CN116287277A - Biomarker of cytidine deaminase activity and application thereof - Google Patents
Biomarker of cytidine deaminase activity and application thereof Download PDFInfo
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- CN116287277A CN116287277A CN202310448538.8A CN202310448538A CN116287277A CN 116287277 A CN116287277 A CN 116287277A CN 202310448538 A CN202310448538 A CN 202310448538A CN 116287277 A CN116287277 A CN 116287277A
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Abstract
The invention discloses a biomarker of cytidine deaminase activity and application thereof. Evaluation of apob 3B enzymatic activity in cell and tissue samples was accomplished by measuring the level of expression of long non-coding RNAs (lncRNA) NEAT1 and MALAT1 at the RNA sites specific for individual apob 3B in these two lncRNA sequences.
Description
Technical Field
The invention belongs to the field of biomedicine, and in particular relates to a biomarker with cytidine deaminase activity and application thereof.
Background
The high degree of heterogeneity is the greatest distinction between tumors and other malignant diseases and is also a source of their difficulty in healing. A large number of tumor genomics studies show that tumors can utilize the Darwin's evolution to complete selection, competition and adaptation at the cellular level, thereby achieving tumorigenesis, microenvironment adaptation, infiltration, metastasis and drug resistance.
The results of the study indicate that enzyme-mediated mutation of the nucleic acid genetic material is one of the major pathways responsible for the heterogeneity of the tumor genome, and that the apodec family (Apolipoprotein B mRNA editing catalytic polypeptide-like) of DNA/RNA deaminases is the active enzyme of this process.
The apodec family enzyme is capable of acting on deoxycytidine in single-stranded DNA, converting it to deoxyuridine by deamination, thereby generating various mutations including C > T mutations at the DNA level. In tumor cells, abnormally activated apopec family enzymes drive the accumulation of heterogeneity and generate refractory drug resistant clones by promoting branch evolution. Abnormal activation of the apodec family was confirmed by large-scale whole genome sequencing in clinical studies and resulted in poor prognosis by driving tumor heterogeneity and drug-resistant clone evolution.
Some research and clinical practice results indicate that other members of the apodec family having enzymatic activity, in particular apodec 3A, have interfering effects on the biochemical means-based activity assay of apodec 3B. In addition, although mutation detection by whole genome sequencing means and methods of extracting single nucleotide mutation spectra are effective in predicting apodec activity, these means are not able to distinguish between apodec 3A and apodec 3B activity while being expensive. This is also a major challenge for apodec 3B-related tumor diagnosis and development of novel targeted therapies.
In summary, there is currently no method that can directly assess apodec 3B specific activity in tumor cells or tissue samples. Thus, there is a strong need in the art to develop biomarkers for specific detection of apodec 3B enzymatic activity in tumor cells or tissue samples.
Disclosure of Invention
The invention aims to provide a biomarker capable of specifically detecting the activity of APOBEC3B enzyme in a tumor cell or tissue sample and application thereof.
In a first aspect of the invention, there is provided the use of a cytidine deaminase apodec 3B enzyme activity marker or a detection reagent thereof, for the preparation of a diagnostic reagent or kit for detecting cytidine deaminase apodec 3B enzyme activity;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof.
In another preferred embodiment, the detection reagent is used to detect whether a C.fwdarw.U change occurs at an RNA editing site (with GRCh38 reference genome coordinates) selected from the group consisting of: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866, or a combination thereof;
and/or the detection reagent is used for detecting whether the editing site (with GRCh38 reference genome coordinates) of the lncRNA MALAT1 selected from the following groups is changed from C to U: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549984, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613, or a combination thereof.
In another preferred embodiment, the diagnostic reagent or kit is also used to evaluate a subject that is positive for cytidine deaminase apodec 3B or whether the subject is suitable for administration of a drug that inhibits cytidine deaminase apodec 3B enzyme activity.
In another preferred embodiment, the subject is a tumor patient.
In another preferred embodiment, the expression level of lncRNA net 1, lncRNA MALAT1, or a combination thereof is detected using fluorescent quantitative PCR to detect cytidine deaminase apodec 3B enzyme activity.
In another preferred embodiment, at least one pair of primers for specifically amplifying lncRNA net 1 and lncRNA MALAT1 is used in the fluorescent quantitative PCR assay, wherein the primers are selected from the group consisting of:
NEAT1_1-F(SEQ ID NO：1)：
5'-GGCACAAGTTTCACAGGCCTACATGGG-3'；
NEAT1_1-R(SEQ ID NO：2)：
5'-GCCAGAGCTGTCCGCCCAGCGAAG-3'；
NEAT1_2-F(SEQ ID NO：3)：
5'-GGAGCCAACCTGCCCTGAAT-3'；
NEAT1_2-R(SEQ ID NO：4)：
5'-CCACAGGCTACCCTCTGCTC-3'；
MALAT1-F(SEQ ID NO：5)：
5'-CTTCCCTAGGGGATTTCAGG-3'；
MALAT1-R(SEQ ID NO：6)：
5'-GCCCACAGGAACAAGTCCTA-3'。
in a second aspect of the present invention, there is provided a kit for detecting the activity of cytidine deaminase apodec 3B enzyme, characterized in that the kit comprises:
(i) RNA of a cytidine deaminase APOBEC3B enzyme activity marker or a detection reagent thereof;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof; and
optionally (ii) a cytidine deaminase apodec 3B enzyme, a promoter thereof, an inhibitor thereof, or a combination thereof.
In another preferred embodiment, the editing site on lncRNA net 1 is selected from the group consisting of: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866;
Wherein the editing site on lncRNA MALAT1 is selected from the group consisting of: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549884, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613.
In another preferred embodiment, the inhibitor is a drug that inhibits the cytidine deaminase apodec 3B enzyme.
In another preferred embodiment, the inhibitor is an antibody or small molecule compound that inhibits the cytidine deaminase apodec 3B enzyme.
In another preferred embodiment, the marker is from a human.
In another preferred embodiment, the marker is from a non-human mammal.
In another preferred embodiment, the detection is directed to a tumor patient, or a tumor susceptible subject.
In another preferred embodiment, the detection is for a non-tumor patient.
In another preferred embodiment, the detection is for an ex vivo sample.
In another preferred embodiment, the ex vivo sample is selected from the group consisting of: a tissue sample, a cell sample, a blood sample, or a combination thereof.
In another preferred embodiment, the detection reagent is coupled to or carries a detectable label.
In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.
In another preferred embodiment, the diagnostic reagent is selected from the group consisting of: primers, probes, sequencing libraries, nucleic acid chips (e.g., DNA chips), or combinations thereof.
In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically binding to polynucleotides (mRNA or cDNA) of any of the markers.
In another preferred embodiment, the kit contains the gene, RNA, and/or cDNA of the marker as a control or quality control.
In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used to determine the cytidine deaminase apodec 3B enzymatic activity.
In another preferred embodiment, the reagents comprise primers, probes, gRNA or a combination thereof, more preferably a primer pair or probe for PCR, qPCR, RT-PCR.
In another preferred embodiment, the detection of the marker can be performed by the following method: sequencing, PCR, or a combination thereof.
In another preferred embodiment, the detection of the marker comprises a quantitative or qualitative detection.
In a third aspect of the present invention, there is provided a detection method characterized by comprising the steps of:
(a) Providing a test sample, wherein the test sample contains a cytidine deaminase apodec 3B enzyme activity marker, and the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof;
(b) Detecting the level (or degree) of the change of C-U of the editing site in the marker in the detection sample, and marking the level as C1; and
(c) Comparing the level C1 of the change of c→u of the editing site in the marker with a control reference value C0;
wherein if the marker detection result in the detection sample meets the following conditions: c1> C0, the cytidine deaminase APOBEC3B enzyme activity in the test sample is indicated to be high.
In another preferred embodiment, the level (or extent) of the change is the ratio of the number of changes C.fwdarw.U in the editing site in the marker to the number of unchanged changes (denoted as C1) in the test sample.
In another preferred embodiment, the level (or extent) of the change is the ratio (expressed as C1) of the amount of change c→u in the editing site in the marker in the test sample to the amount (e.g. moles) of the corresponding lncRNA (lncRNA net 1 and/or lncRNA MALAT 1) in the test sample.
In another preferred embodiment, steps (b) and (c) comprise:
(b) Detecting the ratio of the number of the change of C-U of the editing sites in the marker in the detection sample to the number of the change of the editing sites in the marker, and marking the ratio as C1; and
(c) Comparing the ratio C1 of the quantity of the C-U change of the editing site in the marker relative to the quantity of the unchanged editing site with the quantity of the C-U change of the same site in the control sample relative to the quantity of the unchanged editing site C0;
wherein if the marker detection result in the detection sample meets the following conditions: c1> C0, the cytidine deaminase APOBEC3B enzyme activity in the test sample is indicated to be high.
In another preferred embodiment, said C1> C0 means statistically significantly greater.
In another preferred embodiment, the control reference value C0 is the ratio of the number of changes C.fwdarw.U in the editing site to the number of unchanged changes in the same marker measured in the presence of the cytidine deaminase APOBEC3B of a predetermined or known enzymatic activity.
