CN114934119A - Colorectal cancer diagnosis kit and application thereof - Google Patents
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Abstract
The invention relates to a colorectal cancer diagnosis kit and application thereof, and discovers for the first time through in vivo and in vitro experiments that the overexpression of FIRRE plays a role in promoting tumor progression in colorectal cancer. The direct interaction between the firrre and PTBP1 resulted in translocation of PTBP1 protein from the nucleus to the cytoplasm. The FIRRE and PTBP1 complex in the cytoplasm binds BECN1mRNA, enhancing the stability of BECN1 mRNA. The interaction of the three, firrre, PTBP1, BECN1, affects autophagy, thereby promoting the progression of colorectal cancer. The basic research can provide a new idea for clinical treatment of colorectal cancer, and medicines of target genes can be designed aiming at the axes of FIRRE/PTBP1/BECN1 for treating colorectal cancer, so that a new direction is provided for researching gene therapy.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a colorectal cancer diagnosis kit and application thereof.
Background
Colorectal cancer ranks third in the incidence of tumors worldwide, with up to 53200 deaths annually due to colorectal cancer. The high mortality rate and limited treatment options of colorectal cancer make it important to further investigate the regulatory mechanisms of CRC and it is highly desirable to find biomarkers that can be used for diagnosis and treatment of colorectal cancer.
The onset and progression of colorectal cancer is accompanied by changes in a variety of coding and non-coding genes. LncRNAs are one of non-coding genes and are involved in the development process of colorectal cancer. The function of lncRNAs after binding to proteins is one of the important modes of action. After the LncRNAs are combined with proteins, the LncRNAs are widely involved in the physiological and pathological processes of tumors through various action mechanisms. FIRRE is a conserved lncRNA that maps to the X chromosome, and our studies found that expression of FIRRE is elevated in colorectal cancer. However, studies on the exact function and molecular mechanism of firrre in colorectal cancer have not been reported so far.
Polypyrimidine sequence binding proteins (PTBP 1, also known as hnRNP I), which are important members of the heteronuclear ribonucleoprotein family (hnRNPs), are expressed in elevated colorectal tumors and are associated with poor prognosis. PTBP1, as an RNA binding protein, can interact with lncRNA binding, regulating a variety of biological processes. However, the relationship between firrre and PTBP1 and their role and mechanism in colorectal cancer remains unexplored.
Our studies revealed that overexpression of firrre plays a role in promoting tumor progression in colorectal cancer. The direct interaction between the firrre and PTBP1 resulted in translocation of PTBP1 protein from the nucleus to the cytoplasm. The rre and PTBP1 complex in the cytoplasm binds BECN1mRNA, enhancing the stability of BECN1 mRNA. The interaction of the three, firrre, PTBP1, BECN1, affects autophagy, thereby promoting the progression of colorectal cancer. The influence of the pathway FIRRE-PTBP1-BECN1 on the development of colorectal cancer and the potential clinical application value are not reported, and the lncRNAFIIRRE is proposed to be possibly used as a biomarker for diagnosing the colorectal cancer and a new action target point for treating the colorectal cancer for the first time.
The Chinese patent application: CN113416781A discloses a colorectal cancer diagnosis marker and application thereof, the colorectal cancer diagnosis marker is serum adipokine leptin rs780093 and/or rs900400, the application of the marker in preparing a colorectal cancer detection reagent and a kit containing the marker, the adipokine leptin screened by the invention is highly related to colorectal cancer diagnosis in blood, the pertinence to early colorectal cancer samples is strong, and the application of the biomarker and a corresponding monitoring technology to carry out noninvasive early screening early diagnosis of colorectal cancer is significant. The Chinese patent application: CN107447033B discloses a colorectal cancer diagnosis biomarker and application thereof, and finds that hsa _ circ _0006110 gene expression is closely related to colorectal cancer for the first time, and by detecting the expression of hsa _ circ _0006110 in colorectal tissues of a subject, whether the subject has colorectal cancer or whether the risk of having colorectal cancer exists can be judged more accurately and rapidly, so that a prevention or treatment scheme is provided for a clinician. And the target gene for preparing the medicine for treating the colorectal cancer provides a new treatment target and a new treatment way for treating the colorectal cancer, and compared with the traditional detection means, the diagnosis of the molecular marker is more timely and specific, so that the 5-year survival rate of the colorectal cancer patient is improved, the death rate is reduced, and the application prospect is wide. However, no report is found about the colorectal cancer diagnostic kit and the application thereof.
Disclosure of Invention
The invention aims to provide a colorectal cancer diagnosis kit and application thereof aiming at the defects of the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the present invention provides the use of the diagnostic marker firrre for the manufacture of a diagnostic kit for colorectal cancer.
