CN116676385B - Application of LINC02096 in serving as marker for predicting esophageal cancer immunotherapy effect and prognosis and treatment target - Google Patents
Application of LINC02096 in serving as marker for predicting esophageal cancer immunotherapy effect and prognosis and treatment target Download PDFInfo
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Abstract
The invention provides application of LINC02096 in serving as a marker for predicting the immunotherapy effect and prognosis of esophageal cancer and a therapeutic target. The invention discovers that LINC02096 is exosome LncRNA closely related to the immune treatment effect and prognosis of esophageal squamous carcinoma, can be used as a marker for predicting the immune treatment effect and prognosis of esophageal carcinoma, and can realize the prediction of the immune treatment effect and prognosis of esophageal carcinoma. Further research shows that the LINC02096 knocked out can obviously inhibit the growth of esophageal cancer tumors in vivo; the specific nucleic acid drug targeting LINC02096 has obvious anticancer effect, and can obviously improve the anticancer effect of PD-1 monoclonal antibody when being combined with PD-1 monoclonal antibody.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of LINC02096 in serving as a marker for predicting the immunotherapy effect and prognosis of esophageal cancer and a therapeutic target.
Background
Esophageal cancer is the sixth leading cause of global tumor death, and the incidence and death rate of Chinese esophageal cancer are the first in the world. The pathological types of esophageal cancer are mainly squamous carcinoma and adenocarcinoma, of which 90% of cases are esophageal squamous cell carcinoma (esophageal squamous cell carcinoma, ESCC).
In recent years, the development of immunotherapy breaks the bottleneck of treatment of esophageal cancer and rewrites the treatment pattern of esophageal cancer. Immunotherapy represented by the immune checkpoint inhibitor PD-1 monoclonal antibody is continuously advanced from the posterior line to the two lines and the first line in advanced esophageal cancer, and research such as KEYNOTE-590, ESCORT-1 and the like is performed on exploration of different combination modes in advanced esophageal cancer first-line treatment, including immune combination chemotherapy, double-immune combination chemotherapy, targeted combination chemotherapy and the like. Among them, xu Ruihua teaches that the lead ESCORT-1st study was reported in the American Society of Clinical Oncology (ASCO) annual meeting and published results on the international top journal JAMA, showing that the use of carlizumab in combination with paclitaxel and cisplatin for advanced esophageal cancer first-line treatment significantly improved the progression free survival (6.9 vs 5.6 months) and overall survival (15.3 vs 12.0 months) of the patient. The data of the research is encouraging to promote the application of domestic PD-1 monoclonal antibodies in advanced esophageal cancer, however, only partial patients can benefit from the durable curative effect and long-term survival in immunotherapy despite the results of the existing research, so that the biomarker is explored to realize the accurate screening of the immunotherapy, the crowd which can truly benefit from the immunotherapy is positioned, and the unnecessary cost and the drug toxicity of patients with unresponsiveness are avoided.
The exosomes are vesicles which are secreted by cells and have a size of 30-100 nm, and which contain bioactive substances such as lipids, proteins, mRNA, miRNA, lncRNA, etc., and which can exert biological functions by binding to target cell receptors or transmitting bioactive substances at a level. It has been found that they are capable of modulating host-pathogen interactions, involved in the pathological processes of a wide variety of diseases, such as infectious and inflammatory diseases, neurological diseases and cancers. Exosomes contain abundant biomarkers that can be used to monitor clinical status, therapeutic response, disease progression, etc. Long non-coding RNA (lncRNA) is a class of transcription products of RNA polymerase II that are greater than 200 nucleotide sequences in length, located in the nucleus or cytoplasm. Since lncRNA lack a distinct open reading frame, they do not have the function of encoding proteins, and they were initially considered to be "noise" in the transcription process. However, recent studies have shown that lncRNAs are abnormally expressed in a variety of tumors and play an important regulatory role in the proliferation, migration, apoptosis and metastasis processes of cells. In the tumor growth process, lncRNA can directly participate in the expression regulation of protein coding genes in RNA form through various layers, such as chromatin modification, epigenetic marking, gene transcription control, mRNA combined with the regulation after transcription, and the like.
In the prior art, the plasma exosome lncRNA is used as a biomarker for diagnosing esophageal squamous cell carcinoma, but no report that the plasma exosome lncRNA is used as a biomarker for predicting the immunotherapy effect and prognosis of esophageal squamous cell carcinoma is found.
Disclosure of Invention
The invention aims to solve the technical problem of exploring a technology capable of predicting esophageal cancer patients with good immunotherapy effect, and can be used for clinically guiding esophageal cancer treatment schemes and medication schemes.
It is a primary object of the present invention to provide the use of LINC02096 for predicting the effect and/or prognosis of immunotherapy of esophageal cancer.
It is a further object of the invention to provide the use of LINC02096 as a marker for predicting the effect and/or prognosis of immunotherapy of esophageal cancer.
It is a further object of the invention to provide the use of a reagent for detecting LINC02096 for the preparation of a kit for predicting the immunotherapeutic effect and/or prognosis of esophageal cancer.
It is a further object of the invention to provide the use of LINC02096 as a target for the treatment of esophageal cancer.
It is a further object of the invention to provide the use of LINC02096 as a drug target for the treatment of esophageal cancer.
It is a further object of the invention to provide the use of LINC02096 inhibitors for the manufacture of a medicament for the treatment of esophageal cancer.
It is a further object of the invention to provide the use of LINC02096 inhibitors in combination with PD-1 antibodies for the manufacture of a medicament for the treatment of esophageal cancer.
It is still another object of the present invention to provide a medicament for treating esophageal cancer.
It is a further object of the invention to provide a kit for predicting the effect and/or prognosis of immunotherapy of esophageal cancer.
The invention achieves the aim by the following technical means:
the invention extracts plasma exosome lncRNA of esophageal squamous carcinoma patients with effective and ineffective immunotherapy, analyzes LINC02096 to be exosome LncRNA closely related to the immunotherapy effect and prognosis of esophageal squamous carcinoma, and clarifies the related action mechanism of LINC02096 on the immunotherapy effect and prognosis of esophageal squamous carcinoma, wherein the high expression of LINC02096 reduces infiltration of tumor immune cells and immune killing activity, and LINC02096 promotes expression of immunosuppressive molecules through KMT2A-H3K4me 3.