In another preferred embodiment, the control reference value C0 is the level (or extent) of a change in the editing site from C to U in the same marker measured in the presence of the cytidine deaminase APOBEC3B of predetermined or known enzymatic activity.
In another preferred embodiment, the control reference value C0 is the ratio of the number of changes C.fwdarw.U in the editing site to the number of unchanged changes in the same marker measured in the presence of the enzymatically inactive cytidine deaminase APOBEC 3B.
In another preferred embodiment, the control reference value C0 is the level (or extent) of a change in the editing site from C to U in the same marker measured in the presence of the enzymatically inactive cytidine deaminase APOBEC 3B.
In another preferred embodiment, if the marker detection result in the detection sample meets the following conditions: C0/C1 is less than or equal to 2/3 or C0/C1 is less than or equal to 1/2, preferably C0/C1 is less than or equal to 1/5, more preferably C0/C1 is less than or equal to 1/10, and the cytidine deaminase APOBEC3B enzyme activity in the detection sample is indicated to be higher than the cytidine deaminase APOBEC3B enzyme activity corresponding to the reference value C0.
In another preferred embodiment, the editing site on lncRNA net 1 is selected from the group consisting of: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866, or combinations thereof;
wherein the editing site on lncRNA MALAT1 is selected from the group consisting of: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549984, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613, or a combination thereof.
In another preferred embodiment, the detection method is non-diagnostic, non-therapeutic.
In a fourth aspect of the invention, there is provided an apparatus for assessing the activity of the cytidine deaminase apodec 3B enzyme, the apparatus comprising:
(a) The input module is used for inputting cytidine deaminase APOBEC3B enzyme activity marker data of a sample to be detected from a certain object, wherein the data comprise the level (or degree) of C-U change of an editing site in the marker;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof;
wherein the editing site on lncRNA net 1 is selected from the group consisting of: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866, or combinations thereof;
wherein the editing site on lncRNA MALAT1 is selected from the group consisting of: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549984, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613, or a combination thereof;
(b) The processing module is used for processing the input marker data so as to obtain an evaluation result of the cytidine deaminase APOBEC3B enzyme activity; and
(c) And the output module is used for outputting an evaluation result of the cytidine deaminase APOBEC3B enzyme activity.
In another preferred embodiment, the processing of the processing module includes a processor, which may be programmed to perform automatic calculations.
In another preferred embodiment, the level (or extent) of change is the ratio of the number of changes C.fwdarw.U in the editing site in the marker to the number of unchanged changes in the test sample.
In another preferred embodiment, the level (or extent) of the change is a ratio of the amount of change in the editing site in the marker from C to U in the test sample to the amount (e.g., moles) of the corresponding lncRNA (lncRNA net 1 and/or lncRNA MALAT 1) in the test sample.
In another preferred embodiment, in step (a) comprises:
(a) The input module is used for inputting cytidine deaminase APOBEC3B enzyme activity marker data of a sample to be detected from a certain object, and the data comprise the ratio of the number of changes of C-U of editing sites in the marker to the number of unchanged editing sites.
In a fifth aspect of the present invention there is provided a method of screening for potential compounds, wherein the compounds are compounds that potentially promote or inhibit the enzymatic activity of cytidine deaminase apodec 3B, the method comprising the steps of:
(a) Providing a compound to be screened;
(b) In the test group, cells are cultured in the presence of the compound; and in the control group, the cells were cultured in the absence of the compound and under otherwise identical conditions, and
(c) Detecting the level (or extent) of a C.fwdarw.U change in the edit site in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the test set, designated as C1; and detecting the level (or degree) of the change of C-U of the editing site in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the blank control group, and marking the level as a reference value C0;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof;
if the marker level C1 is significantly greater than the reference value C0, then determining the compound as a potential compound that promotes the enzymatic activity of cytidine deaminase APOBEC 3B;
if the marker level C1 is significantly less than the reference value C0, the compound is determined to be a potential compound that inhibits the enzymatic activity of cytidine deaminase APOBEC 3B.
In another preferred embodiment, the level (or extent) of change in the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the test set is the ratio of the number of changes in the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the test set to the number of unchanged changes (denoted as C1).
In another preferred embodiment, the level (or extent) of change in the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the test set is the ratio (expressed as C1) of the number of changes in the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the test set to the number (e.g., moles) of corresponding lncRNA (lncRNA net 1 and/or lncRNA MALAT 1) in the samples of the test set.
In another preferred embodiment, the level (or extent) of change of the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the blank is the ratio (denoted as C0) of the number of changes of the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the blank to the number of unchanged.
In another preferred embodiment, the level (or extent) of change of c→u in the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the blank is the ratio (expressed as C0) of the number of changes of c→u in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the blank to the number (e.g. mole number) of corresponding lncRNA (lncRNA net 1 and/or lncRNA MALAT 1) in the samples in the blank.
In another preferred embodiment, in step (c) comprises:
(c) Detecting the ratio of the number of changes of C-U changes to the number of unchanged changes of the editing sites in the cytidine deaminase APOBEC3B enzyme activity markers of the cells in the test group, and marking the ratio as C1; detecting the ratio of the quantity of the change of the editing site in the C-U change to the quantity of the non-change in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the blank control group, and marking the ratio as a reference value C0;
in another preferred embodiment, the control reference value C0 is the ratio of the number of changes C.fwdarw.U in the editing site to the number of unchanged changes in the same marker measured in the presence of the cytidine deaminase APOBEC3B of a predetermined or known enzymatic activity.
In another preferred embodiment, the control reference value C0 is the level (or extent) of a change in the editing site from C to U in the same marker measured in the presence of the cytidine deaminase APOBEC3B of predetermined or known enzymatic activity.
In another preferred embodiment, the control reference value C0 is the ratio of the number of changes C.fwdarw.U in the editing site to the number of unchanged changes in the same marker measured in the presence of the enzymatically inactive cytidine deaminase APOBEC 3B.
In another preferred embodiment, the control reference value C0 is the level (or extent) of a change in the editing site from C to U in the same marker measured in the presence of the enzymatically inactive cytidine deaminase APOBEC 3B.
In another preferred embodiment, if the marker detection result in the detection sample meets the following conditions: C0/C1.ltoreq.2/3 or C0/C1.ltoreq.1/2, preferably C0/C1.ltoreq.1/5, more preferably C0/C1.ltoreq.1/10, the compound is suggested to be a potential compound that promotes or inhibits the enzymatic activity of the cytidine deaminase APOBEC 3B.
In another preferred embodiment, the editing site on lncRNA net 1 is selected from the group consisting of: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866, or combinations thereof;
Wherein the editing site on lncRNA MALAT1 is selected from the group consisting of: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549984, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613, or a combination thereof.
In another preferred embodiment, the screening method is an in vitro screening method.
In another preferred embodiment, in step (b), the method further comprises: a promoter positive control group, wherein, in the promoter positive control group, the cells are cultured in the presence of a compound that promotes the activity of cytidine deaminase apodec 3B enzyme and under otherwise identical conditions;
and in step (C), detecting the level (or extent) of the change of C.fwdarw.U in the editing site in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the accelerator positive control group, as a reference value Y0;
wherein, if the marker level C1 is significantly greater than the reference value Y0, the compound is determined to be a compound that promotes the enzymatic activity of cytidine deaminase apodec 3B.
In another preferred embodiment, the level (or extent) of change of C.fwdarw.U in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the accelerator positive control group is the ratio (denoted as Y0) of the number of changes of C.fwdarw.U in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the accelerator positive control group to the number of unchanged.
In another preferred example, the level (or extent) of change of C.fwdarw.U in the edit site in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the accelerator positive control group is a ratio (expressed as Y0) of the number of changes of C.fwdarw.U in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the accelerator positive control group to the number (e.g., mole number) of corresponding lncRNAs (lncRNA NEAT1 and/or lncRNA MALAT 1) in the samples of the test group.
In another preferred embodiment, in step (c) comprises:
(c) The ratio of the number of changes in the editing site from C to U to the number of unchanged changes in the cytidine deaminase APOBEC3B enzyme activity markers of the cells in the accelerator positive control group was detected and recorded as Y0.
In another preferred embodiment, the control reference value Y0 is the ratio of the number of changes C.fwdarw.U in the editing site to the number of unchanged changes in the same marker measured in the presence of a predetermined or known positive control promoter, and a predetermined or known enzyme activity of cytidine deaminase APOBEC 3B.
In another preferred embodiment, the control reference value Y0 is the level (or extent) of a change in C.fwdarw.U at the editing site in the same marker measured in the presence of a predetermined or known positive control promoter, and a predetermined or known enzyme activity of cytidine deaminase APOBEC 3B.
In another preferred embodiment, if the marker detection result in the detection sample meets the following conditions: Y0/C1.ltoreq.2/3 or Y0/C1.ltoreq.1, preferably Y0/C1.ltoreq.1/2, more preferably Y0/C1.ltoreq.1/3, most preferably Y0/C1.ltoreq.1/5, the compound being said to be a potential compound for promoting the enzymatic activity of cytidine deaminase APOBEC 3B.