In a second aspect, the present invention provides a colorectal cancer diagnostic kit comprising a reagent for detecting the expression amount of FIRRE as the only key component of the kit for accomplishing the diagnosis of colorectal cancer.
In a third aspect, the present invention provides a method for assessing the prognosis of colorectal cancer, comprising the steps of: detecting the expression level of FIRRE.
In a fourth aspect, the invention provides an application of FIRRRE in preparing a medicine for preventing and treating colorectal cancer.
In a fifth aspect, the invention provides the use of an inhibitor of rre in the preparation of a medicament for the prevention and treatment of colorectal cancer.
Preferably, the inhibitor of FIRRE is a substance that reduces the expression level of FIRRE.
Preferably, the inhibitor of the FIRRE is selected from a small molecule compound or a biological macromolecule.
Preferably, the biological macromolecule is small interfering RNA, dsRNA, shRNA, micro RNA or antisense nucleic acid which takes the FIRRE protein or the transcript thereof as a target sequence and can inhibit the expression of the FIRRE protein or the gene transcription; or a construct capable of expressing or forming said small interfering RNA, dsRNA, microRNA, antisense nucleic acid.
In a sixth aspect, the invention provides the use of an inhibitor of FIRRE-PTBP1-BECN1 in the manufacture of a medicament for the prevention or treatment of colorectal cancer.
Preferably, said inhibitor of FIRRE-PTBP1-BECN1 is a substance that prevents the binding of the three FIRRE-PTBP1-BECN 1.
The invention has the advantages that:
the subject group found for the first time that overexpression of firrre plays a role in promoting tumor progression in colorectal cancer. The direct interaction between the firrre and PTBP1 resulted in translocation of PTBP1 protein from the nucleus to the cytoplasm. The FIRRE and PTBP1 complex in the cytoplasm binds BECN1mRNA, enhancing the stability of BECN1 mRNA. The interaction of the three, firrre, PTBP1, BECN1, affects autophagy, thereby promoting the progression of colorectal cancer. The basic research can provide a new idea for clinical treatment of colorectal cancer, and medicines of target genes can be designed aiming at the axes of FIRRE/PTBP1/BECN1 for treating colorectal cancer, so that a new direction is provided for researching gene therapy.
Drawings
FIG. 1 shows the results of lncRNA sequencing, in which the colon cancer tissue and the tissue adjacent to the cancer were confirmed by HE staining; panel B chip analysis results volcano plots of known lncRNAs in differentially expressed genes; panel C chip analysis results, scatter plot of known lncRNAs in differentially expressed genes; panel D chip analysis results, heat map of part of known lncRNAs in differentially expressed genes; panel E according to the FDR values, lncRNA is known to express a significantly increased first ten genes in intestinal cancer tissues.
FIG. 2 shows that the expression level of FIRRE is obviously increased in colorectal cancer by TCGA database analysis, and the expression level of FIRRE in intestinal cancer tissues and tissues beside the cancer in ATCGA database is compared; panel B paired comparison of expression levels of firrre in intestinal cancer tissue and tissues adjacent to cancer in TCGA database.
FIG. 3 is the expression of FIRRE in colon cancer cell lines and tissues, AFIRRE in colorectal cancer cell lines as well as normal intestinal epithelial cell lines; panel B expression levels of firrre in colorectal and paracancerous tissues. P < 0.05; p < 0.01; p < 0.001.
FIG. 4 shows that FIRRRE can promote migration, invasion and proliferation of tumor cells, and the influence of different levels of AFIRRE on the migration ability of the cells is shown in the right side of the graph of statistical results; FIG. B shows the effect of different levels of FIRRE on the migration ability of cells, and the statistical results are shown on the right; FIG. C Effect of different expression levels of FIRRE on the proliferative capacity of cells; panel D shows that upon overexpression of rre in cells, plate clonogenic experiments showed a significant increase in the number of over-expressed clones; figure E effect on cell cycle following rre knockdown. P < 0.05; p < 0.01; p < 0.001.
FIG. 5 is a graph of the effect of FIRRE expression on the stage of tumor TNM, overall stage and survival rate in patients with tumors in the TCGA database, illustrating that the expression level of AFIRRE is independent of the age of the patients; panel B expression level and gender of rre are unrelated; panel C expression levels of firrre increased with increasing depth of tumor infiltration, but the difference was not statistically significant; panel D high expression of FIRRE positively correlates with tumor lymph node metastasis; panel E high expression levels of firrre positively correlated with distant metastasis of the tumor, with statistical significance; panel F expression levels of firrre had no effect on the overall stage of the tumor; the high expression of GFIRRE was not clearly correlated with patient survival.