In addition, the invention also discovers that LINC02096 knockout can obviously inhibit the growth of esophageal squamous carcinoma tumor in vivo, and PD-1 antibody combined can further inhibit the growth of esophageal squamous carcinoma tumor in vivo; the specific nucleic acid drug (LINC 02096 inhibitor) targeting LINC02096 has remarkable anticancer effect and can improve the anticancer effect of PD-1 monoclonal antibody.
Based on the experimental results, the following technical schemes are also within the protection scope of the invention.
The invention provides an application of LINC02096 in predicting the immune therapeutic effect and/or prognosis of esophageal cancer, wherein the Gene ID of LINC02096 in NCBI is 107984977.
The invention also provides application of LINC02096 in serving as a marker for predicting the immune treatment effect and/or prognosis of esophageal squamous carcinoma.
The invention also provides application of the LINC02096 detection reagent in preparation of a kit for predicting the immunotherapy effect and/or prognosis of esophageal cancer.
Preferably, the reagent for detecting LINC02096 is a reagent used for qPCR.
The invention also provides application of LINC02096 in serving as an esophageal cancer treatment target.
The invention also provides application of the LINC02096 inhibitor in preparing medicines for treating esophageal cancer.
The invention also provides application of the LINC02096 inhibitor in preparing a synergist of an esophageal cancer immunotherapy drug.
The invention also provides application of the LINC02096 inhibitor combined with the PD-1 antibody in preparing medicines for treating esophageal cancer.
The invention also provides a medicament for treating esophageal cancer, which is characterized by containing an inhibitor of LINC02096.
Preferably, the medicament further comprises a PD-1 antibody.
Preferably, the medicament further comprises pharmaceutically acceptable excipients.
Preferably, the inhibitor of LINC02096 is a specific nucleic acid agent targeting LINC02096.
More preferably, the nucleotide sequence of the LINC 02096-targeting specific nucleic acid agent is 5'-AGCGCACCGAAAGGAAC CAA-3'.
The invention also provides a kit for predicting the immunotherapy effect and/or prognosis of esophageal cancer, which is characterized by containing a reagent for detecting LINC02096.
Preferably, the esophageal cancer is esophageal squamous carcinoma.
The invention has the following beneficial effects:
according to the analysis of the invention, LINC02096 is exosome LncRNA closely related to the immune treatment effect and prognosis of esophageal squamous carcinoma, can be used as a biomarker for predicting the immune treatment effect and prognosis of esophageal carcinoma, realizes the prediction of the immune treatment effect and prognosis of esophageal carcinoma, and has important significance for guiding the treatment and drug regimen selection of clinical esophageal carcinoma patients.
Further research shows that the LINC02096 knocked out can obviously inhibit the growth of esophageal squamous carcinoma tumor in vivo; the specific nucleic acid drug targeting LINC02096 has obvious anticancer effect and can improve the anticancer effect of PD-1 monoclonal antibody.
Drawings
FIG. 1 is a graph showing the results of analysis of the effects of LINC02096 on esophageal squamous carcinoma immunotherapy and prognostic relevance, wherein; FIG. 1A shows the Disease Control Rate (DCR) and Objective Remission Rate (ORR) after treatment with PD-1 mab corresponding to the plasma exosome LINC02096 high and low expression group; FIG. 1B shows the DCR and ORR of LINC02096 high and low expression sets in tumor tissues after treatment with PD-1 mab; FIG. 1C shows total survival (OS) following treatment with PD-1 mab corresponding to the plasma exosome LINC02096 high and low expression group; FIG. 1D shows Progression Free Survival (PFS) following treatment with PD-1 mab corresponding to the plasma exosome LINC02096 high and low expression group.
FIG. 2A is a graph showing the results of analysis of the correlation between LINC02096 expression level and tumor immune cell infiltration and immune killing activity; FIG. 2B shows the expression of LINC02096 knockout cell line immune checkpoint molecules detected by an immune checkpoint chip; FIG. 2C shows the detection of LINC02096 knockout cell line and the expression of immune checkpoint molecule of over-expression cell line by flow cytometry.
FIG. 3A shows the results of an experiment to identify a target protein of LINC02096 that performs a biological function by RNA Pulldown experiment and WB; FIG. 3B is an experimental result of a RIP experiment verifying that LINC02096 specifically binds to KMT 2A; FIG. 3C is an experimental result of MS2-TRAP experiment to verify that LINC02096 specifically binds to KMT 2A; FIG. 3D shows H3K4me3 levels of PD-L1 promoter after ChIP-qPCR detection of LINC02096 knockout; FIG. 3E shows H3K4me3 levels of the IDO-1 promoter after the LINC02096 knockout by ChIP-qPCR.
FIG. 4 is a knockout LINC02096 in vitro CD8 + Effect of T cells on killing activity of esophageal squamous carcinoma cells; wherein, fig. 4A and fig. 4B are graphs of results and statistical graphs of experiments of killing esophageal squamous carcinoma cells by T cells in vitro after crystal violet staining and statistics of LINC02096 knockout; FIG. 4C shows the detection of CD8 + Experimental results of lactate dehydrogenase levels released by tumor cell lysis death in culture medium supernatants of T cells co-cultured with esophageal squamous carcinoma cells; FIG. 4D shows the detection of CD8 + CD8 in culture medium supernatant of co-culture of T cells and esophageal squamous carcinoma cells + Experimental results of interferon IFN-gamma levels secreted by T cells.
FIG. 5A is the effect of knockdown LINC02096 and PD-1 antibodies in combination on tumor growth in esophageal squamous carcinoma; FIG. 5B shows the tumor growth curve, tumor weight, and flow cytometry detection of intratumoral CD8 for huPBMC-NOG-CDX model + T cells and IFN-gamma + CD8 + T cell ratio; FIG. 5C is a flow cytometry detection of intratumoral IFN-gamma + CD8 + Representative plot of T cell ratio; FIG. 5D shows the multiplex immunohistochemical detection of intratumoral CD8 + T cells and GranB + CD8 + T cell ratio.