In another preferred embodiment, in step (b), the method further comprises: an inhibitor-positive control group, wherein, in the inhibitor-positive control group, the cells are cultured in the presence of a compound that inhibits the activity of cytidine deaminase apodec 3B enzyme and under otherwise identical conditions;
and in step (C), detecting the level (or extent) of a change in C.fwdarw.U in the edit site of the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the inhibitor positive control group, as a reference value Z0;
wherein, if the marker level C1 is significantly less than the reference value Z0, the compound is determined to be a compound that inhibits the enzymatic activity of cytidine deaminase apodec 3B.
In another preferred embodiment, the level (or extent) of change of the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells in the inhibitor positive control group is the ratio (denoted as Z0) of the number of changes of the edit site in the cytidine deaminase apodec 3B enzyme activity marker of the cells to the number of unchanged changes.
In another preferred embodiment, the level (or extent) of change of C.fwdarw.U in the edit site in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the inhibitor positive control group is the ratio (expressed as Z0) of the number of changes of C.fwdarw.U in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the inhibitor positive control group to the number (e.g., mole number) of corresponding lncRNAs (lncRNA NEAT1 and/or lncRNA MALAT 1) in the samples of the test group.
In another preferred embodiment, in step (c) comprises:
(c) The ratio of the amount of change in the editing site from C to U to the amount of no change in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the inhibitor positive control group was measured and recorded as Z0.
In another preferred embodiment, the control reference value Z0 is the ratio of the number of changes C.fwdarw.U in the editing site to the number of unchanged changes in the same marker measured in the presence of a predetermined or known positive control inhibitor and a predetermined or known enzyme activity of cytidine deaminase APOBEC 3B.
In another preferred embodiment, the control reference value Z0 is the level (or extent) of a change in C.fwdarw.U at the editing site in the same marker measured in the presence of a predetermined or known positive control inhibitor and a predetermined or known enzyme activity of cytidine deaminase APOBEC 3B.
In another preferred embodiment, if the marker detection result in the detection sample meets the following conditions: Z0/C1 is greater than or equal to 1 or Z0/C1 is greater than or equal to 3/2, preferably Z0/C1 is greater than or equal to 2/1, more preferably Z0/C1 is greater than or equal to 3/1, and most preferably Z0/C1 is greater than or equal to 10, then the compound is suggested to be a potential compound that inhibits the enzymatic activity of cytidine deaminase APOBEC 3B.
In another preferred embodiment, the method further comprises the steps of:
(d) For the potential compounds identified in step (c), their inhibition of tumor cells is further determined.
In another preferred embodiment, said step (d) is an in vitro cell test.
In another preferred embodiment, the cell is a human cell.
In another preferred embodiment, the cell is a non-human mammalian cell.
In another preferred embodiment, the method is an in vitro method.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. Is limited to a space and will not be described in detail herein.
Drawings
FIG. 1 shows Lorentzian curves of RNA differential editing sites detected in T47D cells induced to express APOBEC3B wild-type.
FIG. 2 shows the distribution of all C > U RNA differential editing sites detected in T47D cells induced to express APOBEC3B wild type in the editing ratio variation and average sequencing depth space, with the differential editing sites on NEAT1 and MALAT1 shown in black circular borders.
FIG. 3 shows the results of the editing situation in which five C > U editing sites were detected in a T47D-inducible expression cell line using the microdroplet PCR method.
FIG. 4 shows the results of detection of the interaction of over-expressed APOBEC3A and APOBEC3B with NEAT1 and MALAT1 in T47D cells using the RNA chromatin co-immunoprecipitation (RIP) method.
FIG. 5 shows the results of the knockdown of NEAT1 and MALAT1 using antisense oligonucleotides (ASO) and verification using fluorescent quantitative PCR.
FIG. 6 shows the results of detection of the effect of NEAT1 and MALAT1 knockdown on the activity of APOBEC3A and APOBEC3B recombinant proteins using a fluorescent reporter gene technology based on Cas9-APOBEC family recombinant protein CRISPR editing technology.
FIG. 7 shows the results of knockdown of NEAT1 and MALAT1 in five breast cancer cell lines using shRNA, and verification of NEAT1 and MALAT1 expression levels using fluorescent quantitative PCR.
Fig. 8 shows the results of knockdown of net 1 and MALAT1 in five breast cancer cell lines using shRNA and verification of apodec 3A and apodec 3B expression levels using fluorescent quantitative PCR.
Fig. 9 shows the knockdown of net 1 and MALAT1 using shRNA in five breast cancer cell lines and the effect of knockdown on apodec 3A and apodec 3B total enzyme activity was examined.
Fig. 10 shows knockdown of net 1 and MALAT1 using shRNA in five breast cancer cell lines and detection of the effect of knockdown on apodec 3A enzymatic activity using DDOST 558c > u edit detection (microdroplet digital PCR) as an evaluation tool.
FIG. 11 shows the results of the detection of the degree of editing and the enzymatic activity of APOBEC3B at two differential editing sites mediated by APOBEC3B on NEAT1 (chr 11:65441080 and chr11: 65425652), respectively, in twenty breast cancer cell lines, and for comparison and correlation analysis.
FIG. 12 shows the results of the detection of the degree of editing and the enzymatic activity of APOBEC3B at two differential editing sites (chr 11:65498990 and chr11: 65498321) mediated by APOBEC3B on MALAT1, respectively, in twenty breast cancer cell lines, and the comparison and correlation analysis.
FIG. 13 shows Pearson correlation thermodynamic diagrams of the expression levels of APOBEC3A, APOBEC3B, NEAT and MALAT1 in samples of five tumor patient populations. Wherein the sample source is TCGA database, and the cancers are bladder cancer (BLCA), breast cancer (BRCA), esophageal adenocarcinoma (ESAD), head and Neck Squamous Carcinoma (HNSC) and lung adenocarcinoma (LUAD); transcript expression was determined for RNA sequencing and values were normalized using RPKM methods.
Fig. 14 shows the partitioning of the five tumor patient populations according to the expression profile of apodec 3A, APOBEC3B, NEAT and MALAT1 and the effect of apodec 3A driven genomic mutation profile (SBS 2, SBS 13) was observed.
Detailed Description
The invention aims to provide a biomarker for cytidine deaminase APOBEC3B activity and application thereof. Specifically, through a large number of screening, experimental results show that the main editing sites of the APOBEC3B in the human transcriptome are partial sequences on two lncRNAs, namely NEAT1 and MALAT1, and the NEAT1 and MALAT1 can be selectively edited by the APOBEC 3B; the invention predicts the activity of APOBEC3B by detecting the editing degree of RNA C > U of editing sites in NEAT1 and MALAT 1. On this basis, the present invention has been completed.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.
As used herein, the terms "containing" or "comprising" may be open or closed, i.e., include "consisting of … …" and "containing.
The meaning of all parameters, dimensions, materials and configurations described herein will be readily understood by those skilled in the art. The actual parameters, dimensions, materials, and/or configurations may depend upon the specific application for which the invention is used. It will be appreciated by those skilled in the art that the examples or claims are given by way of example only and that the scope of the invention which can be covered by the embodiments of the invention is not limited to the specifically described and claimed scope within the scope of the equivalents or claims.
All definitions and uses herein should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.
All references, patents and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, and in some cases may contain the entire document.
It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these embodiments.
Cytidine deaminase APOBEC3B
Cytidine deaminase apodec 3B is one of the key enzymes that is aberrantly expressed in tumor cells and can drive tumor evolution by mediating C > U nucleotide mutations. Detection of apodec 3B activity in tumor cells is critical for patient prognosis, prediction of drug resistance development, and applicability of targeted apodec 3B therapies. However, simple means including detection of transcript levels or protein levels of apodec 3B do not predict the activity of apodec 3B in tissue samples well, mainly due to the high homology with apodec 3B and the large interference of apodec 3A with certain co-expression rules on apodec 3B activity. Thus, there is an unmet need for selective detection of apodec 3B activity under clinical conditions. According to the invention, through analysis of the APOBEC3B mediated differential RNA editing sites in transcripts, the non-coding RNA NEAT1 and MALAT1 are found to have APOBEC3B selective C > U editing sites, and the two non-coding RNAs can be selectively combined with the APOBEC3B directly and inhibit the activity of the non-coding RNA NEAT1 and MALAT 1. Furthermore, a method for predicting the activity of apodec 3B in tumor tissues can be designed based on detecting transcript levels of the nea 1 and MALAT1 or specific C > U editing sites on the two non-coding RNAs, thereby providing a class of biomarkers capable of selectively predicting the activity of apodec 3B.
lncRNA
Long non-coding RNAs (lncRNA) are one type of RNA, generally defined as transcripts that are not translated into proteins that are more than 200 nucleotides in length. This definition distinguishes lncRNA from small non-coding RNAs, such as microRNA (miRNA), small interfering RNAs (siRNA), piwi-interacting RNAs (piRNA), snoRNA, and other short RNAs. Long insert/intergenic non-coding RNAs (lincrnas) are lncRNA sequences that do not overlap with the protein-encoding genes.
Long non-coding RNAs include intergenic lincRNA, intronic ncRNA, and sense and antisense lncRNA, each type exhibiting different genomic positions associated with genes and exons.
NEAT1 and lncRNA MALAT1
NEAT1 (English : nuclear Enriched Abundant Transcript 1, also known as VINC, virus Inducible NonCoding RNA) is a long-chain non-coding RNA gene expressed in multiple endocrine tumors.
