Application of LMO7 expression level detection reagent or LMO7 inhibitor related to pancreatic cancer

Abstract

The invention discloses application of an LMO7 expression level detection reagent or an LMO7 inhibitor related to pancreatic cancer, and belongs to the technical field of biological material detection. The expression level of LMO7 is utilized to predict or diagnose whether a pancreatic cancer patient has a metastasis risk, and a medicine for treating the pancreatic cancer patient is selected; the prognosis of the pancreatic cancer patient is predicted, or the sensitivity of the pancreatic cancer patient to afatinib is predicted. Furthermore, the LMO7 inhibitor is used for independently treating the pancreatic cancer or is combined with gemcitabine for treating the pancreatic cancer. An LMO7 expression level detection reagent or an LMO7 inhibitor is utilized to play an important clinical value in diagnosis or treatment of pancreatic cancer.

Description

[0001] Related patents

[0002] This application is a divisional application of the Chinese invention patent application with application number 2023110630139, application date August 23, 2023, and invention name “Application of LMO7 expression level in predicting gemcitabine resistance in pancreatic cancer”. Technical Field

[0003] The present invention belongs to the technical field of biomaterial detection, and in particular, relates to applications of an LMO7 expression level detection reagent or an LMO7 inhibitor related to pancreatic cancer. Background Art

[0004] Pancreatic cancer is a deadly malignancy, and approximately 85% of pancreatic cancer cases are characterized by pancreatic ductal adenocarcinoma (PDAC). The incidence and mortality of PDAC remain high at this stage, and treatment strategies are limited, with a low long-term survival rate.

[0005] Metastasis is the main factor for the high mortality of PDAC. Most patients are found to have metastasis when they are first diagnosed, and the 5-year survival rate of patients with distant metastasis is only 3%. In addition, only 10-20% of PDAC patients are resectable, and most of them will eventually experience local or distant recurrence after surgery and chemotherapy. However, the mechanism of PDAC metastasis is currently unclear, so metastatic PDAC urgently needs markers for early diagnosis.

[0006] Gemcitabine (GEM) is currently the first-line chemotherapy for pancreatic cancer patients, but the overall efficacy is less than 20%, mainly due to chemotherapy resistance. Therefore, drug resistance is also an important factor affecting the overall survival of pancreatic cancer patients, but there is no good program to predict gemcitabine resistance. Summary of the invention

[0007] In order to solve at least one of the above technical problems, the inventors used the Wilcoxon rank sum test to compare the significantly different genes between the sensitive group and the resistant group of gemcitabine-treated PDAC in the GSE140077 data set. Genes whose average gene expression value in the resistant group and the average gene expression value in the sensitive group were greater than 1 and whose p value was less than 0.05 were further screened out, and the metastatic potential characteristics of the genes were analyzed. It was unexpectedly found that the high expression level of the LMO7 gene was strongly correlated with gemcitabine resistance, thereby completing the present invention.

[0008] The present invention provides the use of a detection reagent for LMO7 expression level in preparing a kit for predicting whether a pancreatic cancer patient is resistant to gemcitabine.

[0009] Gemcitabine is a new cytosine nucleoside derivative with a chemical formula of C9H 11 F2N3O4. Like cytarabine, it is activated by deoxycytidine kinase and metabolized by cytosine deaminase after entering the human body. Gemcitabine is a pyrimidine antitumor drug with the same mechanism of action as cytarabine. Its main metabolites are incorporated into DNA in cells and mainly act in the G1 / S phase. However, the difference is that in addition to being incorporated into DNA, difluorodeoxycytidine can also inhibit ribonucleotide reductase, resulting in a decrease in intracellular deoxynucleoside triphosphates; another difference from cytarabine is that it can inhibit deoxycytidine deaminase to reduce the degradation of intracellular metabolites and has a self-enhancing effect. Clinically, gemcitabine is used as a first-line chemotherapy regimen for pancreatic cancer patients. However, pancreatic cancer patients will develop drug resistance after the administration of gemcitabine. Using the technical solution of the present invention, it is possible to predict whether pancreatic cancer patients are resistant to gemcitabine by detecting the expression level of LMO7, thereby achieving the purpose of precise treatment.