FIG. 6 shows the effect of tail intravenous LINC02096-inhibitor and PD-1 monoclonal antibody on esophageal squamous carcinoma tumor volume, tumor weight and flow cytometry detection of intratumoral CD8 in huPBMC-NOG-PDX model + T cells and IFN-gamma + CD8 + T cell ratio; RIME inhibitor in the figure represents LINC02096-inhibitor.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 identification of plasma exosomes LncRNA associated with the Effect and prognosis of esophageal squamous carcinoma immunotherapy
1. Screening candidate plasma exosome LncRNA related to esophageal squamous carcinoma immunotherapy effect and prognosis
1. Collecting a plasma sample:
10 patients with effective and ineffective treatment in esophageal squamous carcinoma PD-1 monoclonal antibody (terep Li Shan antibody) single drug treatment queues (from university of Zhongshan tumor prevention center) were collected, and 20 patients were treated.
Judgment principle of selecting specimens (treatment effective and ineffective): post-treatment imaging of PD-1 mab assessed as either Partial Remission (PR) or Complete Remission (CR) as "effective", and post-treatment imaging of PD-1 mab assessed as "ineffective" for disease Progression (PD).
2. Plasma exosomes LncRNA pooling:
extracting plasma exosomes by using an ultracentrifugation method, carrying out LncRNA library establishment and belief analysis, determining 10 plasma exosomes LncRNA before the patient with ineffective PD-1 monoclonal antibody treatment expression level, taking the 10 plasma exosomes LncRNA as candidate plasma exosomes LncRNA, and further screening and identifying the plasma exosomes LncRNA related to the immune treatment effect and prognosis of esophageal squamous carcinoma.
2. Screening and identifying plasma exosome LncRNA associated with esophageal squamous carcinoma immunotherapy effect and prognosis
1. Analysis of expression level of candidate plasma exosomes LncRNA and treatment effect and prognosis correlation of PD-1 monoclonal antibody
(1) Experimental method
Respectively detecting the expression levels of the 10 candidate plasma exosomes LncRNA in the plasma of 58 esophageal squamous carcinoma patients by using a qPCR method, and calculating the average expression quantity of each candidate plasma exosomes LncRNA; and (3) the patients with the plasma exosome LncRNA expression level higher than the average expression level are classified as a high expression group, the patients with the plasma exosome LncRNA expression level lower than the average expression level are classified as a low expression group, and the treatment effect and the prognosis index of the patients with the LncRNA high-low expression group treated by the PD-1 monoclonal antibody are compared.
The primer sequences of LINC02096 used for qPCR described above are as follows:
LINC02096 forward primer sequence is shown as SEQ ID NO.1, specifically: 5'-TTCTGAGGCCCACGATAAGC-3';
LINC02096 reverse primer sequence is shown as SEQ ID NO.2, specifically: 5'-TGGAGATGTGAGTTGCTGCC-3';
the treatment effect and prognosis index comprise: disease control rate DCR, objective remission rate ORR, total survival OS, progression Free Survival (PFS).
(2) Experimental results
The results show that the expression level of plasma LINC02096 is obviously and negatively related to the curative effect and prognosis of PD-1 monoclonal antibody through verification, as shown in figure 1A. The disease control rate DCR and objective remission rate ORR of the plasma LINC02096 high-expression group are lower than those of the LINC02096 low-expression group (figure 1A), which shows that the effect of the plasma LINC02096 high-expression immunotherapy is poor.
The results primarily suggest that LINC02096 is a key exosome LncRNA with negative correlation to esophageal squamous carcinoma immunotherapy effect and prognosis.
2. Transcriptome sequencing of tumor tissue
And collecting tumor tissues of 29 esophageal squamous carcinoma patients before treatment, extracting RNA, performing a conventional flow of transcriptome sequencing, and analyzing the correlation between the expression level of LINC02096 in the tumor tissues and the treatment effect and prognosis of PD-1 monoclonal antibody.
In this experiment, the sample column in which LINC02096 expression can be detected by transcriptome analysis is a LINC02096 high expression group (5 cases), the sample column in which LINC02096 cannot be detected is a LINC02096 low expression group (24 cases), and the therapeutic effect and prognostic index of the LncRNA high-low expression group are compared: disease control rate DCR, objective remission rate ORR, total survival OS, progression Free Survival (PFS).
(2) Experimental results
The analysis results of the correlation of LINC02096 expression level in tumor tissue and esophageal squamous carcinoma immunotherapy effect and prognosis are shown in FIG. 1B. The results show that the LINC02096 high expression group PD-1 monoclonal antibody in tumor tissues has lower Disease Control Rate (DCR) and Objective Remission Rate (ORR).
In addition, the total survival time OS and the progression-free survival time PFS of the plasma LINC02096 high expression group are both shortened compared with those of the LINC02096 low expression group (fig. 1C and 1D), which proves that the survival prognosis of patients with esophageal squamous cell carcinoma with high expression of plasma LINC02096 is poorer.
In combination with the results, LINC02096 in blood plasma and tumor tissues is highly expressed, PD-1 monoclonal antibody is used for treatment, and the Disease Control Rate (DCR) and Objective Remission Rate (ORR) of the treatment effect are low; and plasma LINC02096 is highly expressed, and the total survival time (OS) and the progression-free survival time (PFS) are obviously shortened when the PD-1 monoclonal antibody is used for treatment.
The results show that LINC02096 is exosome LncRNA closely related to the immune treatment effect and prognosis of the esophageal squamous cell carcinoma, and the expression quantity of LINC02096 is inversely related to the immune treatment effect and prognosis of the esophageal squamous cell carcinoma.
Example 2 Regulation and molecular mechanisms of LINC02096 on tumor immune microenvironment and immune-related factors
To further explore the mechanism by which LINC02096 is responsible for immune therapy non-response and poor prognosis, this example uses bioinformatics to analyze the relevance of LINC02096 to tumor microenvironment.