MALAT1 and lncRNA MALAT1
MALAT-1 (English : metastasis associated lung adenocarcinoma transcript 1, also known as NEAT2, noncoding nuclear-enriched abundant transcript 2) is a long non-coding RNA gene expressed in the nucleus and is highly conserved in mammals. MALAT-1 is involved in regulating various reactions in cells such as nuclear tissues, alternative splicing of mRNA, epigenetic modification of DNA, and the like, and can control gene expression involved in tumor distal metastasis. The RNA abnormal expression is related to diabetes mellitus, cancers and other diseases, and the high expression and the poor survival rate of the cancers are positively related.
lncRNA MALAT1 is an RNA sequence transcribed from the MALAT1 gene.
Marker and method for assessing APOBEC3B enzyme activity
The invention enables the evaluation of the enzymatic activity of apodec 3B by measuring the expression levels of long non-coding RNAs (lncRNA) net 1 and MALAT1, and the degree of editing of the RNA sites specific for individual apodec 3B in these two lncRNA sequences.
The invention discovers that the main editing sites of the APOBEC3B in the human transcriptome are partial sequences on two lncRNAs of NEAT1 and MALAT1 for the first time, and proves that NEAT1 and MALAT1 can be selectively edited by the APOBEC 3B. The invention discovers that NEAT1 or MALAT1 can directly bind to APOBEC3B protein, but not to APOBEC3A for the first time. It was further found that the binding energy of NEAT1 or MALAT1 to APOBEC3B selectively inhibits the enzymatic activity of APOBEC3B and that its expression level in tumor cells is inversely related to the enzymatic activity of APOBEC 3B.
The invention further proves that the RNA C > U editing degree of the editing sites in NEAT1 and MALAT1 is positively correlated with the activity of APOBEC3B, but has low correlation degree with APOBEC3A. Therefore, the degree of editing of these sites can predict the activity of apodec 3B in tumor cells or tissue samples, thereby further inferring the degree of tumor heterogeneity of the samples and the applicability of apodec 3B-related targeted therapeutic strategies.
In order to achieve the purpose of predicting the activity of the APOBEC3B enzyme by measuring the expression quantity of NEAT1 and MALAT1 or the C > U editing degree of the NEAT1 and MALAT1 editing sites, the invention provides the following technical scheme:
the invention provides application of NEAT1 and MALAT1 expression quantity as a biomarker for predicting APOBEC3B enzyme activity in a cell or tissue sample, and also provides application of C > U editing of NEAT1 and MALAT1 editing sites as a biomarker for predicting APOBEC3B enzyme activity. The method can be used as a prediction tool related to the APOBEC3B enzyme activity function, and is applied to tumor diagnosis, treatment strategy formulation and prognosis evaluation.
In the present invention, the means for evaluating the activity of apodec 3B enzyme in a sample predicted by detecting the expression levels of net 1 and MALAT1 comprises: fluorescent quantitative PCR and high-throughput RNA sequencing.
Preferably, the means for assessing apodec 3B enzyme activity in a sample predicted by detecting the expression levels of net 1 and MALAT1 comprises reagents and kits.
Preferably, the method for predicting apodec 3B enzymatic activity in a sample by fluorescent quantitative PCR of the expression levels of net 1 and MALAT1 comprises at least a pair of primers for specifically amplifying net 1 and MALAT1, said primers comprising,
NEAT1_1-F(SEQ ID NO：1)：
5'-GGCACAAGTTTCACAGGCCTACATGGG-3'；
NEAT1_1-R(SEQ ID NO：2)：
5'-GCCAGAGCTGTCCGCCCAGCGAAG-3'；
NEAT1_2-F(SEQ ID NO：3)：
5'-GGAGCCAACCTGCCCTGAAT-3'；
NEAT1_2-R(SEQ ID NO：4)：
5'-CCACAGGCTACCCTCTGCTC-3'；
MALAT1-F(SEQ ID NO：5)：
5'-CTTCCCTAGGGGATTTCAGG-3'；
MALAT1-R(SEQ ID NO：6)：
5'-GCCCACAGGAACAAGTCCTA-3'。
In the invention, the evaluation tool for detecting the APOBEC3B enzyme activity in the NEAT1 and MALAT1 expression level prediction samples by a fluorescent quantitative PCR method comprises all reagents for extracting RNA from tumor tissues and performing reverse transcription and fluorescent quantitative PCR, including reagents (such as Trizol reagent, chloroform, isopropanol and nuclease-free water) for extracting RNA from tumor samples, besides the primer; reverse transcription reagents (e.g., reverse transcriptase, random primers, triphosphates deoxyribonucleotides, and RNase inhibitors) that use RNA as a template; and reagents required by fluorescent quantitative PCR reaction (such as reference genes, specific primers of genes to be detected, fluorescent marked DNA dye, DNA polymerase, corresponding buffers and the like).
Preferably, the above-mentioned fluorescent quantitative PCR method
Preferably, the high throughput RNA sequencing method for detecting apodec 3B enzyme in a predicted sample of the expression levels of net 1 and MALAT1 has the following features: (1) treating the total RNA sample using a ribosome removal method; (2) The library construction method is any cDNA strand specific library construction method; (3) sequencing the number of the effective fragments to be not less than three tens of millions.
In the present invention, the means for measuring the degree of editing of RNA C > U of the editing site in NEAT1 and MALAT1 comprises: high throughput RNA sequencing, microdroplet digital PCR.
Preferably, the means for measuring the degree of RNA C > U editing of editing sites in the net 1 and MALAT1 comprise reagents and kits.
Preferably, the editing site on the measured net 1 is selected from the group (with GRCh38 reference genome coordinates): chr11:65498221, chr11:65498890, chr11:65499884, chr11:65500618, chr11:65500772, chr11:65504613, chr11:65498862, chr11:65499788, chr11:65500899.
Preferably, the editing site on the measured MALAT1 is selected from the group consisting of (with GRCh38 reference genome coordinates): chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549884, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613.
The base types of the reference genome of the sites are C, the base types edited by APOBEC3B are U, and the detected mutation base types are U or T.
Preferably, the method for detecting the editing degree of RNA C > U of the editing site in NEAT1 and MALAT1 by the high-throughput RNA sequencing method comprises the following characteristics: (1) treating the total RNA sample using a ribosome removal method; (2) The library construction method is any cDNA strand specific library construction method; (3) the number of sequencing effective fragments is not less than three tens of millions. (4) Differential editing analysis of editing sites in the net 1 and MALAT1 was performed using the method described in patent application 202211574812.8.
Preferably, the method for detecting the RNA C > U editing degree of the editing site in NEAT1 and MALAT1 by using the microdroplet digital PCR comprises the following characteristics: (1) Designing primers and probes by using a design program matched with a manufacturer of the microdroplet digital PCR instrument; (2) The category of the design program is mutation detection, and two different fluorescein marks are respectively used for marking the reference base type of the editing site in NEAT1 and MALAT1 and the base type edited by APOBEC 3B; (3) The input sequence of the design program comprises sequences of the coordinates of the site to be detected upstream and downstream of the human genome; (4) The reference base type detected is C, and the mutant base type is U or T.
The present invention uses real world tumor sample data to demonstrate that NEAT1 and MALAT1 transcript levels can predict C > T mutation in patient samples with high APOBEC3A/APOBEC3B expression. The results show that apodec 3A is activated in patient samples with high expression of net 1 and MALAT1 due to selective inhibition of apodec 3B activity by these lncRNA, thereby indirectly causing the presentation of a strong genome-level apodec 3A-driven mutation profile in these patient samples.
Therefore, the technical means provided by the invention provides a method for predicting the activity of the APOBEC3B enzyme in the patient sample with high expression of both the APOBEC3A and the APOBEC3B for the first time. This approach would provide diagnostic, concomitant therapy and prognostic tools for novel therapies designed for therapeutic targets around apodec 3B.
Detection kit
Based on the correlation between the cytidine deaminase apodec 3B activity markers net 1 and MALAT1 and the cytidine deaminase apodec 3B activity, the cytidine deaminase apodec 3B activity marker can be used as a judgment marker of the cytidine deaminase apodec 3B activity.
The invention also provides a kit for judging the activity of the cytidine deaminase APOBEC3B, which contains a detection reagent for detecting the gene, mRNA, cDNA, protein or the combination thereof of the cytidine deaminase APOBEC3B activity marker.
In another preferred embodiment, the kit further comprises a label or instructions.
The main advantages of the invention
(1) The method can better embody the actual intracellular activity of apodec 3B relative to a method for detecting the apodec 3B content, which comprises the determination of transcript level or protein level.
(2) The method for measuring the abundance of the selective substrates NEAT1 and MALAT1 of the APOBEC3B and editing the editing situation of the editing sites is the most direct means for reflecting the intracellular enzyme activity of the APOBEC 3B.
(3) Compared with the traditional means, the method can effectively distinguish important interference factors in the APOBEC3B activity measurement, namely the enzyme APOBEC3A in the same family.
(4) The invention discovers that the APOBEC3B enzyme has great application potential and prospect in the specific editing sites of NEAT1 and MALAT1 for the first time and unexpectedly.
(5) The detection method is convenient for quantitative detection.
