CN110205383A - A kind of application of LncRNA marker in lung cancer immune function is detected or assessed - Google Patents

Abstract

The invention relates to the field of molecular biology applications, in particular to the application of a LncRNA marker in the detection or evaluation of lung cancer immune function. The invention discovered for the first time that LncRNA RUNXOR has a significant expression difference in the blood of lung cancer patients. Compared with the normal control group, the test group of lung cancer patients had a high expression of the LncRNA molecular marker RUNXOR, and the expression level of LncRNA RUNXOR was significantly down-regulated after surgery; The expression of RUNX1 regulates the differentiation and immunosuppressive function of MDSCs. Based on this discovery, LncRNA RUNXOR can be used as a molecular marker or target for regulating the immune function of lung cancer in the clinical diagnosis or targeted therapy of lung cancer, and the LncRNA RUNXOR provided by the present invention can be used in lung cancer immune function detection/evaluation products , which is conducive to further elucidating the mechanism of occurrence and development of lung cancer, and has important guiding significance for the treatment of lung cancer.

Description

Application of a lncRNA marker in the detection or evaluation of lung cancer immune function

technical field

The invention relates to the field of molecular biology applications, in particular to the application of a LncRNA marker in the detection or evaluation of lung cancer immune function.

Background technique

The morbidity and mortality of lung cancer rank first among malignant tumors in my country. At present, there are very few biomarkers for early diagnosis, immune function monitoring, and prognosis of lung cancer patients. The 5-year survival rate of patients with advanced lung cancer under traditional treatment is less than 5%. Exploring effective biomarkers for lung cancer is important for improving the treatment effect of lung cancer patients. significance.

Existing studies have found that LncRNA RUNXOR can regulate the expression of RUNX1 at the epigenetic level. In acute myeloid leukemia cells, lncRNA RUNXOR directly binds to the promoter and enhancer of RUNX1 through its 3' end and participates in chromatin remodeling. In addition, LncRNA RUNXOR can also directly recruit RUNX1 protein and bind to the promoter region of RUNX1 gene to promote the expression of RUNX1.

Lung cancer patients are in a state of immunosuppression as a whole, and the proportion of a group of myeloid-derived suppressor cells (MDSCs) with immunosuppressive function in the peripheral blood is significantly increased, while the proportion of Th1 and CTL cells participating in the anti-tumor immune response is significantly decreased. MDSCs are a group of heterogeneous cells composed of immature myeloid cells. In healthy people, these immature bone marrow cells rapidly differentiate into mature granulocytes, macrophages, and DC cells. Under some pathological conditions, such as tumors, inflammation, etc., the differentiation of these immature myeloid cells is blocked, resulting in the massive expansion of MDSCs. MDSCs have immunosuppressive function, can negatively regulate the immune response, effectively inhibit the immune response of T cells, and promote the growth of tumors. The proportion of MDSCs in the peripheral blood of tumor patients is often negatively correlated with the proportion of Th1 and CTL cells. Generally speaking, the weaker the immune function of tumor patients, the more the number of MDSCs in the body, and the lower the number of Th1 and CTL cells. The phenotype of human MDSCs is Lin ^- CD14 ^- CD11b ⁺ CD33 ⁺ CD34 ⁺ HLA-DR ^-/lo , and there are differences in the expression of CD15 and other markers. MDSCs mainly exert their inhibitory effect on T cell-mediated anti-tumor immunity by producing immunosuppressive molecules such as arginase (Arg1), inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS). However, there is no report on the application of LncRNA RUNXOR in the analysis of lung cancer immune function.

Contents of the invention

In order to make up for some deficiencies in the prior art, the present invention provides a new application of LncRNA RUNXOR to solve some problems in the prior art. The LncRNA RUNXOR is located at Gene ID: NM_001001890.2, has a length of 216 kb, and shares exons and introns with the RUNX1 gene.

The present invention also provides a product for analyzing the immune function of lung cancer, which can analyze the expression of LncRNA RUNXOR. Among them, the expression level of LncRNA RUNXOR in the peripheral blood of lung cancer patients was positively correlated with the immune ability of lung cancer patients. Further, the products include kits, probes, chips or preparations.