1. Exploration of the relevance of LINC02096 to the tumor microenvironment
The relevance of LINC02096 and the tumor microenvironment of the esophageal squamous carcinoma patient is analyzed through a biological information technology, and the relevance of LINC02096 and the tumor microenvironment of the esophageal squamous carcinoma patient is analyzed based on TCGA transcriptome data and CIBERSORT algorithm.
As shown in FIG. 2A, it can be seen that LINC02096 expression level and PRF1, GZMA and other tumor immune killing activity indexes have negative correlation trend, and CD8 + Tumor infiltration of cells such as T cells, NK cells, and M1 type macrophages is also inversely related. The results show that the expression level of LINC02096 has a negative correlation trend with tumor immune cell infiltration and immune killing activity.
2. Effect of LINC02096 on immune-related factor expression
The effect of LINC02096 on the expression level of tumor cell immune related molecules is analyzed by an immune check point chip and a flow cytometry technology.
1. Experimental method
(1) Construction of LINC02096 knockout cell line (KO)
LINC02096 knockdown esophageal squamous carcinoma cell lines were constructed using CRISPR technology using two esophageal squamous carcinoma cell lines, KYSE520 and KYSE-70, respectively. The specific process is as follows:
s1, construction of lentiviruses
Constructing a knockdown lentivirus for transfection of an esophageal squamous carcinoma cell line knocked down by LINC02096, wherein the knockdown lentivirus is constructed as follows: pLV_U6-LINC02096 sgRNA01-gRNA Backbone-7SK master-LINC 02096 sgRNA02-gRNA Backbone-EFS-hCAs9-2A-Puro,
the sequence of LINC02096 sgRNA01 (human) is shown as SEQ ID NO.3, and specifically comprises the following steps: 5'-CCCCCACCCGTCACCACCTT-3';
the sequence of LINC02096 sgRNA02 (human) is shown in SEQ ID NO.4, and specifically comprises: 5'-AACAAAATGACTAAGCACCG-3';
meanwhile, the knockdown control lentivirus is constructed for constructing a knockdown control cell line, and the knockdown control lentivirus is constructed as follows: pLV_U6-NC sgRNA01-gRNA backbond-7 SK pro-NC sgRNA02-gRNA backbond-EFS-hCAs 9-2A-Puro,
the NC sgRNA01 (human) has a sequence shown in SEQ ID NO.5, and specifically comprises the following components: 5'-GGAATCCGGAGCTCATGAGG-3';
the sequence of NC sgRNA02 (human) is shown as SEQ ID NO.6, specifically: 5'-TAACCCAGAAGCCCATTCAG-3';
s2, constructing LINC02096 knocked-out esophageal squamous carcinoma cell line by transfection knocked-out lentivirus
At 37℃with 5% CO 2 In the cell culture box of (2), human esophageal squamous carcinoma cell lines KYSE-70 and KYSE520 are cultured, liquid is changed for 1 time every 48 hours, and when the cell density reaches 80 percent, the passages are carried out. Cells grown in log phase were taken at 1X 10 5 Inoculating the individual cells/holes into a 6-hole plate until the cell confluency reaches 30% -40%, constructing a LINC02096 knockdown esophageal squamous carcinoma cell line by using the knockdown lentivirus transfected esophageal squamous carcinoma cell line (KYSE-70, KYSE 520), and simultaneously constructing a knockdown control cell line by using the knockdown control lentivirus transfected esophageal squamous carcinoma cell line (KYSE-70, KYSE 520); puromycinScreening for 1 week, then digesting cells, plating the cells to a 96-well plate, selecting monoclonal holes after a plurality of weeks, extracting cellular RNA, carrying out qPCR experiments, detecting the expression level of LINC02096, and amplifying the cell strain of LINC02096 which is stably knocked out compared with a knocked out control cell line to obtain the esophageal squamous cell carcinoma cell line of LINC02096 which is knocked out.
(2) Construction of LINC02096 overexpressing cell line (OE)
Two esophageal squamous carcinoma cell lines, KYSE520 and KYSE-70, were used respectively, and a LINC02096 over-expression esophageal squamous carcinoma cell line was constructed by using a lentiviral transfection technique. The specific process is as follows:
s1, construction of lentiviruses
And constructing an over-expression lentivirus, and constructing a LINC02096 over-expression esophageal squamous carcinoma cell line by transfection, wherein the element of the over-expression lentivirus after construction is pLenti-EF1a-F2A-Puro-CMV-LINC02096. Meanwhile, the over-expression control slow virus is constructed and used for constructing an over-expression control cell line, and the element after construction of the over-expression control slow virus is pLenti-EF1a-F2A-Puro-CMV-MCS.
S2, constructing LINC02096 over-expressed esophageal squamous carcinoma cell line by transfection over-expressed lentivirus
At 37℃with 5% CO 2 In the cell culture box of (2), human esophageal squamous carcinoma cell lines KYSE-70 and KYSE520 are cultured, liquid is changed for 1 time every 48 hours, and when the cell density reaches 80 percent, the passages are carried out. Cells grown in log phase were taken at 1X 10 5 Inoculating the cells/holes into a 6-hole plate until the cell confluency reaches 30% -40%, using LINC02096 over-expression slow virus to transfect esophageal squamous carcinoma cell lines (KYSE-70, KYSE 520) to construct LINC02096 over-expression esophageal squamous carcinoma cell lines, and simultaneously using the over-expression control slow virus to transfect esophageal squamous carcinoma cell lines (KYSE-70, KYSE 520) to construct an over-expression control cell line; screening puromycin for 1 week, then digesting cells, plating the cells to a 96-well plate, selecting monoclonal holes after a plurality of weeks, extracting cellular RNA, performing qPCR experiment, detecting the expression level of LINC02096, and amplifying the LINC02096 over-expressed cell strain to obtain the LINC02096 over-expressed food, wherein the monoclonal cell strain with significantly increased LINC02096 expression compared with the over-expressed control cell line is successfulSquamous cell carcinoma cell line.