[0010] In some embodiments of the present invention, the LMO7 expression level is the level of LMO7 gene transcription product or the LMO7 protein level.

[0011] The protein encoded by the LIM domain 7 (LMO7) gene contains a calmodulin homology (CH) domain, a PDZ domain, and a LIM domain, and may be involved in protein-protein interactions.

[0012] In some specific embodiments of the present invention, the LMO7 expression level is the level of the LMO7 gene transcription product, and the detection reagent is a primer and / or probe that can specifically bind to at least a portion of the sequence of the LMO7 gene transcription product. In some embodiments of the present invention, the LMO7 gene transcription product is amplified using specific primers to obtain the LMO7 expression level.

[0013] In some preferred embodiments of the present invention, the detection reagent is an RNA sequencing reagent or a gene chip. The transcriptome represents all types of RNA present in cells, including mRNA, rRNA, tRNA, and other various non-coding RNAs. DNA microarrays (DNA Microarrays) technology, also known as gene chip (Genechip) sequencing technology and RNA sequencing technology (RNA sequencing, RNA-seq) are two main technologies in transcriptome analysis. Their main difference is that DNA microarrays are based on hybridization of pre-designed labeled probes with target cDNA sequences, while RNA-seq directly sequences cDNA chains through sequencing technology.

[0014] In other specific embodiments of the present invention, the LMO7 expression level is the LMO7 protein level, and the detection reagent is an antibody that can specifically bind to the LMO7 protein.

[0015] In some embodiments of the present invention, the expression level of LMO7 is detected before gemcitabine is administered to pancreatic cancer patients. If the expression level of LMO7 is higher than a preset threshold, it is predicted that the patient will develop resistance to gemcitabine, and afatinib is recommended as a first-line treatment drug.

[0016] In some embodiments of the present invention, the expression level of LMO7 is detected after gemcitabine is administered to pancreatic cancer patients. If the expression level of LMO7 is higher than a preset threshold, it is predicted that the patient has developed resistance to gemcitabine, and afatinib or other second-line treatment drugs are recommended.

[0017] In some specific embodiments of the present invention, the preset threshold is determined based on a representative value of the LMO7 expression level of non-pancreatic cancer patient samples in a population.

[0018] Furthermore, the representative value is selected from one of the mean, median, mode, maximum value, and third quartile.

[0019] In some embodiments of the invention, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0020] The present invention also discloses the use of a detection reagent for the expression level of LMO7 in the preparation of a kit for selecting a drug for treating pancreatic cancer patients. Specifically, the expression level of LMO7 is detected in pancreatic cancer patients, and if the expression level of LMO7 is higher than a preset threshold, afatinib is selected as a therapeutic drug, and if it is not higher than the preset threshold, gemcitabine is selected as a therapeutic drug.

[0021] The present invention also discloses the following technical solutions:

[0022] The use of a detection reagent for LMO7 expression level in the preparation of a kit for predicting or diagnosing whether a pancreatic cancer patient has a risk of metastasis. If the LMO7 expression level is higher than a preset threshold, the pancreatic cancer cells have a risk of migration and / or invasion, that is, the pancreatic cancer patient has a risk of metastasis.

[0023] The use of a detection reagent for LMO7 expression level in the preparation of a kit for predicting the prognosis of pancreatic cancer patients. If the LMO7 expression level is higher than a preset threshold, the pancreatic cancer patient has a poor prognosis.

[0024] The use of a detection reagent for the expression level of LMO7 in the preparation of a kit for predicting the sensitivity of pancreatic cancer patients to afatinib. If the expression level of LMO7 is higher than a preset threshold, the pancreatic cancer patient has a high sensitivity to afatinib.

[0025] All of the above applications with respect to further limitations or additional technical features regarding detection reagents and preset thresholds are the same as those in the application of the detection reagent for LMO7 expression level in the preparation of a kit for predicting whether a pancreatic cancer patient is resistant to gemcitabine.

[0026] Furthermore, the present invention also relates to:

[0027] Use of LMO7 inhibitors in the preparation of drugs for treating pancreatic cancer.

[0028] Use of LMO7 inhibitor in the preparation of a drug for treating pancreatic cancer in combination with gemcitabine.

