discovery of more miRNAs as biomarkers and therapeutic targets of human diseases, finding a marker capable of accurately diagnosing lung cancer has great significance in optimizing clinical lung cancer diagnosis strategies.
Disclosure of Invention
In view of the above, the present invention aims to provide a lung cancer marker for screening or diagnosing lung cancer, which has the following technical scheme:
in a first aspect, the present invention provides a lung cancer marker comprising: at least one of hsa-miR-10527-5p, hsa-miR-2117, hsa-miR-4457, hsa-miR-6775-5p and hsa-miR-9986.
As one implementation mode, the base sequence of the hsa-miR-10527-5p is shown as SEQ ID NO.1, the base sequence of the hsa-miR-2117 is shown as SEQ ID NO.2, the base sequence of the hsa-miR-4457 is shown as SEQ ID NO.3, the base sequence of the hsa-miR-6775-5p is shown as SEQ ID NO.4, and the base sequence of the hsa-miR-9986 is shown as SEQ ID NO. 5.
The lung cancer markers listed above are microRNAs, and through experimental detection, the expression levels of the markers in serum tissues of a lung cancer patient are statistically different from the expression levels of tissues corresponding to normal people, and the lung cancer markers such as hsa-miR-10527-5p, hsa-miR-2117, hsa-miR-4457, hsa-miR-6775-5p, hsa-miR-9986 and the like can be independently used for detecting and diagnosing lung cancer, and can also be used for jointly detecting and diagnosing lung cancer by more than two markers, and meanwhile, the lung cancer markers can timely reflect the disease states of the lung cancer patient, are particularly suitable for early diagnosis, prognosis, postoperative recurrence detection and the like of lung cancer, avoid complex detection in the past, save time and labor cost, and facilitate clinical doctors to adopt personalized treatment schemes. Moreover, the method for detecting the microRNA markers in the serum samples is very mature, the detection process is simple and convenient and is easy to repeat, the detection investment of hospitals or detection institutions can be greatly reduced by common technicians, meanwhile, the detection samples are easy to obtain, the clinical operability is high, the traumatology is small, the stability of the serum microRNA is good, and the detection is convenient.
In a second aspect, the present invention provides a primer for detecting the above lung cancer marker, the primer comprising: an upstream primer and/or a reverse transcription primer;
wherein, the base sequence of the upstream primer of hsa-miR-10527-5p is shown in SEQ ID NO. 6;
the base sequence of the upstream primer of hsa-miR-2117 is shown in SEQ ID NO. 7;
the base sequence of the upstream primer of hsa-miR-4457 is shown in SEQ ID NO. 8;
the base sequence of the upstream primer of hsa-miR-6775-5p is shown in SEQ ID NO. 9;
the base sequence of the upstream primer of hsa-miR-9986 is shown in SEQ ID NO. 10;
the base sequence of the reverse transcription primer of hsa-miR-10527-5p is shown in SEQ ID NO. 11;
the base sequence of the reverse transcription primer of hsa-miR-2117 is shown in SEQ ID NO. 12;
the base sequence of the reverse transcription primer of hsa-miR-4457 is shown in SEQ ID NO. 13;
the base sequence of the reverse transcription primer of hsa-miR-6775-5p is shown in SEQ ID NO. 14;
the base sequence of the reverse transcription primer of hsa-miR-9986 is shown in SEQ ID NO. 15.
In a third aspect, the invention provides a kit comprising the above primer.
As one embodiment, the kit further comprises; the base sequence of the universal reverse primer of each lung cancer screening or diagnosis marker is shown as SEQ ID NO. 18.
As an embodiment, the kit further comprises: a probe for detecting the lung cancer marker according to claim 1 or 2, wherein the base sequence of the probe is shown in SEQ ID No. 19.
As an embodiment, the kit further comprises: an RNA extraction reagent;
the RNA extraction reagent comprises TRIzol TM LS、Beyozol、PureZOL、Aurum TM At least one of them.