(3) Effect of LINC02096 on immune-related factor expression
The experiment detects the influence of LINC02096 on a series of immune check point molecules by constructing a chip of immune related molecules (immune check point chip), and further verifies the expression level of the related immune check point molecules by a flow cytometry technology.
The experimental procedure for detecting the effect of LINC02096 on a series of immune checkpoint molecules by means of an immune checkpoint chip is as follows: primers of a plurality of (see specifically fig. 2B) immune check point molecular genes such as PD-L1, HLA-B, HLA-C and the like, PCRmix and enzyme-free water are respectively added into a Roche 384-well plate to prepare an immune check point PCR chip, and finally cDNA of a LINC02096 knocked-out esophageal squamous carcinoma cell line (KYSE 520) is added to carry out a qPCR experiment. The expression level of the relevant immune checkpoint molecule was verified by conventional immunoblotting.
Expression levels of PDL1, IDO1 were then verified by conventional flow cytometry techniques:
cells of LINC02096 knockout esophageal squamous carcinoma cell line (KYSE 520) and LINC02096 overexpressing esophageal squamous carcinoma cell line (KYSE 520) were collected, resuspended in appropriate amount of cell staining buffer (1% BSA in PBS), stained with PDL1, IDO1 antibody for 30min at 4deg.C in the absence of light, washed twice, the supernatant was discarded, 200uL of PBS was added to resuspend cells, and detection and analysis were performed with flow cytometry.
2. Experimental results
The expression condition of the immune checkpoint chip for detecting the LINC02096 knockout cell line immune checkpoint molecules is shown as a figure 2B, and the obvious down regulation of the expression level of the immune inhibitory molecules such as PD-L1, IDO1 and the like of tumor cells after LINC02096 knockout is found.
The effect of flow cytometry detection LINC02096 knockout or overexpression on tumor cell PD-L1 and IDO1 expression results are shown in FIG. 2C, and it can be seen that the expression levels of the PD-L1 and IDO1 of the tumor cells after LINC02096 knockout are obviously reduced; the opposite effect was shown in LINC02096 over-expressed cell lines, and the expression levels of PD-L1 and IDO1 were significantly increased, indicating that LINC02096 is a key factor in promoting the expression of tumor cell immunosuppressive molecules such as PD-L1 and IDO1 (FIG. 2C).
The results of FIGS. 2B and 2C demonstrate that expression of LINC02096 can promote expression of tumor cell immune checkpoint molecules such as PD-L1 and IDO-1, and participate in immune escape of tumor cells.
3. Identification of key target proteins of LINC02096 for biological function
1. Screening LINC 02096-bound target proteins
Protein mass spectrometry was combined using the RNA Pulldown method, excluding non-specifically bound proteins from the antisense strand control group, and a key target molecule for LINC02096 function was explored. The specific method comprises the following steps:
(1) RNA Pulldown method
To explore the molecular mechanisms by which LINC02096 inhibits immune cell infiltration and promotes expression of immunosuppressive molecules, we identified proteins that specifically bind to LINC02096.
The operation procedure of the RNA Pulldown kit (Sieimer's fly) was followed: by MEGAscript TM T7Transcription Kit was transcribed in vitro into LINC02096 sense and antisense strands, biotin-labeled using Pierce RNA 3'End Desthiobiotinylation Kit, and RNA pulldown experiments described below were performed using the biotin-labeled LINC02096 sense and antisense strands, respectively. Culturing human esophageal squamous carcinoma cell lines KYSE-70 and KYSE520, extracting cell Protein lysate, performing RNA pulldown experiment according to a Sieimer flight Kit Pierce Magnetic RNA-Protein Pull-Down Kit, collecting eluted Protein, performing LC-MS/MS detection, screening Protein interacted with LINC02096, and verifying through western blot.
(2) Experimental results
The results of the RNA Pulldown experiment and WB validation are shown in FIG. 3A. It can be seen that LINC02096 sense strand (AS) was able to bind to histone methyltransferase KMT2A, whereas antisense strand (S) control was unable to bind to KMT2A, indicating that LINC02096 binding to histone methyltransferase KMT2A is specific. The results indicate that LINC02096 binds to histone methyltransferase KMT 2A.
2. LINC02096-KMT2A binding specificity verification
The specificity of LINC02096-KMT2A binding was further verified by in vitro and in vivo RNA-protein interaction experiments. In vitro and in vivo RNA-protein interaction assays include RNA binding protein immunoprecipitation (RIP assay), MS 2-labeled RNA affinity purification (MS 2-TRAP assay), and the following methods are specific:
(1) Experimental method
A. RNA binding protein immunoprecipitation
RNA binding protein immunoprecipitation experiments on esophageal squamous carcinoma cell lines KYSE520 and KYSE70 were carried out by using a Magna RNA-binding protein immunoprecipitation kit kit to verify the binding specificity of LINC02096-KMT2A, and the specific steps are as follows:
s1, preparing cell lysate: and (3) using RIP lysate to lyse cells on ice, and sub-packaging the lysate for later use.
S2, preparing magnetic beads: after the magnetic beads were resuspended, 50. Mu.L of each sample was washed twice with 500. Mu.L of RIP wash buffer.
S3, magnetic bead-antibody binding: the beads were resuspended in 100. Mu.L of RIP wash buffer, and approximately 5. Mu.L of target or control IgG antibody was added and incubated for 30min at room temperature.
S4, immunoprecipitation of RNA binding protein: RIP immunoprecipitation Buffer was prepared, 900. Mu.L of RIP immunoprecipitation Buffer and 100. Mu.L of cell lysis supernatant were added to each tube, and incubated overnight at 4 ℃.
S5, washing: the beads were recovered, the supernatant discarded, 500. Mu.L of RIP wash buffer was added and the wash repeated 6 times.
S6, preparing proteinase K Buffer (117 mu L of RIP washing Buffer, 15 mu L of 10% SDS and 18 mu L of RNase proteinase K), wherein 150 mu L of each sample is needed; mixing, and incubating at 55deg.C for 30min.