[0029] A pharmaceutical composition comprising an LMO7 inhibitor and gemcitabine.

[0030] In the present invention, the LMO7 inhibitor includes but is not limited to substances capable of inhibiting the transcription level of LMO7 gene and / or the level of LMO7 protein, substances capable of inhibiting the level of LMO7 regulatory genes / proteins, and substances capable of inhibiting the function of LMO7 protein.

[0031] In some embodiments of the present invention, the LMO7 inhibitor is an siRNA capable of silencing the LMO7 gene, wherein the silencing refers to reducing the gene expression level or completely eliminating the gene expression. In some embodiments of the present invention, the siRNA capable of silencing the LMO7 gene is selected from at least one of the following:

[0032] siLM07#1:CTGGATGATTACTCCACAA

[0033] siLM07#2:CATCAAACCGTGCCTACAT

[0034] siLM07#3:CTTCGTAGCATCAGTTGAA

[0035] Preferably, at least one selected from siLM07#1 and siLM07#2.

[0036] Beneficial effects of the present invention

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The technical solution of the present invention uses the expression level of LMO7 to predict the resistance of pancreatic cancer patients to gemcitabine. When the expression level of LMO7 is high, afatinib is recommended, which can effectively improve the treatment effect and safety, and is expected to improve the prognosis. This has important clinical significance for the treatment selection of pancreatic cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The effect of LMO7 gene expression level on PDAC resistance to gemcitabine is shown. Figure 1 A: The box plot shows the expression level of LMO7 gene when gemcitabine was added at different times and doses. The horizontal axis shows the dose of gemcitabine treatment, and the vertical axis shows the expression level of LMO7 gene. Wilcoxon rank sum test was used to compare the difference between 24h and 6h treatment. The red dots are 24h treatment, the blue dots are 6h treatment, and the yellow dots are the untreated control group. ns. indicates p>0.05, ** indicates p<=0.01, and *** indicates p<=0.001. Figure 1 B: The box plot shows the LMO7 gene expression levels of sensitive and resistant cell lines in the GSE140077 data. The horizontal axis is the sensitive and resistant groups, and the vertical axis is the LMO7 gene expression level.

[0040] Figure 2 Shown is the expression of LMO7 in pancreatic cancer detected by immunohistochemistry. Figure 2 A: Pancreatic cancer tumor tissue; Figure 2 B: Neural invasion of pancreatic cancer.

[0041] Figure 3 The relative expression levels of LMO7 in different pancreatic cancer cell lines detected by Western Blot are shown. * indicates p<0.05, ** indicates p<=0.01, and *** indicates p<=0.001.

[0042] Figure 4 It shows that the LMO7 overexpression vector can significantly upregulate the expression of LMO7 in pancreatic cancer cell lines, *** indicates p<=0.001.

[0043] Figure 5 The correlation between LMO7 overexpression and gemcitabine resistance is shown. Wound healing assay and transwell assay showed the migration and invasion abilities of different groups. Figure 5 A: Results of wound healing test and cross-well test in the untreated group; Figure 5 B: Results of wound healing test and cross-well test in sham treatment group; Figure 5 C: Results of wound healing test and cross-well test in the real treatment group; Figure 5 D: Bar graph of wound healing test and cross-well test results of untreated group, sham treatment group and real treatment group. Unpaired t test, * indicates p < 0.05, ** indicates p < = 0.01, *** indicates p < = 0.001.

[0044] Figure 6Survival analysis of TCGA PAAD samples grouped by LMO7 gene expression level is shown. The dotted 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 was statistically significant (p<0.001).

[0045] Figure 7 The silencing effects of three different siRNAs on LMO7 are shown.

[0046] Figure 8 The correlation between LMO7 silencing and gemcitabine resistance is shown. Wound healing assay and transwell assay showed the migration and invasion abilities of different groups. Figure 8 A: Results of wound healing test and cross-well test in the untreated group; Figure 8 B: Results of wound healing test and cross-well test in sham treatment group; Figure 8 C: Results of wound healing test and cross-well test in the real treatment group; Figure 8 D: Bar graph of wound healing test and cross-well test results of untreated group, sham treatment group and real treatment group. Unpaired t test, * indicates p < 0.05, ** indicates p < = 0.01, *** indicates p < = 0.001.

