CN116804221B - Application of LMO7 expression level in prediction of pancreatic cancer gemcitabine resistance - Google Patents
Application of LMO7 expression level in prediction of pancreatic cancer gemcitabine resistance Download PDFInfo
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- CN116804221B CN116804221B CN202311063013.9A CN202311063013A CN116804221B CN 116804221 B CN116804221 B CN 116804221B CN 202311063013 A CN202311063013 A CN 202311063013A CN 116804221 B CN116804221 B CN 116804221B
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Abstract
The application discloses an application of LMO7 expression level in prediction of pancreatic cancer gemcitabine resistance, and belongs to the technical field of biological material detection. According to the application, the drug resistance of pancreatic cancer patients to gemcitabine is predicted by using the expression level of LMO7, and afatinib is recommended to be used when the expression level of LMO7 is higher, so that the treatment effect and the safety can be effectively improved, and the prognosis is expected to be improved, so that the method has important clinical significance for the treatment selection of pancreatic cancer patients.
Description
Technical Field
The application belongs to the technical field of biological material detection, and particularly relates to application of an LMO7 expression level in prediction of pancreatic cancer gemcitabine resistance.
Background
Pancreatic cancer is a fatal malignancy, and about 85% of pancreatic cancer cases are characterized by pancreatic ductal adenocarcinoma (pancreatic ductal adeno-carcinoma, PDAC). The incidence rate and the death rate of PDAC are high at the present stage, the treatment strategy is limited, and the long-term survival rate is low.
Metastasis is a major factor in the high mortality rate of PDACs. Most patients find metastasis at the time of first diagnosis, whereas patients with distant metastasis have a survival rate of only 3% in 5 years. Furthermore, only 10-20% of PDAC patients can be resected, with most patients eventually developing local or distant recurrence after surgery and chemotherapy. However, the mechanism of PDAC metastasis is currently unknown, and thus metastatic PDACs are urgently needed as markers for early diagnosis.
Gemcitabine (gemcitabine, GEM) is a first-line chemotherapy regimen currently used in pancreatic cancer patients, with overall efficacy rates of less than 20%, and is mainly due to chemotherapy resistance. Thus, drug resistance is also an important factor affecting the overall survival of pancreatic cancer patients, but there is no good regimen to predict gemcitabine resistance.
Disclosure of Invention
In order to solve at least one of the above technical problems, the inventors applied WilcoxThe on-rank sum test compares the significant differential genes between the sensitive and resistant groups of gemcitabine-treated PDACs in GSE140077 dataset. Further screening out genes with the ratio of average gene expression value in the drug resistant group to average gene expression value in the sensitive group being more than 1 and p value being less than 0.05, analyzing the transfer potential characteristics of the genes, unexpectedly findingLMO7The high expression level of the gene has a strong correlation with the resistance to gemcitabine, thereby completing the present application.
The application provides application of a detection reagent for LMO7 expression level in preparation of a kit for predicting whether a pancreatic cancer patient is resistant to gemcitabine.
Gemcitabine (Gemcitabine), a novel cytosine nucleoside derivative, having the formula C 9 H 11 F 2 N 3 O 4 . Like cytarabine, it is activated by deoxycytidine kinase and metabolized by cytosine nucleoside deaminase after entering the human body. Gemcitabine is a pyrimidine antitumor drug, the action mechanism is the same as that of cytarabine, and the main metabolite is doped with DNA in cells and mainly acts on the G1/S phase. However, except that difluoro deoxycytidine can inhibit nucleotide reductase in addition to incorporation into DNA, resulting in reduction of intracellular deoxynucleoside triphosphates; the other difference with cytarabine is that it can inhibit deoxycytosine deaminase to reduce the degradation of intracellular metabolites and has self-synergistic effect. Clinically, gemcitabine is used in a first-line chemotherapy regimen for pancreatic cancer patients. However, pancreatic cancer patients develop resistance after administration of gemcitabine. By using the technical scheme of the application, whether pancreatic cancer patients are resistant to gemcitabine can be predicted by detecting the LMO7 expression level, so that the aim of accurate treatment is fulfilled.
In some embodiments of the application, the LMO7 expression level isLMO7Level of gene transcript or LMO7 protein level.