The kit provided by the invention comprises: the primers for detecting the lung cancer markers can detect the lung cancer markers in serum by utilizing a reverse transcription reaction technology and a PCR reaction technology, so that the purpose of screening or diagnosing lung cancer is achieved, the method is mature, the detection process is simple and convenient and easy to repeat, and early diagnosis, prognosis judgment, postoperative recurrence detection and the like of lung cancer are facilitated.
When the kit is used for diagnosing lung cancer, the detection method comprises the following steps:
1) Collecting a serum sample;
2) Extracting total RNA in a sample;
3) Synthesizing cDNA by taking the extracted RNA as a template;
4) Carrying out RT-qPCR reaction on the primer and the probe to obtain the cycle number, namely CT value, of the target primer and the external reference primer in the serum sample to be detected when the fluorescent signals reach a set threshold value;
5) According to the 2-delta Cq calculation method, the delta CT value of the target primer in the serum sample is calculated through the external reference primer.
In a fourth aspect, the invention also provides application of the lung cancer marker or the primer or the kit in preparation of anti-lung cancer drugs.
The lung cancer marker, the primer and the kit provided by the invention are applied to the preparation of the anti-lung cancer medicament, thereby being beneficial to saving time and labor cost and shortening the research and development and production cycle of the anti-lung cancer medicament.
Drawings
FIG. 1 is a graph showing the expression of hsa-miR-10527-5p in serum of lung cancer patients and healthy control groups;
FIG. 2 is a ROC curve of hsa-miR-10527-5 p;
FIG. 3 is a graph showing the expression of hsa-miR-2117 in serum of a lung cancer patient and a healthy control group;
FIG. 4 is an ROC curve of hsa-miR-2117;
FIG. 5 is a graph showing the expression of hsa-miR-4457 in serum of a lung cancer patient and a healthy control group;
FIG. 6 is an ROC curve of hsa-miR-4457;
FIG. 7 is a graph showing the expression of hsa-miR-6775-5p in serum of lung cancer patients and healthy control groups;
FIG. 8 is a ROC curve of hsa-miR-6775-5 p;
FIG. 9 is an expression pattern of hsa-miR-9986 in serum of a lung cancer patient and a healthy control group;
FIG. 10 is a ROC curve of hsa-miR-9986.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. 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
(1) Serum sample collection and preparation
Serum from all volunteers 120 cases of lung cancer patients with confirmed diagnosis is collected, serum from healthy people with physical examination 120 cases is taken from each case, 2ml of peripheral blood is taken from each case, and the obtained serum is centrifuged at 4 ℃ for 2 hours at a speed of 3600g for 10 minutes, and upper serum is taken from each case and stored in a refrigerator at-80 ℃ for standby.
(2) Extraction of RNA and Synthesis of cDNA in serum samples
200uL of serum extracted in the step (1) is taken, 0.1pM nematode cel-miR-54 is added as an external reference, 1ml of TRIzol is added according to the ratio of 1:5, and the mixture is fully and uniformly mixed, and total RNA is extracted according to the scheme provided by the TRIzol. According to the method of the reverse transcription kit, reverse transcription enzyme (Sensiscript Reverse Transcriptase), dNTP (dNTP Mix) and reaction buffer (10× Sensiscript Reverse Transcription Buffer) provided by the kit are added on a common PCR instrument by taking the extracted total RNA (Purified RNA) as a template, and the microRNA marker and the reverse transcription primer mixture (Reverse transcription Mix) of cel-miR-54 are subjected to reverse transcription into cDNA. Preserving at-20 ℃ for standby.
TABLE 1 composition of cDNA Synthesis reaction System
Table 2 reverse transcription primers used
(3) RT-qPCR detection
The cDNA of the serum sample is subjected to reverse transcription, fluorescent real-time quantitative PCR is carried out according to the method of Golden HS TaqMan fluorescent real-time quantitative PCR kit, and Ct values of all samples are read out by a light cycler480 (Roche) fluorescent quantitative PCR instrument. The qRT-RCR application instrument is LightCycler480 (Roche), and the reaction conditions are: (1) pre-denaturation, 95 ℃,5 min; (2) denaturation, 95 ℃,10 seconds; annealing at 60℃for 30 seconds for a total of 40 cycles.