[0047] Fig. 9 The correlation between LMO7 and sensitivity to afatinib is shown. Wound healing assay and transwell assay showed the migration and invasion abilities of different groups. Fig. 9 A: Results of wound healing test and cross-well test in sham treatment group; Fig. 9 B: Results of wound healing test and cross-well test in the real treatment group; Fig. 9 C: Bar graph of wound healing test and cross-well test results of sham treatment group and real treatment group. Unpaired t-test, * indicates p < 0.05, ** indicates p < = 0.01, *** indicates p < = 0.001. DETAILED DESCRIPTION

[0048] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0049] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include numerical values ​​outside the range. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range comprising a numerical value less than 1 or a range comprising a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered as 0.0001, 0.001, 0.01 or 0.1. For a range comprising a single digit less than 10 (e.g., 1 to 5), 1 unit is generally considered as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of numerical values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0050] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0051] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0052] Example

[0053] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0054] 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, and the disclosure and materials cited therein are hereby incorporated by reference.

[0055] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0056] The molecular biology experimental methods not specifically described in the following examples are all carried out in accordance with the specific methods listed in the book Molecular Cloning Experiment Guide (4th Edition) (J. Sambrook, MR Green, 2017), or in accordance with the kits and product instructions. Other experimental methods are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0057] Example 1 Discovery of genes associated with gemcitabine resistance

[0058] The inventors used the Wilcoxon rank sum test to compare the significantly different genes between the sensitive group and the resistant group of PDAC (pancreatic ductal adenocarcinoma) treated with gemcitabine in the GSE140077 dataset. The genes whose average gene expression value in the resistant group was greater than 1 and whose p value was less than 0.05 were further screened out, and the metastatic potential characteristics of the genes were analyzed. Surprisingly, it was found that the high expression of the LMO7 gene was strongly correlated with gemcitabine resistance.

[0059] Furthermore, the inventors observed the expression results of LMO7 in the GSE105083 dataset and unexpectedly found that with the increase of gemcitabine concentration, the expression level of LMO7 in the group treated for 24 hours gradually increased compared with the untreated group ( Figure 1 ). In particular, when the gemcitabine concentration was 30nM, 100nM, 300nM, 3μmol and 10μmol, the expression of LMO7 in the group treated for 24h was significantly increased, while in contrast, the expression of LMO7 in the group treated for 6h was not significantly increased ( Figure 1 A).

[0060] Figure 1 B shows that there was a significant difference in the expression level of LMO7 between the gemcitabine-sensitive group and the gemcitabine-resistant group (p<0.01), indicating that LMO7 is associated with the resistance of PDAC to gemcitabine.

[0061] Example 2 Immunohistochemical Detection of Pancreatic Cancer Tumor Tissue

[0062] 1. Sample acquisition

[0063] Human pancreatic cancer tumor tissue was aseptically isolated from surgical operations of pancreatic cancer patients, prepared into paraffin-embedded blocks, cut into 4-5 μmol thick tissue sections, and attached to glass slides to prepare tissue sections that can be used for immunohistochemical staining.

[0064] 2. Immunohistochemistry

[0065] LMO7 antibody (brand: Sigma, catalog number: HPA020923) was purchased and routine immunohistochemical staining was performed using the antibody at a dilution ratio of 1:200. Figure 2 As shown, LMO7 expression in tumor cells of pancreatic cancer tissue is strong ( Figure 2 A); LMO7 is strongly expressed in the neural infiltration foci of pancreatic cancer ( Figure 2 B).

[0066] 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 Model and Characteristic Experimental Cell Resource Bank (Cell Bank of the Chinese Academy of Sciences).

[0069] The human healthy pancreatic ductal cell line hTERT-HPNE (HPNE) was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA), and the HPNE cell line was cultured according to the method recommended by ATCC.