LIM domain 7 (LIM domain 7,LMO7) The proteins encoded by the genes contain Calmodulin Homology (CH), PDZ and LIM domains, possibly involved in protein-protein interactions.
In some embodiments of the present application,the LMO7 expression level isLMO7The level of gene transcription product, the detection reagent being capable of specifically binding to theLMO7Primers and/or probes for at least a portion of the sequence of the gene transcript. In some embodiments of the application, amplification is performed using specific primersLMO7Gene transcription products, thereby obtaining LMO7 expression levels.
In some preferred embodiments of the application, the detection reagent is an RNA sequencing reagent or a gene chip. Transcriptomes represent the entire class of RNAs present in cells, including mRNA, rRNA, tRNA and other various non-coding RNAs, and the like. DNA microarray (DNA microarray) technology or gene chip (Genechip) sequencing technology and RNA sequencing technology (RNA-seq) are two major technologies in transcriptome analysis. The main difference is that DNA microarrays are based on hybridization of pre-designed labeled probes to the cDNA sequence of interest, whereas RNA-seq directly sequences the cDNA strand by sequencing techniques.
In other embodiments of the application, the LMO7 expression level is LMO7 protein level and the detection reagent is an antibody capable of specifically binding to the LMO7 protein.
In some embodiments of the application, LMO7 expression levels are detected prior to administration of gemcitabine to a pancreatic cancer patient, and if LMO7 expression levels are above a preset threshold, the patient is predicted to develop resistance to gemcitabine, and afatinib is recommended as a first line therapeutic.
In some embodiments of the application, LMO7 expression levels are detected after administration of gemcitabine to a pancreatic cancer patient, and if LMO7 expression levels are above a preset threshold, the patient is predicted to have developed resistance to gemcitabine and afatinib or other second line therapeutic agent is recommended.
In some embodiments of the application, the predetermined threshold is determined from a representative value of LMO7 expression levels in a sample of non-pancreatic cancer patients of the population.
Further, the representative value is selected from one of a mean, a median, a mode, a maximum, and a third quartile.
In some embodiments of the application, the pancreatic cancer is pancreatic ductal adenocarcinoma.
The application also discloses application of the detection reagent for the LMO7 expression level in preparation of a kit for selecting medicines for treating pancreatic cancer patients. Specifically, LMO7 expression levels are detected in pancreatic cancer patients, and afatinib is selected as the therapeutic agent if the LMO7 expression levels are above a preset threshold, and gemcitabine is selected as the therapeutic agent if not above the preset threshold.
The application also discloses the following technical scheme:
use of a detection reagent for LMO7 expression levels in the preparation of a kit for predicting or diagnosing whether a pancreatic cancer patient is at risk of metastasis, if LMO7 expression levels are above a preset threshold, pancreatic cancer cells are at risk of migration and/or invasion, i.e. pancreatic cancer patients are at risk of metastasis.
Use of a detection reagent for LMO7 expression levels in the preparation of a kit for predicting prognosis of a pancreatic cancer patient. If the LMO7 expression level is higher than the preset threshold, the prognosis of the pancreatic cancer patient is poor.
Use of a detection reagent for LMO7 expression levels in the preparation of a kit for predicting sensitivity of a pancreatic cancer patient to afatinib. If the LMO7 expression level is higher than the preset threshold, the pancreatic cancer patient has higher sensitivity to afatinib.
Further defined or additional technical features regarding the detection reagent, the preset threshold value in all the above applications are the same as the detection reagent of LMO7 expression level in the application in the preparation of a kit for predicting whether a pancreatic cancer patient is resistant to gemcitabine.
Still further, the present application relates to:
use of an LMO7 inhibitor in the manufacture of a medicament for the treatment of pancreatic cancer.
Use of an LMO7 inhibitor in the manufacture of a medicament for the treatment of pancreatic cancer in combination with gemcitabine.
A pharmaceutical composition comprising an LMO7 inhibitor and gemcitabine.
In the present application, the LMO7 inhibitor includes, but is not limited to, a substance capable of inhibiting the level of LMO7 gene transcription and/or the level of LMO7 protein, a substance capable of inhibiting the level of LMO7 regulatory genes/proteins, a substance capable of inhibiting the function of LMO7 protein.