Further, the kit includes specific PCR primers or probes for the above-mentioned LncRNA RUNXOR marker. The kit also includes one or more combinations of dNTPs, random primers, reducing agents, RNase inhibitors, reverse transcriptase, MgCl ₂ and PCR buffer, etc., in addition, the kit can also contain standard products and/or reference products. Further, the above kit preferably also includes other auxiliary reagents, the auxiliary reagents are some reagents routinely used in quantitative PCR amplification kits, the characteristics of these reagents and their preparation methods are well known to those skilled in the art The reagents are, for example (but not limited to): negative control substance, positive control substance; it may also include fluorescent quantitative PCR reaction plate, PCR reaction plate sealing film, etc. Further, the chip includes: a solid phase carrier, and oligonucleotide probes fixed on the solid phase carrier in an orderly manner, and the oligonucleotide probes specifically correspond to the above-mentioned part of the LncRNA marker or full sequence. Wherein, the solid phase support includes a glass plate, a silicon plate, a polypropylene membrane, a nitrocellulose membrane, a nylon membrane or a polystyrene membrane.

The invention provides an application of the above-mentioned product in preparing a tool for treating and regulating the immune function of lung cancer. The tools of the present invention can be used to detect the expression levels of multiple genes including LncRNA RUNXOR.

Detection in the present invention includes but not limited to sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology or immunoassay, and nucleic acid amplification technology includes but not limited to polymerase chain reaction, reverse transcription polymerase chain reaction, transcription-mediated Amplification, ligase chain reaction, strand displacement amplification or nucleic acid sequence based amplification.

The present invention also provides the application of the above-mentioned LncRNA RUNXOR in the preparation of a drug for treating and regulating the immune function of lung cancer.

Compared with prior art, the beneficial effect of the present invention is:

The present invention discovered for the first time that LncRNA RUNXOR has a relatively obvious expression difference in the blood of lung cancer patients. Compared with the normal population control group, the test group of lung cancer patients has a highly expressed LncRNA molecular marker RUNXOR, and the expression level of LncRNA RUNXOR is significantly down-regulated after surgery; The expression level of LncRNA RUNXOR in the peripheral blood of patients with lung cancer was significantly negatively correlated with the proportion of MDSCs and the expression level of Arg1, and was significantly positively correlated with the proportion of Th1 and CTL cells. The expression of RUNX1 regulated the differentiation and immunosuppressive function of MDSCs. Based on this discovery, LncRNA RUNXOR can be used as a molecular marker or target for regulating the immune function of lung cancer in the clinical diagnosis or targeted therapy of lung cancer, and the LncRNA RUNXOR provided by the present invention can be used in lung cancer immune function detection/evaluation products , which is conducive to further elucidating the mechanism of occurrence and development of lung cancer, and has important guiding significance for the treatment of lung cancer.

Description of drawings

Figure 1 is a graph showing the expression levels of LncRNA RUNXOR in the peripheral blood of patients with different types of lung cancer in the experimental group, the control group and the experimental group;

Figure 2 is a comparison chart of the expression level of lncRNA RUNXOR in the peripheral blood of patients with lung cancer before and after surgery;

Figure 3 is a comparison chart of MDSCs in the peripheral blood samples of different types of lung cancer patients in the experimental group, the control group and the experimental group;

Figure 4 is a comparative analysis diagram of Th1/CTL in the peripheral blood samples of the experimental group and the control group;

Figure 5 is a comparison chart of the proportion of MDSC and Th1/CTL in the experimental group and the control group;

Figure 6 is a correlation analysis chart of the expression level of LncRNA RUNXOR in the experimental group, the proportion of MDSCs and the level of Arg1;

Figure 7 is a graph showing the correlation between the expression level of LncRNA RUNXOR and the ratio of Th1/CTL in the experimental group. Specific embodiments

Below in conjunction with accompanying drawing, further illustrate the present invention, these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention; The experimental method that does not indicate specific condition in the embodiment, all according to conventional conditions, used in the embodiment The amount of reagents in the kit is only exemplary, and those skilled in the art can make corresponding adjustments according to the actual situation; the reagents and biological materials, unless otherwise specified, can be obtained from commercial channels.

Those skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. In particular embodiments of the invention, the increase or decrease in gene level is typically a decrease compared to a control. The skilled person is able to pick the most relevant controls. This also generally depends on the nature of the disease under study, available samples, etc. Suitable controls include, but are not limited to, similar samples from non-lung cancer patients, the average level of a control group, or a set of clinical data on the average level of the gene product in the sampled tissue. From the above it is clear that the controls may be from the same subject, or from one or more different subjects or derived from clinical data. Optionally, controls are matched on, for example, gender, age, and the like.