(6) The detection method is based on nucleic acid detection and has the advantages of simplicity, convenience, specificity and low price.
Specifically, the invention designs an intracellular activity detection means aiming at the APOBEC3B through an unexpectedly discovered APOBEC3B activity regulation mechanism, namely, the selective negative regulation of non-coding RNA NEAT1 and MALAT1 on the APOBEC 3B. The method can better embody the actual intracellular activity of apodec 3B relative to a method for detecting the apodec 3B content, which comprises the determination of transcript level or protein level. This is because apodec 3B activity is regulated by complex mechanisms within the cell, and its content tends to be disproportionate to activity. The method for measuring the abundance of the selective substrates NEAT1 and MALAT1 of the APOBEC3B and editing the editing situation of the editing sites is the most direct means for reflecting the intracellular enzyme activity of the APOBEC 3B.
In addition, compared with the traditional means, the method can effectively distinguish important interference factors in the activity measurement of the APOBEC3B, namely the enzyme APOBEC3A in the same family. Because apobe 3A cannot bind and edit over 1 and MALAT1, the method provides an effective means of distinguishing apobe 3A from apobe 3B mediated intracellular cytidine deaminase activity.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Materials and methods
Construction of apobic 3B-induced expression T47D cell line: the apodec 3B fragment (nm_ 004900.5) was synthesized using GeneArt service (Thermo Fisher) whole gene, and the coding sequence was subjected to human cell codon optimization prior to synthesis. Therefore, E68 and E225 of APOBEC3B were mutated with QuickChange kit (Agilent), respectively, to obtain an APOBEC3B (E68Q/E225Q) encoding DNA fragment. These two DNA fragments were cloned into pTRIPZ (GE Healthcare) vectors using agoi and ClaI (BspDI), respectively, to give lentiviral-induced expression plasmids. 293TN (Clonetech) cells were co-transfected with the above expression plasmids using helper plasmids pMD2.G and psPAX2, and the medium was changed once after 24 hours. And the culture supernatants were collected twice 48 and 72 hours after transfection. After 0.44 μm microfiltration, lentiviruses were concentrated using the standard protocol of Peg-IT kit (System Biosciences). T47D cells were transduced with concentrated lentivirus, medium was changed 48 hours after transduction, and 1. Mu.g/ml puromycin was added to the medium 24 hours later and the cells were kept under culture at this selection pressure. Exogenous apodec 3B was induced using 1 μg/ml doxycycline and monitored using immunoblotting, the primary antibody used was the Abcam EPR18138 monoclonal antibody.
High throughput sequencing: RNA extraction was performed using Roche MagnaPure 96 instrument and supporting reagents; in a preferred embodiment, the A260/A280 value of the genomic DNA sample is between 1.8 and 2.0, and the RIN value of the RNA sample is more than 8. For genomic DNA samples, standard whole genome sequencing library building means were employed, and 30 Xsequencing depth sequencing in PE100 format was performed using a BGISeq-500 instrument. For RNA samples, after reducing the rRNA content in the library by using a ribosome removal method, a strand specific sequencing library is constructed by using a first strand method, and then a BGI-seq500 instrument is used for carrying out sequencing work in a PE100 mode, wherein the sequencing data volume of each sample is six tens of millions of fragments, and each sample set comprises an experimental treatment condition and four biological repetitions.
Detection of differential RNA editing sites: using the method described in patent application 202211574812.8.
Construction of stable knockdown cell lines: methods of knocking down NEAT1 and MALAT1 using short hairpin RNA (shRNA) are described in Zhang, P., cao, L., zhou, R., yang, X.and Wu, M.,2019.The lncRNA Neat1 promotes activation of inflammasomes in macrophages.Nature communications,10 (1), p.1495, and Qu, D., sun, W.W., li, L., ma, L., sun, L., jin, X, li, T., hou, W.and Wang, J.H.,2019.Long noncoding RNA MALAT1 releases epigenetic silencing of HIV-1replication by displacing the polycomb repressive complex 2from binding to the LTR promoter.Nucleic acids research,47 (6), pp.3013-3027. Wherein, the liquid crystal display device comprises a liquid crystal display device,
The sequence of the shRNA used by NEAT1 is (SEQ ID NO: 7):
5'-GAGTTACCATCCCGTCCTCTAGGATCCTAGAGGACGGGATGGTAACTC-3'；
the shRNA used in MALAT1 has the sequence (SEQ ID NO: 8):
5'-AAGACCTTGAAATCCATGACGCTCGAGCGTCATGGATTTCAAGGTCTT-3'；
the non-targeting shRNA sequence used for the control was (SEQ ID NO: 9):
5'-TTCTCCGAACGTGTCACGTATCTCGAGATACGTGACACGTTCGAGAA-3'。
the shRNA is subcloned into a pLKO.1 lentiviral shRNA expression vector. Lentiviruses were prepared using helper plasmids pMD2.G and psPAX2 with the plKO.1 vector encoding shRNA, transduced into the target cell line. Target cells were screened with puromycin.
Fluorescent quantitative PCR detection of net 1 and MALAT 1: detection was performed using a PCR premix kit Thunderbird SYBR qPCR Mix (manufactured by TOYOBO Co.) containing SYBR Green dye in combination with an ABI 7900HT instrument. Wherein the PCR primer and sequence for NEAT1 detection are,
NEAT1_1-F(SEQ ID NO：1)：
5'-GGCACAAGTTTCACAGGCCTACATGGG-3'；
NEAT1_1-R(SEQ ID NO：2)：
5'-GCCAGAGCTGTCCGCCCAGCGAAG-3'；
NEAT1_2-F(SEQ ID NO：3)：
5'-GGAGCCAACCTGCCCTGAAT-3'；
NEAT1_2-R(SEQ ID NO：4)：
5'-CCACAGGCTACCCTCTGCTC-3'；
MALAT1-F(SEQ ID NO：5)：
5'-CTTCCCTAGGGGATTTCAGG-3'；
MALAT1-R(SEQ ID NO：6)：
5'-GCCCACAGGAACAAGTCCTA-3'；
GAPDH-F(SEQ ID NO：10)：
5'-GGGAAATCGTGCGTGACAT-3'；
GAPDH-R(SEQ ID NO：11)：
5'-GTCAGGCAGCTCGTAGCTCTT-3'。
wherein the reaction temperature program conditions were 95℃for 5 minutes followed by 40 cycles of 95℃for 30 seconds, 55℃for 30 seconds, and 72℃for 30 seconds. In terms of data analysis, the relative amounts of NEAT1 and MALAT1 transcripts were assessed using the ΔΔCT method using GAPDH as an internal control.
Microdroplet digital PCR after extracting whole RNA from the cell sample using Trizol reagent, reverse transcription was performed using High Capacity cDNA Reverse Transcription reverse transcription kit (Thermo Scientific). 40ng of the resulting cDNA was combined with 2. Mu.L of the designed primer and probe and mixed with a microdroplet digital PCR premix (ddPCR Supermix for Probes No UTP, bio-Rad Co.) to a final volume of 25. Mu.L. The primers and probes were purchased from Bio-Rad, and were designed using software on a matched line. After water-in-oil droplets were generated using a QX200 droplet generator (Bio-Rad Co.), the reaction system was transferred to a C1000 Touch droplet digital PCR apparatus (Bio-Rad Co.), and amplified using the following procedure: 95℃for 5 minutes followed by 40 cycles of 94℃for 30 seconds, 53℃for 60 seconds, 98℃for 10 minutes with an additional temperature gradient of 2℃per second. The resulting droplets were read using a QX200 droplet reader, wherein FAM fluorescent markers were wild-type transcripts and HEX fluorescent markers were mutant transcripts. The wild-type transcripts were analyzed for sample ratios using QuantaSoft software (Bio-Rad).
Co-immunoprecipitation assay for apodec 3B enzyme activity: the method adopts an immune coprecipitation method to separate intracellular APOBEC3B and determine the enzyme activity thereof. This method can distinguish apodec 3B activity from apodec 3A activity, but is complex and requires fresh cell and tissue samples, thus limiting its use in clinical conditions. The specific method comprises the following steps: 100 micrograms of cell lysate was added to 1 milliliter of co-immunoprecipitation buffer (containing 20mM Tris pH8.0, 250mM NaCl,1mM DTT,1mM EDTA) and 1 microgram of apodec 3B antibody (Abcam ab 184990) was added and incubated overnight at 4 ℃ and immunoprecipitation was adsorbed using 20 microliter Dynabeads Protein A/G microbeads (Thermo Scientific company) in combination with this patch and the microbeads were washed using co-immunoprecipitation buffer. The resulting microbeads were assayed for biochemical activity of apobe 3B using a BspH1 biosensor assay described in Zhang, y.h., guo, x.c., zhong, j.b., zhong, d.x., huang, x.h., fang, z.y., zhang, c.and Lu, Y.J.,2022.Discovery of APOBEC Cytidine Deaminases Inhibitors Using a BspH1 Restriction Enzyme-Based biosensor. Chemistry select,7 (21), p.e202201456.