In some embodiments of the application, the LMO7 inhibitor is an siRNA capable of silencing an LMO7 gene, wherein silencing refers to allowing reduced or no expression of the gene expression level. In some embodiments of the application, the siRNA capable of silencing an LMO7 gene is selected from at least one of:
siLM07#1：CTGGATGATTACTCCACAA
siLM07#2：CATCAAACCGTGCCTACAT
siLM07#3：CTTCGTAGCATCAGTTGAA
preferably, at least one selected from silm07#1 and silm07#2.
The beneficial effects of the application are that
Compared with the prior art, the application has the following beneficial effects:
according to the technical scheme, the drug resistance of pancreatic cancer patients to gemcitabine is predicted by using the expression level of LMO7, and afatinib is recommended to be used when the expression level of LMO7 is higher, so that the treatment effect and the safety can be effectively improved, the prognosis is expected to be improved, and the method has important clinical significance for the treatment selection of pancreatic cancer patients.
Drawings
FIG. 1 showsLMO7Effects of gene expression levels and PDAC on resistance to gemcitabine. Fig. 1A: the box plot shows the time and dose of gemcitabine additionLMO7Gene expression level. The abscissa shows the dose of gemcitabine treatment and the ordinate shows the dose of gemcitabine treatmentLMO7Gene expression level. Comparing the difference between treatment 24h and 6h using Wilcoxon rank sum test, red dots for treatment 24h, blue dots for treatment 6h, yellow dots for untreated control group, ns. for p>0.05 represents p<=0.01,/represents p<=0.001. Fig. 1B: the box plot shows the sensitive and drug resistant cell lines in the GSE140077 dataLMO7Gene expression level. The sensitive and resistant groups are on the abscissa and the ordinateLMO7Gene expression level.
FIG. 2 shows the immunohistochemical detection of expression in LMO7 pancreatic cancer. Fig. 2A: pancreatic cancer tumor tissue; fig. 2B: pancreatic cancer nerve infiltration foci.
Fig. 3 shows the relative expression amounts of LMO7 in different pancreatic cancer cell lines by Western Blot, with p <0.05, p < = 0.01, p < = 0.001.
Fig. 4 shows that LMO7 overexpression vectors were able to significantly up-regulate LMO7 expression in pancreatic cancer cell lines, representing p < = 0.001.
FIG. 5 shows the correlation between LMO7 overexpression and resistance to gemcitabine. Wound healing and cross-hole tests showed different groups of migration and invasion capacities. Fig. 5A: results of untreated group wound healing test and cross-well test; fig. 5B: results of the sham wound healing test and the cross-hole test; fig. 5C: results of true treatment group wound healing test and cross-hole test; fig. 5D: results of wound healing assays and cross-well assays for untreated, sham, and true treated groups are bar graphs. Unpaired t-test, p <0.05, p < = 0.01, p < = 0.001.
FIG. 6 shows a pressLMO7Survival analysis of TCGA PAAD samples of gene expression level groupings. The dashed line represents the LMO7 low expression group, the solid line represents the LMO7 high expression group, and the horizontal axis represents the follow-up time. The Logrank test is statistically significant (p<0.001）。
Figure 7 shows the silencing effect of 3 different sirnas on LMO 7.
Figure 8 shows the correlation between LMO7 silencing and resistance to gemcitabine. Wound healing and cross-hole tests showed different groups of migration and invasion capacities. Fig. 8A: results of untreated group wound healing test and cross-well test; fig. 8B: results of the sham wound healing test and the cross-hole test; fig. 8C: results of true treatment group wound healing test and cross-hole test; fig. 8D: results of wound healing assays and cross-well assays for untreated, sham, and true treated groups are bar graphs. Unpaired t-test, p <0.05, p < = 0.01, p < = 0.001.
Fig. 9 shows the correlation between LMO7 and afatinib sensitivity. Wound healing and cross-hole tests showed different groups of migration and invasion capacities. Fig. 9A: results of the sham wound healing test and the cross-hole test; fig. 9B: results of true treatment group wound healing test and cross-hole test; fig. 9C: results of the wound healing test and the cross-hole test for sham and true treatment groups are bar graphs. Unpaired t-test, p <0.05, p < = 0.01, p < = 0.001.