[0070] According to the instructions of the whole protein extraction kit (Pierce Chemical), the whole protein of pancreatic cancer tumor tissue was extracted, and the loading buffer was added to boil and denatured for 5 minutes after quantification by BCA method; 40 μg of total protein was subjected to 8-10% SDS-PAGE gel electrophoresis, and then electrotransferred to PVDF membrane, blocked with 5% skim milk for 2 hours; incubated with anti-LMO7 antibody at a dilution ratio of 1:1000 at 4°C overnight, incubated with secondary antibody at a dilution ratio of 1:5000 at room temperature for 2 hours, and washed with PBST 3 times, each time for 10 minutes. Scanned by gel image analysis system, GAPDH was used as internal reference, and the ratio of LMO7 to internal reference gray value was calculated for semi-quantitative detection.

[0071] The results are as follows Figure 3 As shown, LMO7 is highly expressed in human pancreatic cancer cell lines and lowly expressed in human healthy pancreatic ductal cell lines. Moreover, among pancreatic cancer cell lines, the expression level is lowest in MiaPaca-2 and highest in CFPAC-1.

[0072] Example 4 Verification of the correlation between LMO7 and gemcitabine resistance

[0073] To verify the correlation between LMO7 and gemcitabine resistance, the inventors conducted wound healing assays and transwell experiments. In the experiment, the inventors used untreated pancreatic cancer cell line MiaPaca-2 as a blank control group (untreated) to observe its metastasis and invasion status. Pancreatic cancer cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Vicente, Montreal, QC, Canada), supplemented with 10% fetal bovine serum (FBS), 100U / mL penicillin and 100μg / mL streptomycin, and cultured in a 37°C humidified incubator containing 95% air and 5% carbon dioxide.

[0074] The LMO7 overexpression vector (LMO7-OE) and LMO7 empty vector (LMO7-OE#NC) plasmids were purchased from Fenghui Biotechnology Co., Ltd., China, and the correctness was verified by sequencing.

[0075] The expression of LMO7 in pancreatic cancer cell lines was upregulated by Lipofectamine 3000 (Invitrogen, USA) using LMO7-OE plasmid. Figure 4 Cell counting kit-8 (CCK-8) was provided by Dojindo (Tokyo, Japan).

[0076] The experiment was divided into three groups:

[0077] Untreated group: MiaPaca-2 was transfected with LMO7-OE, and non-overexpressing cell line (NC) was used as blank control.

[0078] Sham treatment group (LMO7-OE#NC+gemcitabine): MiaPaca-2 cells were transfected with LMO7-OE#NC at 3×10 3 The cells were seeded in 96-well cell culture plates at a density of 100 μL of culture medium. After 12 h, the medium containing solvent (DSMO) or different concentrations of gemcitabine (3 μmol, 6 μmol) was added.

[0079] Treatment group (LMO7-OE + gemcitabine): MiaPaca-2 cells were transfected with LMO7-OE at 3×10 3 The cells were seeded in 96-well cell culture plates at a density of 100 μL of culture medium. After 12 h, the medium containing solvent (DSMO) or different concentrations of gemcitabine (6 μmol, 12 μmol) was added.

[0080] 1. Wound healing assay

[0081] The inventors used wound healing assay to evaluate the migration ability of pancreatic cancer cells. Different groups of cells were inoculated in 6-well plates (5×105 / well). After growing to 80% to 90% confluence, the monolayer cells were scraped with a 200 μL pipette tip, and the separated cells were removed with PBS. The cells remaining in the 6-well plate were cultured with serum-free DMEM, and cell proliferation was inhibited. At 0h and 48h, scratch images were taken with an inverted fluorescence microscope (Japan), and the relative area of ​​the wound was measured using ImageJ software. Each wound healing experiment was repeated 3 times and repeated 3 times independently.

[0082] 2. Transwell Experiment

[0083] The inventors used Transwell filters (BD Biosciences Pharmaceuticals) to perform transwell assays to determine cell migration and invasion abilities. 4 After culturing the cells in the culture medium for 24 h, 2.5×10 4 Cells were inoculated in 200 μL serum-free medium. Complete medium (500 μL) was added to the bottom chamber. After incubation at 37°C for 24 h, cells above the cell membrane were wiped with a cotton swab, and cells that passed through the cell membrane were collected and stained with 0.5% crystal violet for 30 min to evaluate the migration ability of the cells.