S7, RNA purification: the supernatant was collected and 400. Mu.L of phenol was added to each tube: chloroform: isoamyl alcohol extraction, adding 5 μl of salt solution I, 15 μl of salt solution II, 5 μl of precipitation enhancer, 850 μl of absolute ethanol, and standing overnight;
s8, cleaning the sample with 80% ethanol, airing at room temperature, dissolving 20 mu L of enzyme-free water, and carrying out PCR or sequencing.
B. MS 2-labeled RNA affinity purification
S1 ESCC cells (KYSE 70 cell line) were co-transfected with pcDNA3.1-MS2 or pcDNA3.1-MS2-LINC02096 and MCP-3xFLAG plasmid.
After S2.48 hours, the cells were washed with PBS and the liquid was blotted off, the plates were placed on ice and irradiated with 254nm UV light at 400mJ/cm 2 Living cells are irradiated.
S3, using WB and IP lysate (containing protease phosphatase inhibitor) to lyse cells on ice for 15 min, centrifuging at 13,000Xg and 4 ℃ for 10min.
S4 immunoprecipitation was performed with anti-FLAG M2 affinity gel (Sigma-Aldrich, USA) to bind FLAG-tagged proteins.
S5, washing the anti-FLAG M2 affinity gel 3 times by using PBS buffer, adding 40 microliters of 1 XSDS-PAGE loading buffer, and heating at 95 ℃ for 10min to denature proteins.
S6, analyzing the FLAG-MCP-MS2-RNA protein complex by western blotting.
(2) Experimental results
FIG. 3B is an experimental result of a RIP experiment verifying that LINC02096 specifically binds to KMT 2A; it can be seen that KMT2A can be combined with LINC02096 endogenous in the cell, corresponding nucleic acid bands can be identified through RT-PCR and agarose gel electrophoresis, and IgG control group can not be combined with LINC02096, and the reverse verification that LINC02096 can be combined with KMT 2A;
FIG. 3C is an experimental result of MS2-TRAP experiment to verify that LINC02096 specifically binds to KMT 2A. It can be seen that MS 2-labeled LINC02096 was able to bind to KMT2A in cells, whereas MS 2-labeled control RNA was unable to bind to KMT2A in cells. MS2-TRAP experiments demonstrated that LINC02096 can bind to KMT2A in vivo.
3. LINC02096-KMT2A interaction regulates transcription of tumor cell immunosuppressive molecules through H3K4me3
The experiment researches the LINC02096-KMT2A interaction to regulate the transcription of tumor cell immunosuppressive molecules through H3K4me3 by combining with ChIP through a messenger assay.
(1) ChIP-qPCR experiment
Detection was performed using the ChIP kit. KYSE-70, KYSE520, LINC02096 knockout cell line KYSE-70, LINC02096 knockout cell line KYSE520 genome DNA are fixed, lysed and sheared by ultrasound. After detecting the shearing effect by agarose gel electrophoresis, respectively adding an anti-H3K 4me3 antibody, a control IgG antibody and magnetic beads into the supernatant, incubating and washing, performing DNA purification to obtain a purified product, designing primers according to a section with biological prediction binding, performing qRT-PCR quantification, and verifying whether the trimethylated histone (H3K 4me 3) of the third subunit IV is enriched in PD-L1 and IDO-1 gene promoters;
the primer sequences of the PD-L1 gene and IDO-1 gene promoters used in qRT-PCR quantitative analysis are as follows:
the forward primer sequence of the PD-L1 gene promoter is shown as SEQ ID NO.7, and specifically comprises the following components: PD-L1 Gene promoter Forward primer: 5'-GCCCATTCACTAACCCAAAGC-3';
the reverse primer sequence of the PD-L1 gene promoter is shown as SEQ ID NO.8, and specifically comprises the following components: PD-L1 Gene promoter reverse primer: 5'-AAAGAACTTCCCATCCCGAGC-3'.
The IDO-1 gene promoter primer sequence is as follows:
the sequence of the forward primer of the IDO-1 gene promoter is shown as SEQ ID NO.9, and specifically comprises the following steps: IDO-1 gene promoter forward primer: 5'-CGTTGTTCCATGTCTTCACT-3';
the reverse primer sequence of the IDO-1 gene promoter is shown as SEQ ID NO.10, and specifically comprises the following components: IDO-1 gene promoter reverse primer: 5'-CTTTGGGAATCAGTTCAAGG-3'.
(2) Experimental results
The experimental results are shown in FIG. 3D and FIG. 3E, wherein FIG. 3D is the H3K4me3 level of the PD-L1 promoter after LINC02096 knockout by ChIP-qPCR detection; FIG. 3E shows H3K4me3 levels of the IDO-1 promoter after the ChIP-qPCR assay knockdown of LINC02096. It can be seen that after LINC02096 is knocked out, the level of H3K4me3 of the immunosuppressive molecules PD-L1 and IDO-1 promoters is obviously reduced, and the transcription level is inhibited. The results indicate that LINC02096-KMT2A interactions promote immunosuppressive molecule expression through H3K4me 3.
In view of the experimental results, LINC02096 regulates and controls the transcription level of the target gene immune checkpoint molecule through KMT2A-H3K4me 3.
Example 3 knockout of LINC02096 in vitro CD8 + Effect of T cells on killing Activity of esophageal squamous carcinoma cells
1. Experimental method
Isolation of human peripheral blood CD8 + T cells were co-cultured with LINC02096 knocked out esophageal squamous carcinoma cells, KYSE70 or KYSE520, at a ratio of 10:1 for 12 hours, medium supernatants were collected against non-LINC 02096 knocked out esophageal squamous carcinoma cells, KYSE70 or KYSE520, interferon IFN- γ levels were detected using ELISA kit (ab 174443, abcam) according to manufacturer's instructions, and Lactate Dehydrogenase (LDH) detection kit (ab 102526, abcam) was used to detect LDH levels. The remaining surviving tumor cells were subjected to crystal violet staining and the cell numbers were counted.