Detailed Description
Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited in this application are incorporated by reference, particularly as if they were set forth in the relevant terms of art. If the definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition of the term provided in the present application controls.
The numerical ranges in the present application are approximations, so that it may include the numerical values outside the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or process, and are not related to whether or not such other component, step or process is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising", "including" or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.
In order to make the technical problems, technical schemes and beneficial effects solved by the application more clear, the application is further described in detail below with reference to the embodiments.
Examples
The following examples are presented herein to demonstrate preferred embodiments of the present application. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the application, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the application.
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 application belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the claims.
The molecular biology experiments described in the following examples, which are not specifically described, were performed according to the specific methods listed in the "guidelines for molecular cloning experiments" (fourth edition) (j. Sambrook, m.r. Green, 2017) or according to the kit and product specifications. Other experimental methods, unless otherwise specified, are all conventional. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
EXAMPLE 1 discovery of genes associated with gemcitabine resistance
The inventors compared the significant differential genes between the sensitive and resistant groups of gemcitabine-treated PDAC (pancreatic ductal adenocarcinoma) in GSE140077 dataset using Wilcoxon rank sum test. Further screening out genes with the ratio of the average gene expression value in the drug resistant group to the average gene expression value in the sensitive group being more than 1 and the p value being less than 0.05, analyzing the transfer potential characteristics of the genes, surprisingly finding,LMO7the high expression of the gene has strong correlation with gemcitabine resistance.
Further, the inventors observed GSE105083 datasetLMO7Unexpectedly, it was found that as the gemcitabine concentration increased, the treatment time was 24h in the groupLMO7The expression level was gradually increased relative to the untreated group (untreated) (fig. 1). In particular at gemcitabine concentrations of 30nM, 100nM, 300nM, 3. Mu. Mol and 10. Mu. Mol, in groups with a treatment time of 24hLMO7In contrast to the 6h treatment time group, the expression of (a) was significantly increasedLMO7The increase in expression of (a) was not significant (fig. 1A).
Figure 1B shows that in the gemcitabine-susceptible group and the drug-resistant group,LMO7there is a significant difference in expression levelp<0.01 Indicated by the fact thatLMO7Is associated with resistance of PDAC to gemcitabine.
EXAMPLE 2 immunohistochemical detection of pancreatic cancer tumor tissue
1. Sample acquisition
In the surgery of pancreatic cancer patients, human pancreatic cancer tumor tissues are aseptically separated, paraffin embedded blocks are prepared, 4-5 mu mol thick tissue slices are cut and stuck on a glass slide, and the tissue slices which can be used for immunohistochemical staining are prepared.
2. Immunohistochemical detection
LMO7 antibody (brand: sigma, cat# HPA 020923) was purchased and conventional immunohistochemical staining was performed using the antibody at a dilution ratio of 1:200. The results are shown in FIG. 2, where LMO7 expression of tumor cells in pancreatic cancer tumor tissue was strong (FIG. 2A); LMO7 expression was strong in pancreatic cancer neuroinvasive foci (fig. 2B).
EXAMPLE 3 Western blot detection of LMO7 protein expression
Cell line acquisition:
human pancreatic cancer cell lines (CFPAC-1, PANC-1, BXPC-3, miaPaca-2), all from the national academy of sciences cell bank.
A human healthy pancreatic ductal cell line, hTERT-HPNE (HPNE), purchased from American type culture Collection (ATCC; rockville, medical doctor, USA), was cultured according to the procedure recommended by ATCC.
Extracting total protein of pancreatic cancer tumor tissue according to total protein extraction kit (Pierce Chemical), quantifying by BCA method, adding loading buffer, boiling, and denaturing for 5min; taking 40 mug total protein, performing 8-10% SDS-PAGE gel electrophoresis, and then performing electrotransfer to a PVDF membrane, and sealing with 5% skimmed milk for 2 hours; incubation overnight at 4℃with anti-LMO 7 antibody at a dilution ratio of 1:1000, incubation with secondary antibody at a dilution ratio of 1:5000 for 2h at room temperature, PBST wash 3 times for 10min each. Scanning by a gel image analysis system, taking GAPDH as an internal reference, calculating the ratio of LMO7 to the gray value of the internal reference, and performing semi-quantitative detection.