FIG. 6 shows the direct binding of FIRRE to PTBP1 protein, panel A PTBP1 RNA expression in FIRRE negative control and over-expressed cells; panel B expression of PTBP1 RNA in the cells of the FIRRE negative control group and knockdown group; panel C expression at PTBP1 protein level in the firrre overexpression group (upper panel) and PTBP1 protein level in the firrre knockdown group (lower panel); panel D FIRRE RNA probe synthesized by in vitro transcription, antisense and sense; FIG. E RNA pull down experiment resulted in RNA-protein complexes that were tested for enrichment of PTBP1 protein by WB (using anti-PTBP 1 antibody); in FIG. F, the enrichment of PTBP1 protein in the RNA-protein complex obtained from RNA pull down test is detected by silver staining test (using anti-PTBP 1 antibody), and the band indicated by red arrow is PTBP 1; FIG. G RIP experiment, detecting the effect of the magnetic beads bound to the antibody on the pull-down of PTBP1 protein by WB; FIG. H RIP experiment pull-down RNA after PCR using gel electrophoresis to detect FIRRE RNA enrichment; panel I RIP experiment pull-down RNA was purified and qPCR was used to detect FIRRE RNA enrichment. Panel J in vitro transcription synthesis of RRD fragments; panel K RNA pull down experiment confirmed the binding of RRD and PTBP 1.
FIG. 7 is a graph of the effect of FIRRE on the subcellular localization of PTBP1 protein, plotting the effect of AFIRRE expression level on the subcellular localization of PTBP1, at a scale bar of 5 μm; panel B cellular localization of PTBP1 protein after overexpression of FIRRE by RKO cells; panel C cellular localization of PTBP1 protein following overexpression of FIRRE by HCT116 cells; panel D cellular localization of PTBP1 protein following knockdown of FIRRE expression; RKO cells on the left and HCT116 cells on the right; panel E subcellular localization of rre was detected in RKO overexpressing rre knockdown PTBP1 cells.
FIG. 8 shows that the protein-RNA complex formed by FIRRE RNA, PTBP1 protein and BECN1mRNA, and the protein-RNA complex predicted by the Credit analysis that the target gene of PTBP1 protein was BECN 1; panel B in vitro synthesis of BECN1mRNA fragments; figure C Sanger sequencing verifies the sequence of the synthetic fragment; panel D RNA pull down experiment of PTBP1 protein with fragment BECN 1-F1; FIG. E RIP assay of BECN1 RNA amplified by conventional PCR using anti-PTBP 1 pull-down, PCR product gel electrophoresis detection; panel F RIP experiment with anti-PTBP 1 pull-down BECN1 RNA enrichment Using qPCR assay results.
FIG. 9 is a graph of the enhancement of BECN1mRNA stability by FIRRE via PTBP1, which depicts the expression level of BECN1mRNA when AFIRRE is knocked down; panel B expression level of BECN1 protein when the expression of FIRRE was reduced; the CFIRRE expression level is reduced, and a BECN1 protein expression level gray value statistical chart is shown; FIG. D RKO cells, PTBP1 RNA expression levels following siPTBP1 transfection; panel E expression of PTBP1 RNA in HCT116 cells following transfection of siPTBP 1; FIG. F expression of PTBP1 at the protein level following transfection of siPTBP1 in RKO and HCT116 cells; FIG. G shows the presence of BECN1mRNA after actinomycin D treatment of recombinant RKO cells for 60 min; FIG. H expression of BECN1mRNA from recombinant HCT116 cells after 60min actinomycin D treatment.
FIGS. 10 and 11 show that FIRRRE is able to promote autophagy activity, and that protein expression levels of p62(SQSMT1) and LC3 are shown after AFIRRE knockdown; the ratio of WB result grayscale values of LC3II and LC3I and WB result grayscale value of p62 with FIRRE strike on the right; panel B immunofluorescence results against LC3 with rre knockdown, RKO cells on the left and HCT116 cells on the right; panel C expression levels of BECN1, p62 and LC3 were detected by knocking down FIRRE and PTBP1, respectively or in combination; panel D cells were transfected with bifluorescent virus and examined for unobstructed autophagy by knock-down of FIRREE and PTBP1, alone or in combination, under confocal microscopy.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
Example 1
First, experiment method
Increased expression of the first part of the FIRE in colorectal cancer
1.1.LncRNA sequencing experiment process
Three pairs of intestinal cancer and paracarcinoma tissues are selected, total RNA is extracted from the tissues, and the concentration, purity and integrity of the RNA are detected by Nanodrop2000 and RNA agarose gel electrophoresis. After the removal of rRNA, mRNA is fragmented into fragments of about 200bp by metal ions. cDNA is synthesized by reverse transcription, and information analysis is carried out after the Illumina machine sequencing.