2. Experimental results
The experimental results are shown in FIG. 4. Wherein, fig. 4A and fig. 4B are graphs of results and statistical graphs of experiments of killing esophageal squamous carcinoma cells by T cells in vitro after crystal violet staining and statistics of LINC02096 knockout; FIG. 4C shows the detection of CD8 + Experimental results of lactate dehydrogenase levels released by tumor cell lysis death in culture medium supernatants of T cells co-cultured with esophageal squamous carcinoma cells; FIG. 4D shows the detection of CD8 + CD8 in culture medium supernatant of co-culture of T cells and esophageal squamous carcinoma cells + Experimental results of interferon IFN-gamma levels secreted by T cells.
It can be seen that when LINC02096 is knocked out, CD8 + Tumor cells surviving co-culture of T cells with esophageal squamous carcinoma cells decreased (fig. 4A, fig. B), lactate dehydrogenase LDH released by lysis of killed tumor cells increased (fig. 4C), CD8 + The elevated levels of interferon IFN-gamma secreted by T cells (FIG. 4D) demonstrate that LINC02096 knockdown enhances the killing activity of CD8+ T cells against esophageal squamous carcinoma cells, helping to inhibit survival of esophageal squamous carcinoma cells in the tumor microenvironment.
Examples 4-6 below explore the therapeutic strategy of targeting LINC02096 in humanized immunized mice and anticancer effect of combined immunotherapy.
Example 4 Effect of LINC02096 knockout on tumors in esophageal squamous carcinoma bodies
1. Experimental grouping
A. Control group: KYSE520+IgG;
B. PD-1 mab experimental group: KYSE520+PD-1 monoclonal antibody;
C. knock-out LINC02096 experimental group: LINC02096 knockdown KYSE520+IgG;
D. knockdown LINC02096 in combination with PD-1 mab experimental group: LINC02096 knockdown KYSE520+PD-1 monoclonal antibody.
2. Experimental method
1. Construction of LINC02096 knockout esophageal squamous carcinoma cell line
LINC02096 knockout esophageal squamous carcinoma cell line (KYSE 520) was constructed using CRISPR technology and the procedure was as in example 2.
2. Construction of immune System humanized cell line transplantation tumor model huPBMC-NOG-CDX
Human PBMC (from healthy volunteers) were transplanted by tail vein injection in NOG immunodeficient mice (Beijing Veitzerland) at an injection rate of 1X 10 7 Only, a mouse model of immune system humanization (huPBMC-NOG) was constructed. The line KYSE520 from esophageal squamous carcinoma cells KYSE520 or LINC02096 knockout KYSE520 was inoculated subcutaneously in huPBMC-NOG according to the above experimental group, and the injection amount was 1×10 7 Only, huPBMC-NOG-CDX models were constructed.
3. Animal experiment
After the huPBMC-NOG-CDX model described above was developed, solid tumors formed at the injection site were measured every 3 days using digital calipers and tumor volumes were calculated using the following equation: tumor volume = length x width 2 2; in mm 3 . When the tumor grows to about 100mm 3 At the same time, according to the above group, the corresponding treatments are given respectively: igG and PD-1 antibody were administered 1 time every 3 days at a dose of 200 μg/mouse by intraperitoneal injection for 3 total treatments; tumor volume was measured every 3 days when tumor volume of any one mouse reached 2000mm 3 At the same time, all the test mice are euthanized by cervical dislocation, the tumor volumes are counted, and the treatment effect differences among different treatment groups are compared. The body weight of the mice was examined every 3 days during the course of the experiment, and was not significantly reduced (10% of body weight was not reduced), suggesting no severe immune rejection.
And (II) detecting the proportion of each immunocyte subgroup in the tumor by using flow cytometry and multiple immunohistochemistry.
(1) Flow cytometry
Placing tumor tissue in RPMI-1640 medium, and separating tumor tissueAnd (5) separating and cutting into small blocks, and mechanically dissociating by using a dissociator. Collagenase I, DNA enzyme was added and incubated at 37℃for 30 minutes. The digested tumor tissue was collected, the single cell suspension was filtered through a single cell screen to a centrifuge tube, and erythrocytes were lysed by addition of buffered ammonium chloride. The cell is dyed by using different flow antibodies, and the marker on the surface of the cell membrane is dyed first, and the marker in the cell plasma is dyed after the membrane is broken. Analysis of CD4 in tumors further using flow cytometry + T cells, CD8 + The proportion of cell components such as T cells, macrophages and the like, and simultaneously co-staining a CD8+ T cell activation index (IFN-gamma), and evaluating the activation condition of the CD8+ T cells in tumor tissues.
(2) Multiplex immunohistochemistry
Fixing mouse tumor tissue in paraformaldehyde solution, embedding in paraffin, slicing, co-staining with corresponding antibody (CD 8, PD-L1, granzyme B), photographing with confocal microscope, and evaluating CD8 in tumor tissue + T cells and Granzyme B + Infiltration of killer T cells.
3. Experimental results
The experimental results are shown in fig. 5, wherein fig. 5A shows the effect of LINC02096 knockout and PD-1 antibody combination on tumor growth in esophageal squamous cell carcinoma; FIG. 5B shows huPBMC-NOG-CDX model tumor growth curves and flow cytometry for intratumoral IFN-gamma detection + CD8 + T cell ratio; FIG. 5C is a flow cytometry detection of intratumoral IFN-gamma + CD8 + T cell ratio; FIG. 5D shows the detection of GranB in tumor by multiplex immunohistochemistry + CD8 + T cell ratio.
As can be seen from the tumor growth curves of fig. 5A and 5B, knockout LINC02096 significantly inhibited tumor growth in esophageal squamous carcinoma, combined with PD-1 mab tumor growth in esophageal squamous carcinoma was further inhibited.
It can be seen from fig. 5B, 5C, and 5D that knockout of LINC02096 significantly increases CD8 + T cells and IFN-gamma + CD8 + T or GranB + CD8 + T cell ratio, combined PD-1 mab CD8 + T cells, IFN-gamma + CD8 + T or GranB + CD8 + T cellThe ratio of (2) becomes further large. The result shows that the LINC02096 can be knocked out to obviously increase CD8 + Infiltration of T cells in tumors and activity of the T cells to kill tumors further illustrate that LINC02096 participates in immune escape of esophageal squamous carcinoma by inhibiting T cell killing tumors. The LINC 02096-targeted therapeutic means and the anticancer mode of combined immunotherapy have anticancer prospect.