The results are shown in FIG. 3, where LMO7 was highly expressed in human pancreatic cancer cell lines and low in human healthy pancreatic ductal cell lines. Furthermore, the expression level was the lowest in MiaPaca-2 and the highest in CFPAC-1 among the pancreatic cancer cell lines.
Example 4LMO7Verification of correlation with resistance to gemcitabine
To verifyLMO7Correlation with resistance to gemcitabine, the inventors performed wound healing assays and cross-well experiments. In experiments, the inventors observed the metastatic and invasive status of untreated pancreatic cancer cell line MiaPaca-2 as a blank (untracked) group, and pancreatic cancer cells in Dulbecco's Modified Eagle Medium (DMEM) (Weissent, montreal, QC, canada) 10% fetal bovine serum (fetal bovine serum), 100U/mL penicillin and 100. Mu.g/mL streptomycin were added and incubated in a 37℃humidified incubator containing 95% air and 5% carbon dioxide.
LMO7 over-expression vector (LMO 7-OE) and LMO7 empty vector (LMO 7-OE#NC) plasmids were purchased from China Peak Biotech company and were confirmed to be correct by sequencing.
LMO7 expression in pancreatic cancer cell lines was upregulated by liposome 3000 (Invitrogen, usa) using LMO7-OE plasmid (as shown in fig. 4). Cell counting kit-8 (CCK-8) is provided by Dojindo (Tokyo, japan).
The experiments were divided into three groups:
untreated group: miaPaca-2 was transfected with LMO7-OE and non-overexpressing cell line (NC) was used as a blank.
Sham treatment group (LMO 7-oe#nc+gemcitabine): miaPaca-2 was transfected with LMO7-OE #NC at 3X 10 per well 3 Is plated in 96-well cell culture plates and 100. Mu.L of medium. After 12h, medium containing solvent (DSMO) or gemcitabine (3. Mu. Mol, 6. Mu. Mol) at various concentrations was added.
True treatment group (LMO 7-oe+gemcitabine): transfection of MiaPaca-2 with LMO7-OE at 3X 10 per well 3 Is plated in 96-well cell culture plates and 100. Mu.L of medium. After 12h, medium containing solvent (DSMO) or gemcitabine (6. Mu. Mol, 12. Mu. Mol) at various concentrations was added.
1. Wound healing experiments
The inventors used wound healing experiments to evaluate the ability of pancreatic cancer cells to migrate. Different groups of cells were seeded in 6-well plates (5X 10) 5 /hole). After growing to 80% -90% confluence, single-layer cells were scraped with 200 μl pipette tips and the isolated cells were removed with PBS. Cells remaining in the 6-well plate were cultured with serum-free DMEM, and cell proliferation was inhibited. Scratch images were photographed with an inverted fluorescence microscope (japan) at 0h and 48h, respectively, and the relative areas of the wound surfaces were measured with ImageJ software. Each wound healing test was repeated 3 times and independently 3 times.
2. Hole crossing experiment
The inventors performed a trans well filter (BD bioscience pharmaceutical company) for a trans well experiment to determine cell migration and invasion capacity. In serum-free X10 4 After culturing in the medium of cells for 24 hours, 2.5X10 are grown 4 Individual cells were inoculated in 200 μl of serum-free medium. To the bottom chamber was added complete medium (500 μl). After incubation at 37 ℃ for 24 hours, cells above the cell membrane were rubbed with a cotton swab, cells passing through the cell membrane were collected, and stained with 0.5% crystal violet for 30min to evaluate the migration ability of the cells.
For cell invasion experiments Matrigel (BD biosciences pharmaceutical company) was added to cover the membrane of the Tanswell filter. The remainder of the experiment was identical to the wound healing experiment except that the incubation time was 48 h. 3 views were randomly picked per well and photographed and the migrated and affected cells were counted using cell counting kit-8 (CCK-8, dojindo) at 100 x magnification. All experiments were repeated 3 times.