Example 1:

(1) Collection and processing of samples

The peripheral blood samples of 100 lung cancer patients (53 cases of lung adenocarcinoma, 24 cases of squamous cell carcinoma, and 23 cases of small cell lung cancer) were collected as the experimental group, and 100 healthy peripheral blood samples were collected as the control group. In addition, peripheral blood samples were collected from 40 patients with lung cancer before and after surgical treatment, and these samples met the following criteria: (1) the patient was diagnosed with lung cancer for the first time and received surgical treatment for the first time; (2) the postoperative sample was 7 days after surgery collection. All above-mentioned sample providers gave informed consent, and the acquisition of samples was approved by the organizational ethics committee.

Centrifuge the peripheral venous blood collected in 2 ml K ₂ EDTA anticoagulant tube at 18°C and 2000 rpm/min for 5 minutes to separate plasma and blood cells, resuspend the blood cells in 6-8 ml ACK, let stand at room temperature for 10 minutes, and then centrifuge at 500 g for 5 minutes; Discard the supernatant, wash the cell pellet twice with 10 ml of PBS buffer, centrifuge at 500g for 5 min each time, discard the supernatant, shake the cell pellet evenly, and set aside.

(2) qRT-PCR detection of the expression of LncRNA RUNXOR molecules

Add 1ml Trizol (Invitrogen) to the treated blood cells, invert and mix several times, extract total RNA, and synthesize cDNA by reverse transcription (Toyobo ReverTra Aca qPCR RT Kit Kit (Code No.: FSQ-101)). Using cDNA as a template, LncRNA RUNXOR primers were added for PCR. Detection was performed on a Bio Rad (CFX96) fluorescent quantitative PCR instrument. The relative expression of the target gene was calculated by β-actin.

The forward primer sequence of the LncRNA RUNXOR is shown in SEQ ID NO.1, namely: 5'-CCTGTTCACGGTCCAAACTGG-3'; the reverse primer sequence of the LncRNA RUNXOR is shown in SEQ ID NO.2, namely: 5'-CGGCAAGATCACAGTCCCTAGC -3'; the forward primer sequence of the β-actin is shown in SEQ ID NO.3, namely: 5'-CACGAAACTACCTTCAACTCC-3', the reverse primer sequence of the β-actin is shown in SEQ ID NO.4, Namely: 5'-CATACTCCTGCTTGCTGATC-3'.

Figure 1 is a graph showing the expression levels of LncRNA RUNXOR in the peripheral blood of patients with different types of lung cancer in the experimental group, the control group, and the experimental group; among them, Figure A is a comparison chart of the expression levels of LncRNA RUNXOR in the peripheral blood of the experimental group and the control group, and Figure B It is a comparison chart of the expression level of lncRNA RUNXOR in the peripheral blood of patients with different types of lung cancer in the experimental group. It can be seen from Figure 1 that compared with the control group, the expression of lncRNA RUNXOR in the experimental group was significantly increased, and the expression levels of LncRNA RUNXOR in the peripheral blood of patients with different types of lung cancer were significantly increased. Figure 2 is a comparison chart of the expression level of lncRNA RUNXOR in the peripheral blood of lung cancer patients before and after surgery; it can be seen from Figure 2 that the expression level of lncRNA RUNXOR in the peripheral blood of lung cancer patients was significantly reduced one week after the initial surgical treatment.

Example 2: Detection of Arg1 expression level in MDSCs, Th1 cells, CTL cell ratio and MDSCs

(1) The proportion of MDSCs whose peripheral blood phenotype was CD33 ⁺ CD11b ⁺ CD14 ^- HLA-DR ^- in the experimental group and the control group.

Add PBS to resuspend the cell pellet at 1×10 ⁶ cells/100 μL, add PE-anti-human-CD14, PE/CY5-anti-human-CD11b, APC-anti-human-HLA-DR and FITC-anti-human- 0.25 μg each of CD33 antibody or corresponding isotype antibody. After mixing for 30 minutes at 4°C in the dark, the cells were washed with PBS and resuspended in 200 μL of PBS, and the proportion of MDSCs in peripheral blood was detected by flow cytometry (FCM).