Example 5 specific nucleic acid drug targeting LINC02096 and anticancer Effect of Combined immunotherapy on esophageal squamous carcinoma
1. Experimental grouping
A. Control group: non-specific ASO sequence control inhibitor+igg;
B. PD-1 mab experimental group: non-specific ASO sequence control inhibitor+pd-1 mab;
C. LINC02096-inhibitor experiment group: specific nucleic acid drug ASO+IgG targeting LINC 02096;
D. LINC02096-inhibitor combined PD-1 mab experimental group: specific nucleic acid drug ASO+PD-1 monoclonal antibody targeting LINC02096.
2. Experimental method
1. Construction of a specific nucleic acid drug targeting LINC 02096:
designing and screening a specific nucleic acid drug ASO (LINC 02096-inhibitor) for targeted inhibition of LINC02096, and packaging into an exosome to obtain the LINC02096-inhibitor nucleic acid drug.
The specific nucleic acid drug ASO sequence of the targeted LINC02096 is shown in SEQ ID NO.11, and specifically comprises the following steps: 5'-AGCGCACCGAAAGGAACCAA-3'.
ASO (Antisense oligonucleotide) is antisense oligonucleotide, and after special chemical modification, the ASO can play an effective lncRNA inhibition role in an in-vivo model.
The non-specific ASO sequence control inhibitor is a disordered non-specific nucleotide sequence.
2. Construction of humanized-humanized tumor tissue graft (PDX) model
S1, constructing a PDX model, cutting esophageal squamous carcinoma tumor tissues from a patient into proper volumes, and immediately inoculating the esophageal squamous carcinoma tumor tissues into the subcutaneous of a NOG immunodeficiency mouse.
S2, whenSuccessfully established PDX (P1) reaches about 500mm 3 At this time, mice were sacrificed, tumors were cut into appropriate volumes, transplanted onto a second batch of mice (P2), the procedure was repeated, and tumor tissue was transplanted onto a third batch of mice (P3), resulting in mice with P3 grafts.
S3, finally, the PBMC of the healthy volunteers are transplanted by tail vein injection by using the mice with the P3 implant, wherein the injection amount is 1 multiplied by 10 7 Construction of an immune System humanized mouse model (huPBMC-NOG-PDX)
3. Animal experiment
The corresponding treatments are respectively given according to the above groups: igG and PD-1 antibody were administered 1 time every 3 days at a dose of 200 μg/mouse by intraperitoneal injection for 3 total treatments; the effect on PDX tumor growth was observed in a humanized-human tumor tissue graft (PDX) model by tail vein injection of a specific nucleic acid drug ASO targeting LINC02096 or a non-specific ASO sequence control inhibitor, injected once every 3 days, 10nmol each time. Tumor volume and mouse body weight were measured every 3 days when tumor volume of any mouse reached 2000mm 3 At the same time, all the test mice are euthanized by cervical dislocation, the tumor volumes are counted, and the treatment effect differences among different treatment groups are compared.
The proportion of each immunocyte subset in the tumor was detected by flow cytometry, and the experimental method was the same as in example 3.
3. Experimental results
The experimental results are shown in fig. 6, and as can be seen from fig. 6, LINC02096-inhibitor can obviously inhibit the growth of tumors in esophageal squamous carcinoma bodies, and PD-1 monoclonal antibody can further inhibit the growth of tumors in bodies; LINC02096-inhibitor can increase CD8 + T cells and IFN-gamma + CD8 + T cell ratio, combined PD-1 mab CD8 + T cells and IFN-gamma + CD8 + The proportion of the T cells is further increased, which indicates that LINC02096-inhibitor can increase the activity of killing tumor by the T cells, LINC02096-inhibitor has obvious anticancer effect, and LINC02096-inhibitor can improve the anticancer effect of PD-1 monoclonal antibody.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. Application of a reagent for detecting LINC02096 in preparing a kit for predicting the immunotherapy effect of esophageal cancer.
2. The application of the LINC02096 inhibitor in preparing medicines for treating esophageal cancer is characterized in that the inhibitor is a specific nucleic acid medicine ASO, and the sequence of the specific nucleic acid medicine ASO is shown in SEQ ID NO.11, specifically 5'-AGCGCACCGAAAGGAACCAA-3'.
3. The application of the LINC02096 inhibitor in preparing a synergist of an esophageal cancer immunotherapy drug is characterized in that the inhibitor is a specific nucleic acid drug ASO, and the sequence of the specific nucleic acid drug ASO is shown as SEQ ID NO.11, specifically 5'-AGCGCACCGAAAGGAACCAA-3'.
4. The application of LINC02096 inhibitor combined with PD-1 antibody in preparing medicine for treating esophageal cancer is characterized in that the inhibitor is specific nucleic acid medicine ASO, and the sequence of the specific nucleic acid medicine ASO is shown as SEQ ID NO.11, specifically 5'-AGCGCACCGAAAGGAACCAA-3'.
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| CN111714629A (en) * | 2020-05-13 | 2020-09-29 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Application of medicine composition in preparing medicine for treating tumor insensitive to PD-1 antibody immunotherapy |
| CN111763740A (en) * | 2020-08-13 | 2020-10-13 | 中国医学科学院肿瘤医院 | System for predicting treatment effect and prognosis of neoadjuvant radiotherapy and chemotherapy of esophageal squamous carcinoma patient based on lncRNA molecular model |
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| CN108588220A (en) * | 2018-04-26 | 2018-09-28 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma long-chain non-coding RNA LINC01419 molecular markers and its application |
| CN109735620A (en) * | 2019-01-10 | 2019-05-10 | 中山大学肿瘤防治中心 | A kind of application of molecular target in esophageal squamous cell carcinoma prognosis evaluation and treatment |
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| CN111714629A (en) * | 2020-05-13 | 2020-09-29 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Application of medicine composition in preparing medicine for treating tumor insensitive to PD-1 antibody immunotherapy |
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