TCGA database data mining and analysis
Data used in this study included clinical data for colorectal cancer as well as mRNA expression data.
1.3. Patient screening and clinical data collection
Colorectal cancer patients who received radical surgery treatment at the Jinshan Hospital affiliated to the university of Fudan in 2015 to 2017 according to established grouping standards and exclusion standards.
1.4. Detecting the expression of FIRRE RNA in intestinal cancer cells and normal intestinal epithelial cells
When the cell fusion degree reaches 90%, sucking out the culture medium, washing the cells by PBS, sucking out PBS, sucking the cell solution into an RNase-Free microfuge tube after digestion, centrifuging for 5min at 300 Xg, carefully removing the supernatant, and keeping the precipitate; and then extracting cell RNA according to the steps of the total RNA extraction kit of the Tiangen cultured cell and detecting the expression condition of the FIRRE in the cell by a qPCR experiment.
1.5. Detecting the expression of FIRRE RNA in intestinal cancer and tissues beside the cancer
Rinsing scissors with DEPC water for 3 times, shearing 20mg of colorectal cancer and tissues beside the cancer, extracting tissue RNA according to a total RNA extraction kit of the tissue of the rhizomatous animal, and carrying out qPCR (quantitative polymerase chain reaction) to detect the expression condition of FIRRE in the tissues beside the cancer and the intestinal cancer.
The second part of FIRRE promotes proliferation, migration and invasion of colorectal cancer cells, and affects lymphatic metastasis and distant metastasis of CRC patients
Third part 2.1 detection of biological function of tumor cells
Constructing a FIRRE overexpression lentivirus packaging and stable transfer cell strain, simultaneously constructing a FIRRE knockdown cell strain by transcribing Antisense oligonucleotide (ASO), and verifying the successful construction of the cell overexpression and knockdown cell strain by qPCR and WB. And then carrying out CCK8, cell migration/invasion test, plate cloning test, cell cycle detection and other methods to verify the function of the FIRRE in the colorectal cancer.
2.2. Exploring the role of FIRREE in colorectal cancer disease by TCGA database and clinical data analysis of collected patients
Mechanism discussion of FIRRE promoting colorectal cancer progression in the third part
From bioinformatics analysis tools, Starbase 2.0 and cataPID, we know that FIRRRE may function in conjunction with PTBP 1. To confirm the direct binding effect between the rre and PTBP1, the mechanism of the role of rre in colorectal cancer was explored by in vitro synthesis of gene fragments, RNA pull down and RNA co-immunoprecipitation (RIP), immunofluorescence, RNA Fluorescence In Situ Hybridization (FISH), protein immunoblotting (WB), qRT-PCR, etc.
Second, results and analysis
Elevated expression levels of FIRRE in colorectal cancer
RNA sequencing results of colorectal cancer and tissues beside the cancer show that the expression level of FIRRE in the cancer tissues is obviously higher than that of the tissues beside the cancer; the expression level of FIRRE in colon cancer cell strains RKO, HCT116, HT29, SW620 and SW480 is obviously higher than that in normal intestinal epithelial cell strains FHC; the qPCR detection is carried out on 20 intestinal cancer tissues and tissue specimens beside the cancer, and the result shows that the expression level of FIRRE is higher in the intestinal cancer tissues than in the tissues beside the cancer.
FIRRE promotes migration, invasion and proliferation of tumor cells; associated with lymph node metastasis and distant metastasis of tumor patients
The FIRRE over-expression stable cell strain and the FIRRE knockdown cell strain are successfully constructed in RKO and HCT116 cells; in the FIRRE over-expressed RKO and HCT116 cells, the migration, invasion and proliferation capacities of the cells are stronger than those of a negative control group; in the FIRRE-knocked-down RKO and HCT116 cells, the migration, invasion and proliferation capacities of the cells are obviously weaker than those of a negative control group; flow cytometry results suggest: when the firrre is knocked down, the cells generate S-phase block; the TCGA database mining result shows that the expression level of FIRRE is independent of the age and sex of patients, patients with high expression of FIRRE present more serious lymph node metastasis and distant metastasis, but the expression level of FIRRE is not obviously related to the infiltration depth of tumors, the overall stage of the tumors and the survival period; clinical data analysis of patients suggests that high levels of firrre often imply tumor invasion, lymphatic metastasis and distant metastasis; FIRRE is associated with the overall stage of the tumor, and patients with high expression of FIRRE have higher overall tumor stage grade; however, FIRRRE does not affect the tumor site and the size of the tumor.