According to the 2-delta Cq calculation method, calculating delta CT value of the serum sample by taking cel-miR-54 as an external reference, and according to a formula delta CT (detection) =CT (microRNA, detection) -CT (external reference, detection); Δct (calibration) =ct (lung cancer, calibration) -CT (control, calibration); Δct=Δct (detection) - Δct (calibration), based on the obtained CT value(s), and carrying out relative quantitative detection on the target gene according to the 2-delta CT value.
Data are sorted by adopting a 2-delta CT method, data analysis and graphic processing are analyzed by adopting GraphPad Prism 8.3.0 software, all data are detected by adopting a Two-driven Student's test statistical method, the data are expressed by mean value +/-standard deviation, and P <0.05 is statistically significant.
TABLE 3 real-time fluorescent quantitative PCR reaction system
TABLE 4 Lung cancer markers, upstream primers and probe sequences of this example
Example 2 comparison of hsa-miR-10527-5p expression in serum of Lung cancer patients with Normal persons
Serum 112 cases of lung cancer patients with confirmed diagnosis and serum 112 cases of healthy people with physical examination are collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, with informed consent of all volunteers, and the results are shown in FIGS. 1-2.
As shown in fig. 1-2, hsa-miR-10527-5P expression levels in serum of lung Cancer patients (Cancer) were significantly down-regulated (P < 0.0001) compared to normal (Control) (fig. 1); the area under the curve corresponding to the ROC curve was 0.6985 (95% CI:0.6326-0.7644, P < 0.0001) (FIG. 2), the sensitivity was 71.67%, and the specificity was 58.33%.
The experimental results of this example show that the expression of hsa-miR-10527-5p in the serum of lung cancer patient is down-regulated compared with that of normal human, and the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-10527-5p on lung cancer patients showed auc= 0.6984, sensitivity of 71.67% and specificity of 58.33%.
Example 3 comparison of hsa-miR-2117 expression in serum of Lung cancer patient and Normal person
Serum 112 cases of lung cancer patients with confirmed diagnosis and serum 112 cases of healthy people with physical examination are collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 3 to 4.
Significant upregulation of hsa-miR-2117 expression levels in serum of lung Cancer patients (Cancer) over normal (Control) (P < 0.0001) (FIG. 3); the area under the curve corresponding to the ROC curve was 0.7267 (95% CI:0.6628-0.7905, P < 0.0001) (FIG. 4), the sensitivity was 70.83%, and the specificity was 64.17%.
Experimental results in the embodiment show that the hsa-miR-2117 in the serum of the lung cancer patient is up-regulated compared with the expression of normal people, and the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-2117 on lung cancer patients showed auc= 0.7267, sensitivity of 70.83% and specificity of 64.17%.
Example 4 comparison of hsa-miR-4457 expression in serum of Lung cancer patient with Normal person
Serum 112 cases of lung cancer patients with confirmed diagnosis and serum 112 cases of healthy people with physical examination are collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 5 to 6.
Significant downregulation of hsa-miR-4457 expression levels in serum of lung Cancer patients (Cancer) compared with normal (Control) (P < 0.0001) (FIG. 5); the area under the curve corresponding to the ROC curve was 0.7147 (95% CI:0.6482-0.7813, P < 0.0001) (FIG. 6), the sensitivity was 70.83%, and the specificity was 51.67%.
The experimental results of the embodiment show that the hsa-miR-4457 in the serum of the lung cancer patient has lower expression compared with the normal human group, and the difference has statistical significance. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-4457 on lung cancer patients showed auc= 0.7147, sensitivity of 70.83% and specificity of 51.67%.
Example 5 comparison of hsa-miR-6775-5p expression in serum of Lung cancer patients with Normal persons
Serum 112 cases of lung cancer patients with confirmed diagnosis and serum 112 cases of healthy people with physical examination are collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 7 to 8.
Significant downregulation of hsa-miR-6775-5P expression levels in serum of lung Cancer patients (Cancer) compared to normal (Control) (P < 0.0001) (FIG. 7); the area under the curve corresponding to the ROC curve was 0.838 (95% CI:0.7887-0.8873, P < 0.0001) (FIG. 8), the sensitivity was 80.83%, and the specificity was 70.83%.