Example 1: discovery of RNA editing hotspots in breast cancer cell lines by APOBEC3B
In T47D cells constructed by slow viruses and used for inducing and expressing APOBEC3B, 1 mug/ml doxycycline is used for inducing and expressing exogenous APOBEC3B protein for 72 hours, and the cells are marked as an induction expression group; and using solvent DMSO as a control, marking as a control group, and verifying the induced expression of the APOBEC3B by using an immunoblotting method. Four biological replicates of each of the two conditioned experimental groups were performed to extract DNA and RNA samples, and 30X depth whole genome DNA sequencing and 60M reads strand specific RNA sequencing were performed, respectively, using the high throughput sequencing means described above. Differential RNA editing site analysis was performed using the method described in patent application 202211574812.8. The results obtained are shown in Table 1.
Table 1: the results obtained from RNA sequencing and differential RNA editing site analysis were performed in apodec 3B-induced expression T47D cell lines.
Taking the C > U differential RNA editing sites (false positive rate FDR <0.05, minimum depth DP of site is > 10) obtained by analysis, setting the total read depth of the C > U editing of the APOBEC3B induced expression group as 100%, calculating the ratio of the depth of each C > U editing site, and calculating the ratio of the site to the total RNA editing activity of the APOBEC 3B. And drawing a Lorentz curve by taking the duty sequence of the total RNA editing activity of the APOBEC3B at the C > U editing site as an ordinate and the RNA sequencing depth sequence occupied by the editing site. In this curve, if there is no specificity in the distribution of apodec 3B at the transcriptome editing site, the curve should be close to the y=x function; whereas the higher the curvature of the curve, the higher the specificity of apodec 3B at the transcriptome editing site. As shown in FIG. 1, the results indicate that the assigned coefficient of base for the full transcriptome editing activity of APOBEC3B is as high as 0.54, suggesting that APOBEC3B has significant selectivity for RNA editing sites.
And marking the gene of the editing site of the C > U differential editing site by using genome annotation of GENCODE 38 th edition. If one differential editing site corresponds to a plurality of genes, all the corresponding genes are listed respectively. And taking the editing proportion change of all C > U differential editing sites, which is influenced by APOBEC3B induced expression, as an ordinate and the abundance of the C > U editing sites in RNA sequencing as a bubble chart, wherein the bubble size shows that the sites occupy the total RNA editing activity ratio of the APOBEC 3B. As shown in FIG. 2, the results indicate that non-coding RNAs NEAT1 and MALAT1 are the major editing substrates for APOBEC 3B. Wherein all C > U differential editing sites on NEAT1 account for 7.9% of the total RNA editing activity of APOBEC3B, and all C > U differential editing sites on MALAT1 account for 1.8% of the total RNA editing activity of APOBEC 3B.
The RNA sequencing, differential editing site detection and later data analysis described above were repeated using lentiviral constructed T47D cell lines that induced expression of apodec 3B inactivating mutation (E68Q/E225Q). As a result, as shown in Table 1, only 2 differential C > U editing sites were detected by inducing expression for 72 hours of APOBEC3B inactivating mutation, and neither was located in NEAT1 and MALAT1 genes.
On T47D cell lines constructed by using lentiviruses and used for inducing and expressing APOBEC3B inactivating mutation (E68Q/E225Q), 9 NEAT1 and 9 APOBEC3B mediated differentiation sites detected by using an RNA sequencing method are subjected to verification analysis by using a digital droplet PCR method, primers and probes required for experiments are designed by using a droplet digital PCR online design program of Bio-Rad company, the experimental category is mutation detection (mutation detection), and the input sequence is 100bp sequences on the upstream and downstream of the site to be detected. The coordinates of the sites to be detected were as given in the GRCh38 reference genome and the mutation types were all C > T mutations as given in Table 2. The experiments were carried out using the materials and methods section described above. As shown in table 2, these candidate differential editing sites all showed a dependence on apodec 3B-induced expression, i.e. the degree of C > U editing was only increased when apodec 3B was highly expressed. This result verifies that these C > U edits at the sites over net 1 and MALAT1 are mediated by apodec 3B.
TABLE 2
From these results, it can be seen that NEAT1 and MALAT1 are the main transcriptome-selective editing subjects of APOBEC3B in the breast cancer cell model.
Example 2: effect of APOBEC 3B-induced expression on specific sites on NEAT1 and MALAT1
In this example, the effect of apodec 3B-induced expression on editing sites on net 1 and MALAT1 was examined using a microdroplet digital PCR method. The design of the primers and probes required for the experiment was performed using the microdroplet digital PCR in-line design program from Bio-Rad, the class of experiments was mutation detection (mutation detection), and the input sequence was 100bp upstream and downstream of the site to be detected. The coordinates of the site to be detected are given as the GRCh38 reference genome: chr11:65425652 (NEAT 1), chr11:65441080 (NEAT 1), chr11:65431271 (NEAT 1), chr11:65498990 (MALAT 1) and chr11:65498321 (MALAT 1), all of the types of mutations detected were C > T mutations. The experiments were carried out using the materials and methods section described above.
T47D cell lines constructed by slow viruses and used for inducing and expressing APOBEC3B wild type and inactivating mutation (E68Q/E225Q) are taken, DMSO control or 1 mu g/ml doxycycline is used for inducing cells for 72 hours, total RNA of samples is extracted, and a microdroplet digital PCR method is carried out after reverse transcription to detect the site mutation. As a result, as shown in FIG. 3, all the differential C > U editing sites on NEAT1 and MALAT1 tested exhibited a dependence on the activity of APOBEC3B enzyme and were therefore direct substrates for editing of APOBEC3B enzyme.
Example 3: NEAT1 and MALAT1 interact with the intracellular phase of APOBEC 3B.
This example uses RNA co-immunoprecipitation to detect whether NEAT1 forms an RNA-protein complex with MALAT 1. First, flag-tagged apodec 3A and apodec 3B recombinant proteins were overexpressed in T47D cells. The recombinant protein-encoding vector backbone consisted of pCMV6-Entry, purchased from origin Inc., encoding Myc-DDK (Flag) -tagged full-length APOBEC3A (NM-145699, RC220995) and APOBEC3B (NM-004900, RC222712), respectively. The two vectors were transfected into T47D cells using Lipofectamine 3000 transfection reagent, the total vector DNA amount was 1.2 μg per well total of six well plates, and cells were fixed with 1% neutral formaldehyde for 5 min 48 hours after transfection. Cells were collected and RNA co-precipitation experiments were performed using Magna Nuclear RIP (cross-linked) kit (Merck). All experimental parameters were chosen following the manufacturer's instructions except for the antibody, which was a Flag-M2 (F1804) mouse anti-Flag monoclonal antibody from Sigma. The NEAT1 and MALAT1 content co-precipitated by the Flag-M2 monoclonal antibody or negative control antibody was analyzed using the aforementioned fluorescent quantitative PCR detection means, and normalized to the respective input controls. The results are shown in FIG. 4, which shows that NEAT1 and MALAT1 both form complexes with the Flag-tagged recombinant APOBEC3B expressed in cells and are immunoprecipitated by Flag-M2 monoclonal antibody. This phenomenon was not observed in T47D cells transfected with Flag-tagged recombinant APOBEC 3A. In contrast, the negative control antibodies failed to co-precipitate with either NEAT1 or MALAT1 in all groups. It follows that NEAT1 and MALAT1 can selectively form RNA-protein complexes with APOBEC 3B.
Example 4: transient knockdown of NEAT1 and MALAT1 effects on APOEBC3A and APOBEC3B enzymatic activity.
This example uses Martin, A.S., salamango, D.J., serebrenik, A.A., shban, N.M., brown, W.L. and Harris, R.S.,2019.A panel of eGFP reporters for single base editing by APOBEC-Cas9 editome complexes, scientific reports,9 (1), p.497. Reported fluorescent reporter gene technology based on Cas9-APOBEC family recombinant protein CRISPR editing technology. The technology uses an overexpression vector comprising an enhanced green fluorescent protein (eGFP) encoding the L202S mutation as an editing substrate for Cas 9-apodec family recombinant proteins. After co-transfection of the Cas 9-apodec family recombinant protein vector, the corresponding L202S-eGFP targeting gRNA and the L202S-eGFP encoding vector in cells, the Cas 9-apodec family recombinant protein vector can selectively mutate amino acid residue 202 of eGFP from serine to lysine under the direction of the gRNA, thereby activating eGFP to generate green fluorescence.
The experimental Cas9-APOBEC family recombinant protein vector is selected from A3A-Cas9n-UGI-NLS, A3Bi-Cas9n-UGI and A3Bi-CTD-Cas9n-UGI-NLS, and respectively encodes recombinant Cas9-APOBEC3A, recombinant Cas9-APOBEC3B and recombinant Cas9-APOBEC3B-CTD (C-terminal domain). The sgRNA encoding vector is selected from eGFP-L202-gRNA (eGFP targeting) and Non-specific-sgRNA (Non-targeting control) vectors; the L202S-eGFP Reporter gene vector is an eGFP-L202-Reporter vector. The above vectors are all available from Addgene Inc.