TABLE 1 relationship of FIRRE expression levels to patient clinical characteristics
FIRRE can bind directly to PTBP1 protein and PTBP1 protein moves from nucleus to cytoplasm
The belief-generating analysis predicted that the firrre might bind to PTBP1 protein. Firstly, the expression level of FIRRE in the recombinant cells can not influence the expression of PTBP1 RNA and protein. The FIRRE sense strand (sense) and antisense strand (antisense) gene fragments are successfully synthesized by in vitro transcription, the result of an RNA pull down experiment indicates that the sense can enrich more PTBP1 protein compared with the antisense, and the silver staining experiment further confirms the result; the results of PCR detection of RIP experiment enriched RNA suggest that the anti-PTBP 1 group can enrich more FIRRE RNA compared with the control anti-IgG group, and the qPCR results also suggest that the anti-PTBP 1 group can enrich more FIRRE RNA. In the FIRRE over-expression cells, the protein localization of PTBP1 was detected by immunofluorescence, and the result showed that the PTBP1 protein was localized in the cytoplasm when FIRRE was over-expressed; in the rre knockdown cells, PTBP1 is mostly concentrated in the nucleus; WB test was performed on isolated cell nuclear plasma protein, and the results suggested that the distribution of PTBP1 protein in the cytoplasm was increased when FIRRE was overexpressed. The RNAFISH results suggest that when the expression level of PTBP1 is reduced, the subcellular localization of FIRRE is changed and the FIRRE is transferred from the nucleus to the cytoplasm.
Binding of PTBP1 protein to BECN1mRNA in the cell paste, enhancing BECN1mRNA stability, promoting autophagy activity
The target gene that PTBP1 was predicted to likely bind by the biological assay software to be BECN1mRNA and the binding site likely to be located at the 3' end of BECN 1. In vitro synthesis of BECN1mRNA fragment, RNA pull down experiment, results suggest: the BECN1mRNA fragment was able to enrich for more PTBP1 protein than the control group; RIP experiments with anti-PTBP 1 and anti-IgG antibodies confirmed that the anti-PTBP 1 group was able to enrich more FIRRE RNA than the control group anti-IgG. qPCR results suggested that when rre was knocked down, the expression level of BECN1 was also significantly reduced, whether at the RNA or protein level. Treatment of cells knocked down for firrre alone or in combination with PTBP1 with actinomycin D showed a significant decrease in BECN1mRNA levels when firrre was knocked down alone, while no significant change in BECN1mRNA levels occurred when firrre and PTBP1 were knocked down in combination. Due to the important role of BECN1 in autophagy, the expression of the autophagy-important molecules p62 and LC3 was also examined at the time of FIRRE knockdown. The results suggest that when the level of FIRRE is reduced, the expression levels of p62 and LC3B are significantly reduced; LC3 immunofluorescence detected autophagosome formation, suggesting that autophagosome formation decreases when the expression level of FIRRE decreases.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (10)
1. Application of a diagnosis marker FIRRE in preparing a colorectal cancer diagnosis kit.
2. A colorectal cancer diagnostic kit, characterized in that the kit comprises a reagent for detecting the expression level of firrre, wherein the reagent is used as the only key component for diagnosing colorectal cancer.
3. A method for assessing the prognosis of colorectal cancer, comprising the steps of: detecting the expression level of FIRRE.
Application of FIRRE in preparing medicine for preventing and treating colorectal cancer.
Application of an inhibitor of FIRRE in preparing a medicine for preventing and treating colorectal cancer.
6. The use according to claim 5, wherein the inhibitor of FIRRE is a substance that reduces the expression level of FIRRE.
7. The use according to claim 6, wherein the inhibitor of FIRRE is selected from a small molecule compound or a biological macromolecule.
8. The use of claim 7, wherein the biological macromolecule is a small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid targeting the FIRRE protein or its transcript and capable of inhibiting the expression of the FIRRE protein or the transcription of the gene; or a construct capable of expressing or forming said small interfering RNA, dsRNA, microRNA, antisense nucleic acid.
Use of an inhibitor of FIRRE-PTBP1-BECN1 in the manufacture of a medicament for the prevention or treatment of colorectal cancer.
10. The use as claimed in claim 9 wherein the inhibitor of FIRRE-PTBP1-BECN1 is a substance which prevents the binding of FIRRE-PTBP1-BECN 1.
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