The experimental results of this example show that hsa-miR-6775-5p in serum of lung cancer patients is down-regulated compared with normal human expression, and the difference is statistically significant. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-6775-5p in lung cancer patients showed auc=0.838 (fig. 8), sensitivity 80.83% and specificity 70.83%.
EXAMPLE 6 comparison of hsa-miR-9986 expression in serum of Lung cancer patient and Normal person
Serum 112 cases of lung cancer patients with confirmed diagnosis and serum 112 cases of healthy people with physical examination are collected. Quantitative analysis was performed by the method of example 1 with the exception of peripheral blood, respectively, with informed consent of all volunteers, and the results are shown in FIGS. 9 to 10.
As shown in fig. 9-10, hsa-miR-9986 expression levels in serum of lung Cancer patients (Cancer) were significantly down-regulated (P < 0.0001) compared to normal (Control) (fig. 9); the area under the curve corresponding to the ROC curve was 0.7684 (95% CI:0.7088-0.828, P < 0.0001) (FIG. 10), the sensitivity was 75.83%, and the specificity was 64.17%.
Experimental results in the embodiment show that the hsa-miR-9986 in the serum of the lung cancer patient has lower expression compared with normal human, and the difference has statistical significance. ROC curve analysis of diagnostic efficacy of circulating hsa-miR-9986 on lung cancer patients showed auc= 0.7684 (fig. 10), sensitivity of 75.83% and specificity of 64.17%.
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, and alternatives falling within the spirit and principles of the invention.
Claims (8)
1. A marker for lung cancer, comprising: at least one of hsa-miR-10527-5p, hsa-miR-2117, hsa-miR-4457, hsa-miR-6775-5p and hsa-miR-9986.
2. The lung cancer marker of claim 1, wherein the base sequence of hsa-miR-10527-5p is shown in SEQ ID No.1, the base sequence of hsa-miR-2117 is shown in SEQ ID No.2, the base sequence of hsa-miR-4457 is shown in SEQ ID No.3, the base sequence of hsa-miR-6775-5p is shown in SEQ ID No.4, and the base sequence of hsa-miR-9986 is shown in SEQ ID No. 5.
3. A primer for detecting the lung cancer marker of claim 1 or 2, the primer comprising: an upstream primer and/or a reverse transcription primer;
wherein, the base sequence of the upstream primer of hsa-miR-10527-5p is shown in SEQ ID NO. 6;
the base sequence of the upstream primer of hsa-miR-2117 is shown in SEQ ID NO. 7;
the base sequence of the upstream primer of hsa-miR-4457 is shown in SEQ ID NO. 8;
the base sequence of the upstream primer of hsa-miR-6775-5p is shown in SEQ ID NO. 9;
the base sequence of the upstream primer of hsa-miR-9986 is shown in SEQ ID NO. 10;
the base sequence of the reverse transcription primer of hsa-miR-10527-5p is shown in SEQ ID NO. 11;
the base sequence of the reverse transcription primer of hsa-miR-2117 is shown in SEQ ID NO. 12;
the base sequence of the reverse transcription primer of hsa-miR-4457 is shown in SEQ ID NO. 13;
the base sequence of the reverse transcription primer of hsa-miR-6775-5p is shown in SEQ ID NO. 14;
the base sequence of the reverse transcription primer of hsa-miR-9986 is shown in SEQ ID NO. 15.
4. A kit, comprising: the primer according to claim 3.
5. The kit of claim 4, further comprising; the base sequence of the universal reverse primer of each lung cancer screening or diagnosis marker is shown as SEQ ID NO. 18.
6. The kit of claim 4, further comprising: a probe for detecting the lung cancer marker according to claim 1 or 2, wherein the base sequence of the probe is shown in SEQ id No. 19.
7. The kit of claim 4, further comprising: an RNA extraction reagent;
the RNA extraction reagent comprises TRIzol TM LS、Beyozol、PureZOL、Aurum TM At least one of them.
8. Use of a lung cancer marker according to claim 1 or 2 or a primer according to claim 3 or a kit according to any one of claims 4 to 7 for the preparation of an anti-lung cancer medicament.
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