As a result, as shown in FIG. 5, in the NC group, tumors grew with the lapse of time (0 to 48 h), and in the NC groupLMO7Upon overexpression, tumor cell lines were enhanced in migration and invasiveness (fig. 5A, 5D). After gemcitabine administration, the tumor still grew and drug resistance occurred, even though gemcitabine dose was increased, neither migration nor invasiveness of the tumor cell lines was reduced (fig. 5B, 5D). When (when)LMO7Overexpression was also used with gemcitabine, and tumors had more migration and invasion than the gemcitabine-alone treated group (fig. 5C, 5D). This further proves thatLMO7Overexpression results in resistance of PDACs to gemcitabine.
Example 5LMO7Relationship of expression level to poor prognosis
According toLMO7Expression levels of genes the inventors split samples of TCGA-PAAD into two groups according to the median of the expression levels and performed Kaplan-Meier survival analysis.
The results show that the data obtained from the above-mentioned method,LMO7high expression groups were significantly correlated with poor prognosis (FIG. 6), i.e.LMO7Patients with higher expression levels than median have a shorter survivalLMO7Expression levelPatients with a lower median survival period are relatively long.
EXAMPLE 6 inhibitionLMO7Relationship of expression level to gemcitabine resistance
Example 4 verification ofLMO7Overexpression may enhance gemcitabine resistance, as demonstrated by the inventors in this exampleLMO7Whether the reduced expression of (c) enhances the therapeutic effect of gemcitabine.
The inventors are directed toLM07The gene designs 3 kinds of siRNA, and the sequences are as follows:
siLM07#1：CTGGATGATTACTCCACAA
siLM07#2：CATCAAACCGTGCCTACAT
siLM07#3：CTTCGTAGCATCAGTTGAA
verified, the siLM07#3 pair is foundLM07The silencing effect of the gene was poor (as shown in FIG. 7).
The experiments were also divided into three groups, respectively:
untreated group: CFPAC-1 cell lines were subjected to silm07#1 and silm07#2LM07Silencing of genes CFPAC-1 cell line without gene silencing was used as a blank (NC).
Sham treatment group (silmo7#nc+gemcitabine): using the CFPAC-1 cell line without gene silencing, 3X 10 cells per well 3 Is plated in 96-well cell culture plates and 100. Mu.L of medium. After 12h, medium containing solvent (DSMO) or gemcitabine (0.2. Mu. Mol, 0.4. Mu. Mol) at various concentrations was added.
True treatment group (silmo7#1+gemcitabine): CFPAC-1 cell line with siLM07#1LM07Silencing of genes at 3X 10 per well 3 Is plated in 96-well cell culture plates and 100. Mu.L of medium. After 12h, medium containing solvent (DSMO) or gemcitabine (0.2. Mu. Mol, 0.4. Mu. Mol) at various concentrations was added.
Wound healing and cross-well experiments were performed using the same procedure as in example 4.
As a result, as shown in FIG. 8, it can be seen from the graph that in the untreated group (untracked), silencing was performed by using siLM07#1 and siLM07#2, relative to the NC groupLMO7GeneAfter that, the migration and invasion ability of the tumor were reduced (fig. 8A and 8D). If gene silencing is not performed, gemcitabine alone (0.2. Mu. Mol or 0.4. Mu. Mol) is used, migration and invasion capacity is not reduced (FIGS. 8B, 8D); in contrast, gemcitabine treatment (0.2. Mu. Mol or 0.4. Mu. Mol) while silencing the LMO7 gene significantly attenuated the migratory invasive capacity of the tumor (FIGS. 8C, 8D).
The results show that the LMO7 inhibitor can reduce the drug resistance of PDAC patients to gemcitabine and improve the treatment effect.
Example 7LMO7Relationship between expression level and afatinib sensitivity
The experiments were divided into two groups:
sham treatment group (LMO 7-oe#nc+afatinib): miaPaca-2 was transfected with LMO7-OE #NC at 3X 10 per well 3 Is plated in 96-well cell culture plates and 100. Mu.L of medium. After 12h, medium containing solvent (DSMO) or different concentrations of afatinib (4. Mu. Mol, 8. Mu. Mol) was added.
True treatment group (LMO 7-oe+afatinib): transfection of MiaPaca-2 with LMO7-OE at 3X 10 per well 3 Is plated in 96-well cell culture plates and 100. Mu.L of medium. After 12h, medium containing solvent (DSMO) or different concentrations of afatinib (2. Mu. Mol, 4. Mu. Mol) was added.