To examine the effect of the knockdown of NEAT1 and MALAT1 on the activity of APOBEC family proteases, the knockdown of NEAT1 and MALAT1 was performed using antisense nucleotides (Antisense Oligonucleotides, ASO), respectively. ASO sequences such as Adriaens, C., standaert, L., barra, J., latil, M., verfaillie, A, kalev, P, boeckx, B, wijnhoven, P.W., radali, E., vermi, W.and Leucci, E.,2016.p53 induces formation of NEAT1 lncRNA-containing paraspeckles that modulate replication stress response and chemiositity.Nature medium, 22 (8), pp.861-868, and Amodio, N., stamat, M.A., juli, G., morelli, E, fulciti, M., manzoni, M., taiana, E, agnelli, L., cantaf, M.E.G., romeo, E.and Raidi, L. 2018.Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple, and African, M.1947.32. Wherein, the liquid crystal display device comprises a liquid crystal display device,
the NEAT1 targeting ASO sequence is (SEQ ID NO: 12):
5'-CTCACACGTCCATCT-3'；
MALAT1 targeting ASO sequence is (SEQ ID NO: 13):
5'-ACATTGCCTCTTCATT-3'。
the non-targeting ASO sequence used as control was (SEQ ID NO: 14):
5'-CATACTATATGACAG-3'。
the ASOs described above were all constructed using the GapmeRs company locked nucleotide (Locked Nucleic Acid, LNA) technology.
This example was performed in the T47D cell line. In each well of the six-well plate, a recombinant protein vector according to Cas 9-apodec family: sgRNA encoding vector: the ratio of L202S-eGFP was 3:1:2, mixed well at a total of 1.2. Mu.g, and ASO was added at a final concentration of 20 nM. Transfection complexes were constructed using Lipofectamine 2000 transfection reagent and T47D cells were added. At 72 hours post-transfection, hoechst33342 viable cell nuclear dye was added and the proportion of cells expressing green fluorescent protein to total cell number was calculated using the PE operatta CLx high content system. The results of this experiment are shown in fig. 5 and 6, and the results show that the apopec family enzyme-targeted ASO selected in this example can effectively knock down the net 1 and the MALAT1 compared with the non-targeted ASO. On the premise that the effectiveness of APOBEC family recombinant proteins on eGFP targeted editing is verified by using a negative control ASO and a non-targeted sgRNA vector, the activity of recombinant Cas9-APOBEC3B or recombinant Cas9-APOBEC3B-CTD can be improved by knocking down NEAT1 and MALAT1. This phenomenon was not observed in cells transfected with Cas 9-apodec 3A encoding vector. Thus, the simultaneous knocking-down of NEAT1 and MALAT1 has an effect of improving the enzyme activity of APOBEC3B in cells, which indicates that NEAT1 and MALAT1 selectively inhibit the activity of APOBEC3B in cells.
Example 5: effect of stable knockdown of NEAT1 and MALAT1 on APOBEC3A and APOBEC3B Activity
This example was carried out in five cell lines for which apodec 3A and apodec 3B expression are known. Wherein HCC2218 cell line expresses only APOBE3A, T47D and MCF7 express only APOBE 3B, and BT474 and HCC202 cells are both APOBE3A and APOBE 3B highly expressed. The stable knockdown NEAT1 and MALAT1 cell strains are constructed by adopting a slow virus mediated shRNA technology, a reference material and method part is specifically developed, and experiments are developed after stable rotation construction and subculture are completed for three times on all cell strains. Cell lines stably knocked down NEAT1 and MALAT1 were verified using fluorescent quantitative PCR. As shown in fig. 7, the results demonstrate that lentiviral-mediated NEAT1 and MALAT 1-targeted shRNA is effective in knocking down the expression of NEAT1 and MALAT 1.
The expression levels of the apodec 3A and apodec 3B genes were detected separately using fluorescent quantitative PCR in cell lines stably knocked down by over 1 and MALAT 1. As shown in fig. 8, stable knockdown of the net 1 and the MALAT1 cannot affect the expression levels of apodec 3A and apodec 3B.
The above stable knockdown NEAT1 and MALAT1 cell lines were assayed for APOBEC3B total activity using the method of Zhang, Y.H., guo, X.C., zhong, J.B., zhong, D.X., huang, X.H., fang, Z.Y., zhang, C.and Lu, Y.J.,2022.Discovery of APOBEC Cytidine Deaminases Inhibitors Using a BspH1 Restriction Enzyme-Based biosensor. Chemistry select,7 (21), p.e. 202201456. As shown in fig. 9, the results demonstrate that stable knockdown of the net 1 and MALAT1 had an elevating effect on APOBECB enzyme activity of APOBEC 3B-only expressing cell lines (T47D and MCF 7) or APOBEC3A/B co-expressing cell lines, while having no effect on APOBEC 3B-only expressing cell line (HCC 2218). The knockdown of NEAT1 and MALAT1 was predicted to selectively reduce intracellular APOBEC3B activity.
In view of the findings of the previous studies that aporec 3B has a reverse regulatory function on the activity of aporec 3A, it is predicted that elevation of aporec 3B activity by net 1 and MALAT1 would attenuate the activity of aporec 3A. To verify this hypothesis, the apobe 3A specific editing site DDOST1 558c > u reported previously was used as a predictor of its activity, and the effect of stable knockdown of net 1 and MALAT1 on DDOST1 558c > u editing was examined using a droplet-wise digital PCR method. As shown in fig. 10, the results demonstrate that stable knockdown of net 1 and MALAT1 reduced apodec 3A activity in apodec 3A and apodec 3B co-expressed cell lines (BT 474 and HCC 202). This phenomenon, however, could not be observed in cell lines expressing only apodec 3A (HCC 2218), indicating that the increased apodec 3B activity mediated by the net 1 and MALAT1 knockdown negatively affected the activity of apodec 3A.
Example 6 relationship between the degree of editing of specific sites on NEAT1 and MALAT1 and the activity of APOBEC3B in different cell lines.
The present example uses the identified C > U editing sites mediated by APOBEC3B in NEAT1 and MALAT1, and the correlation between the degree of C > U editing and the biochemical activity of APOBEC3B enzyme at these sites was examined by digital droplet PCR method. In this experiment, the biochemical activity of the apodec 3B enzyme was tested using the apodec 3B co-immunoprecipitation combined with BspH1 biosensor method, specific methods being referred to in the "detailed description column". Wherein the biochemical activity of the APOBEC3B enzyme is expressed by the signal ratio of TAMRA and FAM fluorescent groups in the probe molecule, and the higher the ratio is, the stronger the activity of the APOBEC3B enzyme is. Since the method requires fresh cell and tissue samples, the application range is narrow in clinical research.
The proportion of mutated bases and the biochemical activity of APOBEC3B enzyme were determined in twenty breast cancer cell lines (SKBR 3, HCC2218, MDA-MB-436, MDA-MB-157, MDA-MB-175, MCF-10-A, HCC1806, MCF7, CAL51, BT20, BT549, BT474, MDA-MB-231, EFM1924, MDA-MB-453, MDA-MB-468, T47D, HCC1143, MDA-MB-486, HCC 202), respectively, for the two NEAT1 differential RNA editing sites mediated by APOBEC3B (chr 11:65441080 and chr11: 65425652), and a scatter plot was drawn. The results are shown in FIG. 11. The results indicate that in APOThe degree of editing of the BEC 3B-dependent differential RNA editing site has good correlation with the biochemical activity of APOBEC3B enzyme, and the square R of the Pearson correlation coefficient 2 All are about 0.6. Thus, it is predicted that the enzymatic activity of apodec 3B in tumor cells can be well predicted by examining these differentiated RNA editing sites.
Using the same experimental procedure, the correlation of two MALAT 1-differentiated RNA editing sites (chr 11:65498990 and chr11: 65498321) with the biochemical activity of APOBEC3B enzyme was examined in twenty breast cancer cell lines, and the results are shown in FIG. 12. Similar to the differential editing site on NEAT1, the APOBEC 3B-mediated differential RNA editing site of MALAT1 can well predict the enzymatic activity of APOBEC3B in tumor cells.
Example 7: expression levels of NEAT1 and MALAT1 predictive of the ability of APOBEC3A and APOBEC3B to be active in tumor tissues of clinical patients
This example uses the The Cancer Genome Atlas (TCGA) real world clinical data of the american cancer institute (NCI) for the ability of the net 1 and MALAT1 to predict the degree of apodec 3A driven C > T genomic mutation. According to previous studies, tumor species were selected for abnormal expression of apodec 3A and apodec 3B and contained a large number of these two enzyme-mediated mutations, respectively bladder cancer (BLCA), breast cancer (BRCA), esophageal adenocarcinoma (esophagus adenocarcinoma, ESAD), head and neck squamous carcinoma (head and neck squamous cancer, HNSC), lung adenocarcinoma (lung adenocarcinoma, LUAD). Calling a patient sample containing whole genome or whole exome DNA sequencing data and providing matched transcriptome RNA sequencing in a TCGA database, wherein 409 of bladder cancer patients meets the condition; 1007 patients with breast cancer; 182 people with esophageal adenocarcinoma; 494 with head and neck squamous carcinoma; lung adenocarcinoma 506.
Transcriptome RNA sequencing data was taken from each patient in transcriptome RNA sequencing, and expression data (RPKM normalization data) for apodec 3A, APOBEC3B, NEAT1 and MALAT1 were extracted. The Pearson correlation was used to analyze the correlation of these genes in patient expression and to map the thermodynamic diagram. As shown in FIG. 13, there was no obvious correlation between NEAT1/MALAT1 and APOBEC3A/APOBEC3B in terms of the expression level. While there is a partial correlation between apodec 3A and apodec 3B, and between net 1 and MALAT 1.