[0084] For the cell invasion assay, Matrigel (BD Biosciences) was added to cover the membrane of the Tanswell filter. The rest of the experiment was the same as the wound healing assay except that the incubation time was 48 h. Three fields of view were randomly selected from each well to take pictures, and the migrating and invading cells were counted using a cell counting kit-8 (CCK-8, Dojindo) at 100× magnification. All experiments were repeated 3 times.

[0085] The results are as follows Figure 5 As shown in Figure 2, in the NC group, tumors grew over time (0-48 h), and when LMO7 was overexpressed, the migration and invasiveness of tumor cell lines were enhanced ( Figure 5 A. Figure 5 D). After the use of gemcitabine, the tumor continued to grow and developed drug resistance. Even when the gemcitabine dose was increased, the migration and invasiveness of the tumor cell line were not weakened ( Figure 5 B. Figure 5 D). When LMO7 overexpression was used together with gemcitabine, the tumors underwent more migration and invasion compared to the group treated with gemcitabine alone ( Figure 5 C. Figure 5 D). This further demonstrates that LMO7 overexpression can lead to gemcitabine resistance in PDAC.

[0086] Example 5 Relationship between LMO7 expression level and poor prognosis

[0087] According to the expression level of the LMO7 gene, the inventors divided the TCGA-PAAD samples into two groups according to the median of the expression level and performed Kaplan-Meier survival analysis.

[0088] The results showed that the high expression of LMO7 was significantly associated with poor prognosis ( Figure 6 ), that is, patients with LMO7 expression levels higher than the median had a shorter survival period, while patients with LMO7 expression levels lower than the median had a relatively longer survival period.

[0089] Example 6 Relationship between inhibition of LMO7 expression level and gemcitabine resistance

[0090] Example 4 verified that overexpression of LMO7 can enhance gemcitabine resistance. In this example, the inventors verified whether reduced expression of LMO7 can enhance the therapeutic effect of gemcitabine.

[0091] The inventors designed three siRNAs for the LM07 gene, and their sequences are as follows:

[0092] siLM07#1:CTGGATGATTACTCCACAA

[0093] siLM07#2:CATCAAACCGTGCCTACAT

[0094] siLM07#3:CTTCGTAGCATCAGTTGAA

[0095] After verification, it was found that siLM07#3 had a poor silencing effect on the LM07 gene (e.g. Figure 7 shown).

[0096] The experiment was also divided into three groups:

[0097] Untreated group: siLM07#1 and siLM07#2 silenced the LM07 gene in the CFPAC-1 cell line, and the CFPAC-1 cell line without gene silencing was used as a blank control (NC).

[0098] Sham treatment group (siLMO7#NC+gemcitabine): CFPAC-1 cells without gene silencing were used, with 3×10 3 The cells were seeded in 96-well cell culture plates at a density of 100 μL of culture medium. After 12 h, the medium containing solvent (DSMO) or different concentrations of gemcitabine (0.2 μmol, 0.4 μmol) was added.

[0099] Treatment group (siLMO7#1+gemcitabine): siLM07#1 was used to silence the LM07 gene in the CFPAC-1 cell line, with 3×103 The cells were seeded in 96-well cell culture plates at a density of 100 μL of culture medium. After 12 h, the medium containing solvent (DSMO) or different concentrations of gemcitabine (0.2 μmol, 0.4 μmol) was added.

[0100] The wound healing test and transwell test were carried out in the same manner as in Example 4.

[0101] The results are as follows Figure 8 As shown in the figure, it can be seen that in the untreated group, compared with the NC group, after silencing the LMO7 gene using siLM07#1 and siLM07#2, the migration and invasion abilities of the tumor were weakened ( Figure 8 A. Figure 8 D). Without gene silencing, only gemcitabine (0.2 μmol or 0.4 μmol) was used for treatment, and the migration and invasion abilities were not weakened ( Figure 8 B. Figure 8 D); On the contrary, when LMO7 gene was silenced and gemcitabine was used for treatment (0.2 μmol or 0.4 μmol), the migration and invasion ability of tumors was significantly weakened ( Figure 8 C. Figure 8 D).

[0102] The above results indicate that the use of LMO7 inhibitors can reduce the resistance of PDAC patients to gemcitabine and improve the treatment effect.