Wound healing and cross-well experiments were performed using the same procedure as in example 4.
As a result, as shown in fig. 9, similarly, in the MiaPaca-2 cell line, when afatinib alone was used, there was no significant decrease in tumor growth and migration and invasiveness with increasing afatinib dose (fig. 9A, fig. 9C). While whenLMO7The use of afatinib-treated cell lines for gene overexpression reduced migration and invasiveness (FIGS. 9B, 9C), demonstrating thatLMO7High gene expression may make PDAC cell lines sensitive to afatinib.
The results show that LMO7 is related to gemcitabine resistance, PDAC treatment by afatinib can be a better treatment scheme, the treatment effect and the safety are both remarkably improved, and prognosis is expected to be improved, so that the method has important clinical significance for treatment selection of PDAC patients.
All documents mentioned in this disclosure are incorporated by reference in this disclosure 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 application as defined in the appended claims.
Claims (10)
- Use of a detection reagent for lmo7 expression levels in the preparation of a kit for predicting whether a pancreatic cancer patient is resistant to gemcitabine.
- 2. The use according to claim 1, wherein the LMO7 expression level is the level of LMO7 gene transcript or LMO7 protein level.
- 3. The use according to claim 2, wherein the LMO7 expression level is the level of an LMO7 gene transcript and the detection reagent is a primer and/or probe capable of specifically binding to at least a portion of the sequence of the LMO7 gene transcript.
- 4. The use according to claim 3, wherein the detection reagent is an RNA sequencing reagent.
- 5. The use according to claim 1, wherein the LMO7 expression level is LMO7 protein level and the detection reagent is an antibody capable of specifically binding to the LMO7 protein.
- 6. The use according to claim 1, wherein the LMO7 expression level is detected prior to administration of gemcitabine to a patient with pancreatic cancer, and if the LMO7 expression level is above a predetermined threshold, the patient is predicted to develop resistance to gemcitabine, and afatinib is recommended as a first line therapeutic.
- 7. The use according to claim 1, wherein the LMO7 expression level is detected after administration of gemcitabine to a patient with pancreatic cancer, and if the LMO7 expression level is above a preset threshold, the patient is predicted to have developed resistance to gemcitabine, and afatinib is recommended as a second line therapeutic agent.
- 8. The use according to claim 6 or 7, wherein the preset threshold is determined from a representative value of LMO7 expression levels in a sample of non-pancreatic cancer patients.
- 9. The use according to claim 8, wherein the representative value is selected from one of a mean, a median, a mode, a maximum, a third quartile.
- 10. The use according to any one of claims 1 to 7, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114778844A (en) * | 2022-03-07 | 2022-07-22 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Use of PLD1 as molecular marker for evaluating sensitivity of tumor patient to chemotherapeutic drugs |
| CN116004631A (en) * | 2023-01-18 | 2023-04-25 | 中山大学 | siRNA for inhibiting P311 gene, composition and application thereof |
| CN116479130A (en) * | 2023-04-21 | 2023-07-25 | 中国药科大学 | Coiled-coil forming protein kinase ROCK1 as pancreatic cancer tumor marker and application thereof |
Family Cites Families (3)
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| BR112018075660A2 (en) * | 2016-06-13 | 2019-04-09 | Chemocentryx, Inc. | Pancreatic Cancer Treatment Methods |
| CN115418403A (en) * | 2022-08-24 | 2022-12-02 | 中国医学科学院北京协和医院 | Method and reagent for diagnosing pancreatic cystic tumor |
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Patent Citations (3)
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| CN114778844A (en) * | 2022-03-07 | 2022-07-22 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Use of PLD1 as molecular marker for evaluating sensitivity of tumor patient to chemotherapeutic drugs |
| CN116004631A (en) * | 2023-01-18 | 2023-04-25 | 中山大学 | siRNA for inhibiting P311 gene, composition and application thereof |
| CN116479130A (en) * | 2023-04-21 | 2023-07-25 | 中国药科大学 | Coiled-coil forming protein kinase ROCK1 as pancreatic cancer tumor marker and application thereof |
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