The combined mutation detection (joint mutation calling) results of whole genome or whole exome sequencing of all patients were called for and downloaded as VCF format files. Mutation maps of each patient were analyzed using the non-Negative Matrix Factorization (NMF) clustering method reported previously, in which single base substitutions (single base substitution, SBS) No. 2 (SBS 2) and No. 13 (SBS 13) were apodec 3A dominated C > U mutations. All patients were classified into two categories, with the RNA expression levels of net 1 or MALAT1 ranked as the first 25% defined as high-expressive and the rest as low-expressive. On this basis, these patients were further classified into eight classes according to the expression of APOBEC3A and APOBEC3B, wherein the RNA expression levels of APOEBEC3A and APOBEC3B were ranked as the first 25% as defined as high-expressors, and the eight classes of patients were mapped with eight percent of SBS2 and SBS13 mutations as total mutations. As shown in fig. 14, in the patients with apodec 3A and apodec 3B high expression, the high expressers of the net 1 or MALAT1 showed high levels of SBS2 and SBS13 mutations relative to the low expressers, with statistical differences, and replicates were obtained in the patients of all tumor species.
Because of the complex negative regulatory relationship between apodec 3A and apodec 3B and the obvious co-expression of the two genes, the single use of the transcriptional level of the two genes to predict apodec 3A-driven C > U mutation has obvious disadvantages and requires further refinement of patient classification. The results of this example indicate that NEAT1 and MALAT1 are two biomarkers that effectively predict APOBEC 3A-driven C > U mutations in APOBEC3A/APOBEC3B high expressing patients. In connection with the previous embodiments, the NEAT1 and MALAT1 are likely to activate APOBEC3A indirectly by directly inhibiting APOBEC3B, and are embodied in real-world clinical samples. NEAT1 and MALAT1 are used as biomarkers, can be applied to novel tumor treatment methods of targeting APOBEC3A or APOBEC3B in the future, and provide matched diagnosis, treatment and prognosis tools.
Discussion of the invention
Apodec 3B is the only member of the apodec family of enzymes that is commonly highly expressed and abnormally activated in a variety of tumors, and its expression level is positively correlated with the degree of mutation of the C > T sequence in about 50% of human tumors. High expression of apodec 3B in tumors is associated with drug resistance development and poor clinical prognosis, and therefore, evaluating the activity of apodec 3B in tumor cells and tissue samples by accurate means is of great scientific and practical significance.
In addition to acting on single-stranded DNA, studies have shown that the C-terminal domain of apodec 3B is highly homologous to the known RNA editing enzyme apodec 3A, and thus C > U editing by cytidine deamination at the RNA level is also highly likely. The N-terminal domain of APOBEC3B and RNA can form a high molecular weight complex, thereby playing a role in regulating the editing activity of APOBEC3B on DNA and RNA. The RNA editing capability mediated by APOBEC3B can change the amino acid coding sequence corresponding to the transcript, and plays a key role in tumor heterogeneity, drug-resistant clone generation and tumor specific new antigen generation to a certain extent.
By evaluating the enzymatic activity of the APOBEC3B in tumor tissues and cells, the specific molecular mechanism and regulation mode of the APOBEC3B in the RNA editing function can be further explored, thereby providing ideas for new drug resistance generation and tumor immunotherapy development.
The invention provides an effective marker, an evaluation method, an evaluation kit and evaluation equipment for monitoring the activity of the APOBEC3B enzyme in a tumor cell model or a patient sample, thereby providing a powerful tool for the research works such as basic research, tumor disease diagnosis, treatment strategy selection, prognosis evaluation, drug screening and the like related to the APOBEC3B, and having important significance.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. The use of a cytidine deaminase apodec 3B enzyme activity marker or a detection reagent thereof for preparing a diagnostic reagent or kit for detecting cytidine deaminase apodec 3B enzyme activity;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof.
2. The use of claim 1, wherein the detection reagent is used to detect whether a c→u change occurs at an RNA editing site (with GRCh38 reference genome coordinates) selected from the group consisting of incrna nea 1: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866, or a combination thereof;
and/or the detection reagent is used for detecting whether the editing site (with GRCh38 reference genome coordinates) of the lncRNA MALAT1 selected from the following groups is changed from C to U: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549984, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613, or a combination thereof.
3. The use of claim 1, wherein the expression level of lncRNA net 1, lncRNA MALAT1, or a combination thereof is detected using fluorescent quantitative PCR to detect cytidine deaminase apodec 3B enzyme activity.
4. The use according to claim 3, wherein at least one pair of primers for specifically amplifying lncRNA net 1 and lncRNA MALAT1 is used in the fluorescent quantitative PCR assay, said primers being selected from the group consisting of:
NEAT1_1-F(SEQ ID NO：1)：
5'-GGCACAAGTTTCACAGGCCTACATGGG-3'；
NEAT1_1-R(SEQ ID NO：2)：
5'-GCCAGAGCTGTCCGCCCAGCGAAG-3'；
NEAT1_2-F(SEQ ID NO：3)：
5'-GGAGCCAACCTGCCCTGAAT-3'；
NEAT1_2-R(SEQ ID NO：4)：
5'-CCACAGGCTACCCTCTGCTC-3'；
MALAT1-F(SEQ ID NO：5)：
5'-CTTCCCTAGGGGATTTCAGG-3'；
MALAT1-R(SEQ ID NO：6)：
5'-GCCCACAGGAACAAGTCCTA-3'。
5. A kit for detecting the enzymatic activity of cytidine deaminase apodec 3B, characterized in that the kit comprises:
(i) RNA of a cytidine deaminase APOBEC3B enzyme activity marker or a detection reagent thereof;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof; and
optionally (ii) a cytidine deaminase apodec 3B enzyme, a promoter thereof, an inhibitor thereof, or a combination thereof.
6. The kit of claim 5, wherein the editing site on lncRNA net 1 is selected from the group consisting of: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866;
wherein the editing site on lncRNA MALAT1 is selected from the group consisting of: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549884, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613.
7. The kit of claim 5, wherein the detection of the marker comprises a quantitative or qualitative detection.
8. A method of detection comprising the steps of:
(a) Providing a test sample, wherein the test sample contains a cytidine deaminase apodec 3B enzyme activity marker, and the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof;
(b) Detecting the level (or degree) of the change of C-U of the editing site in the marker in the detection sample, and marking the level as C1; and
(c) Comparing the level C1 of the change of c→u of the editing site in the marker with a control reference value C0;
wherein if the marker detection result in the detection sample meets the following conditions: c1> C0, the cytidine deaminase APOBEC3B enzyme activity in the test sample is indicated to be high.
9. An apparatus for assessing cytidine deaminase apodec 3B enzyme activity, the apparatus comprising:
(a) The input module is used for inputting cytidine deaminase APOBEC3B enzyme activity marker data of a sample to be detected from a certain object, wherein the data comprise the level (or degree) of C-U change of an editing site in the marker;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof;
Wherein the editing site on lncRNA net 1 is selected from the group consisting of: chr11:65425652, chr11:65444937, chr11:65428056, chr11:65441080, chr11:65431271, chr11:65436573, chr11:65436866, chr11:65442018, chr11:65443866, or combinations thereof;
wherein the editing site on lncRNA MALAT1 is selected from the group consisting of: chr11:65498861, chr11:65498821, chr11:65498890, chr11:6549984, chr11:65500618, chr11:65500772, chr11:65501742, chr11:65504613, or a combination thereof;
(b) The processing module is used for processing the input marker data so as to obtain an evaluation result of the cytidine deaminase APOBEC3B enzyme activity; and
(c) And the output module is used for outputting an evaluation result of the cytidine deaminase APOBEC3B enzyme activity.
10. A method of screening for potential compounds, wherein the compounds are compounds that potentially promote or inhibit the enzymatic activity of cytidine deaminase apodec 3B, the method comprising the steps of:
(a) Providing a compound to be screened;
(b) In the test group, cells are cultured in the presence of the compound; and in the control group, the cells were cultured in the absence of the compound and under otherwise identical conditions, and
(c) Detecting the level (or extent) of a C.fwdarw.U change in the edit site in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the test set, designated as C1; and detecting the level (or degree) of the change of C-U of the editing site in the cytidine deaminase APOBEC3B enzyme activity marker of the cells in the blank control group, and marking the level as a reference value C0;
wherein the cytidine deaminase apodec 3B enzyme activity marker is selected from the group consisting of: lncRNA net 1, lncRNA MALAT1, or a combination thereof;
if the marker level C1 is significantly greater than the reference value C0, then determining the compound as a potential compound that promotes the enzymatic activity of cytidine deaminase APOBEC 3B;
if the marker level C1 is significantly less than the reference value C0, the compound is determined to be a potential compound that inhibits the enzymatic activity of cytidine deaminase APOBEC 3B.
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| JASON A. WEST: "The Long Noncoding RNAs NEAT1 and MALAT1 Bind Active Chromatin Sites", 《MOLECULAR CELL》, vol. 55, pages 791 - 802 * |
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