[0103] Example 7 Relationship between LMO7 expression level and afatinib sensitivity

[0104] The experiment was divided into two groups:

[0105] Sham treatment group (LMO7-OE#NC+afatinib): MiaPaca-2 cells were transfected with LMO7-OE#NC at 3×10 3 The cells were seeded in 96-well cell culture plates at a density of 100 μL of culture medium. After 12 h, medium containing solvent (DSMO) or different concentrations of afatinib (4 μmol, 8 μmol) was added.

[0106] Treatment group (LMO7-OE + afatinib): MiaPaca-2 cells were transfected with LMO7-OE at a concentration of 3 × 10 3 The cells were seeded in 96-well cell culture plates at a density of 100 μL of culture medium. After 12 h, medium containing solvent (DSMO) or different concentrations of afatinib (2 μmol, 4 μmol) was added.

[0107] The wound healing test and transwell test were carried out in the same manner as in Example 4.

[0108] The results are as follows Fig. 9 Similarly, in the MiaPaca-2 cell line, when afatinib alone was used, there was no significant decrease in tumor growth, migration, and invasiveness with increasing afatinib doses ( Fig. 9 A. Fig. 9 C). When LMO7 gene was overexpressed, the cell lines treated with afatinib weakened their migration and invasion ( Fig. 9 B. Fig. 9 C), indicating that high expression of the LMO7 gene makes PDAC cell lines sensitive to afatinib.

[0109] The above results indicate that LMO7 is associated with gemcitabine resistance, and the use of afatinib to treat PDAC may be a better treatment option, with significantly improved therapeutic efficacy and safety, and is expected to improve prognosis, which has important clinical significance for the treatment options of PDAC patients.

[0110] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Application of the LMO7 expression level detection reagent in the preparation of any of the following kits: A kit for predicting or diagnosing whether a pancreatic cancer patient has a risk of metastasis; A kit for selecting a drug for treating a patient with pancreatic cancer; a kit for predicting the prognosis of patients with pancreatic cancer; A test kit for predicting sensitivity to afatinib in patients with pancreatic cancer.

2. The use according to claim 1, characterized in that: The LMO7 expression level refers to the level of LMO7 gene transcription product or LMO7 protein level.

3. The use according to claim 2, characterized in that: The LMO7 expression level is the level of the LMO7 gene transcription product, and the detection reagent is a primer and / or a probe that can specifically bind to at least a portion of the sequence of the LMO7 gene transcription product.

4. The use according to claim 3, characterized in that: The detection reagent is an RNA sequencing reagent.

5. The use according to claim 1, characterized in that: The LMO7 expression level is the LMO7 protein level, and the detection reagent is an antibody that can specifically bind to the LMO7 protein.

6. The use according to claim 1, characterized in that: For a kit for predicting or diagnosing whether a pancreatic cancer patient has a risk of metastasis, if the expression level of LMO7 is higher than a preset threshold, the pancreatic cancer patient has a risk of metastasis; For a kit for selecting a drug for treating a pancreatic cancer patient, the expression level of LMO7 is detected in the pancreatic cancer patient, and if the expression level of LMO7 is higher than a preset threshold, afatinib is selected as the treatment drug, and if it is not higher than the preset threshold, gemcitabine is selected as the treatment drug; For a kit for predicting the prognosis of a pancreatic cancer patient, if the LMO7 expression level is higher than a preset threshold, the pancreatic cancer patient has a poor prognosis; For the kit used to predict the sensitivity of pancreatic cancer patients to afatinib, if the LMO7 expression level is higher than the preset threshold, the pancreatic cancer patient has a higher sensitivity to afatinib.

7. The use according to claim 6, characterized in that: The preset threshold is determined based on the representative value of the LMO7 expression level of non-pancreatic cancer patient samples in the population.

8. The use according to claim 8, characterized in that: The representative value is selected from one of the mean, median, mode, maximum value, and third quartile.

9. Use of an LMO7 inhibitor in the preparation of a medicament for treating pancreatic cancer alone or in combination with gemcitabine.

10. A pharmaceutical composition comprising an LMO7 inhibitor and gemcitabine.

Images