Figure 3 is a comparison chart of MDSCs in the peripheral blood samples of the experimental group, the control group, and different types of lung cancer patients in the experimental group; as shown in Figure 3, the expression of MDSCs in the experimental group was significantly higher than that in the control group, and different types of lung cancer The proportion of MDSCs in the peripheral blood of patients was significantly increased.

(2) Detection of peripheral blood Th1 and CTL cells in the experimental group and the control group

Use Ficoll density gradient centrifugation to isolate PBMCs from lung cancer patients/healthy peripheral blood samples, then add them to 24-well plates at 1.0×10 ⁶ /well, add 50ng/mL PMA, 1μg/mL ionomycin, and then add 1 μg/mL ml BFA was incubated at 37°C, 5% CO ₂ for 4h. Cells were collected and stained with fluorescent antibodies. After staining with PE/CY5-anti-human-CD3 and FITC-anti-human-CD8 mAbs for 30 minutes, the membrane was ruptured, and then stained with PE-anti-human-IFN-γmAb for 45 minutes. Detection was performed using flow cytometry (FCM). Figure 4 is a comparative analysis diagram of Th1/CTL in the peripheral blood samples of the experimental group and the control group; as shown in Figure 4, the ratio of Th1/CTL in the experimental group was significantly lower than that in the control group.

(3) Detection of the expression level of Arg1 in peripheral blood MDSCs in the experimental group and the control group

The qRT-PCR detection method was performed as the qRT-PCR method described in Example 1 (2). Wherein, the 18S forward primer sequence is shown in SEQ ID NO.5, namely: 5'-CGGACAGGATTGACAGATTG-3', and the 18S reverse primer sequence is shown in SEQ ID NO.6, namely: 5'-GCCAGAGTCTCGTTCGTTATC-3'; Wherein, the forward primer sequence of Arg1 is shown in SEQ ID NO.7, namely: 5'-CCTTTGCTGACATCCCTAAT-3', and the reverse primer sequence of Arg1 is shown in SEQ ID NO.8, namely: 5'-GATTCTTCCGTTCTTCTTGACT-3' .

Figure 5 is a comparison chart of the proportion of MDSCs and Th1/CTL in the experimental group and the control group; as shown in Figure 5, the proportion of MDSCs is significantly negatively correlated with the proportion of Th1/CTL.

Example 3: Correlation analysis between the expression level of LncRNA RUNXOR in peripheral blood and the proportion of MDSCs, Th1 cells, CTL cells and the expression level of Arg1 in MDSCs

GraphPad Prism 5.0 software was used for statistical processing, the data were normally distributed, and Student's t-test was used. Correlation analysis uses Spearman's correlation coefficient analysis. P<0.05 means the difference is statistically significant. Figure 6 is a correlation analysis chart of LncRNA RUNXOR expression level and MDSCs ratio and Arg1 level in the experimental group; Figure 7 is a correlation analysis chart of LncRNA RUNXOR expression level and Th1/CTL ratio in the experimental group; as shown in Figure 6 and 7, by correlation Through sex analysis, it was found that the expression level of LncRNA RUNXOR in the peripheral blood of patients with lung cancer was significantly positively correlated with the proportion of MDSCs and the expression of Arg1, the main effector molecule of MDSCs, and was significantly negatively correlated with the proportion of Th1/CTL.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

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Claims (7)

1. The application of LncRNA RUNXOR markers, characterized in that the application is the application of LncRNA RUNXOR in the preparation of lung cancer immune function detection/assessment information products. 2. The application according to claim 1, wherein the product comprises a kit, a probe, a chip or a preparation for analyzing the expression of LncRNA RUNXOR. 3. application according to claim 2, is characterized in that, described test kit comprises the specific PCR primer or probe of LncRNA RUNXOR label. 4. application according to claim 3, is characterized in that, described test kit also comprises a kind of in dNTP, random primer, reducing agent, RNase inhibitor, reverse transcriptase, _MgCl and PCR buffer etc. or a combination of several. 5. The application according to claim 2, wherein the chip comprises: a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier in an orderly manner, and the oligonucleotide probes Specifically corresponding to part or all of the sequence of LncRNA RUNXOR. 6. The application according to claim 1, characterized in that the application is the application of LncRNA RUNXOR in the preparation of a tool for treating and regulating the immune function of lung cancer. 7. The application according to claim 1, characterized in that, the application is the application of LncRNA RUNXOR in the preparation of a drug for regulating the immune function of lung cancer.