CN113855675A

CN113855675A - Kit and medicine based on gallbladder cancer marker

Info

Publication number: CN113855675A
Application number: CN202110905119.3A
Authority: CN
Inventors: 薛晨; 何玉婷
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2021-12-31
Also published as: CN108931633A; CN108931633B

Abstract

The invention discloses a marker PIM1 for diagnosing and prognosticating gallbladder cancer, and also discloses a kit for diagnosing and prognosticating gallbladder cancer, which can be used for diagnosing, predicting, detecting or screening human gallbladder cancer cell spreading, clinical gallbladder cancer stage or gallbladder cancer patient prognosis. The invention also discloses application of the PIM1 protein inhibitor in preparing anti-gallbladder cancer drugs. The invention shows that the PIM1 has very important significance in clinical practice, can possibly become a new generation of tumor biological diagnosis marker and a molecular target for tumor treatment, opens up the research of a brand new target of gallbladder cancer diseases mainly based on PIM1, and also opens up a new way for developing new anti-tumor drugs.

Description

Kit and medicine based on gallbladder cancer marker

Technical Field

The invention relates to the technical field of A61P 35/00.

Background

Gallbladder cancer (GBC) is the most common form of biliary malignancy, accounting for 80% -95% of biliary malignancies. In the past decades, gallbladder cancer treatment strategies have not made substantial progress, and due to the lack of typical symptoms, the early diagnosis rate is only 19.1%, and most patients are already in the middle and late stages when they are clearly diagnosed. Surgical resection remains the mainstay of treatment for patients with gallbladder cancer to date, however surgical resection is not suitable for patients with advanced stage cancer. Gallbladder cancer is not sensitive to radiotherapy, chemotherapy and other treatment measures, so the overall prognosis is very poor, and foreign epidemiological studies show that the 5-year survival rate of the gallbladder cancer after operation is only 5 percent. In addition, the tendency to relapse or metastasize after surgery largely determines the poor prognosis of advanced gallbladder cancer. It has been reported that the occurrence of gallbladder cancer is caused by complicated genetic and environmental factors, however, the molecular mechanism of gallbladder cancer progression is not clear. Therefore, it is crucial to explore biomarkers and effective treatment strategies for early detection of confirmed gallbladder cancer.

The expression level and functional role of PIM family, including PIM1, PIM2 and PIM3, PIM1 in gallbladder cancer are not clear, which requires much research.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art.

In order to explore the expression level of PIM1 in gallbladder cancer, a tissue chip technology and an immunohistochemical technology are adopted to explore the expression level of PIM1 protein in gallbladder cancer tissues and adjacent paracarcinoma tissue specimens, and the results are further verified in the gallbladder cancer tissues and gallbladder cancer cells by a western blot technology. Investigation of expression of PIM1 at the protein level showed that PIM1 expression was significantly higher in gallbladder cancer than in paracarcinoma tissues.

In order to further explore the relationship between the expression of the PIM1 and the clinical characteristics and prognosis of the gallbladder cancer, the inventor proves that the expression level of the PIM1 in a sample with the TNM stage III-IV is obviously higher than that of a sample with the stage I-II; the inventor also proves that the expression level of the PIM1 in a sample with tumor distant metastasis is obviously higher than that in a sample without tumor distant metastasis, and the invention proves that the PIM1 molecule can be used for preparing, predicting and screening a kit for human gallbladder cancer spread and tumor clinical staging.

The inventor also carries out Kaplan-Meier survival analysis, and the result shows that the survival time of the patient with the high expression group of the PIM1 gene is obviously shorter than that of the patient with the low expression group of the PIM1 gene, and the increase of the expression of the PIM1 during the disease period has close correlation with the clinical prognosis of the gallbladder cancer. The expression of the PIM1 can be used as a reference judgment index of gallbladder cancer prognosis survival time and a prognosis survival risk prediction reference.

In order to further explore the role of the PIM1 gene in the occurrence and development of gallbladder cancer, the inventor adopts PIM1 interfering RNA and a PIM1 specific inhibitor SGI-1776 to respectively inhibit the expression of PIM1 in gallbladder cancer cells and analyze the influence on the proliferation, migration, invasion and apoptosis of the gallbladder cancer cells, and proves that the expression of PIM1 is inhibited, the proliferation, migration and invasion capacities of the gallbladder cancer cells are obviously reduced, the apoptosis is obviously increased, and a gallbladder cancer marker PIM1 can also be used as a molecular target for screening and preparing a medicament for diagnosing, relieving or treating the gallbladder cancer.

The invention also applies GBC-SD cells of PIM1 knocked out by lentivirus to be inoculated subcutaneously of nude mice, which proves that the influence of PIM1 expression on the in-vivo tumor forming capability of the gallbladder cancer cells is inhibited, the inventor finds that compared with a control group without knocking out, the tumor volume and the fluorescence expression of an experimental group with PIM1 knocked out are obviously reduced, and the invention proves that the gallbladder cancer marker PIM1 can also be used as a molecular target for screening and preparing medicaments for diagnosing, relieving or treating the gallbladder cancer. In a first aspect, the invention discloses a human PIM1 protein, and an application of the protein as a marker for diagnosing and/or prognosing gallbladder cancer.

In a second aspect, the invention discloses the use of human PIM1 protein for the preparation of a kit for the diagnosis and/or prognosis of gallbladder cancer; correspondingly, the invention provides a kit, which comprises an antibody of the anti-human PIM1 protein and is used for diagnosing and/or prognosing gallbladder cancer; the antibody may be a monoclonal or polyclonal antibody. The kit can be used for diagnosing, predicting, detecting or screening human gallbladder cancer cell spreading, gallbladder cancer clinical stage or gallbladder cancer patient prognosis.

In a third aspect, the invention provides a use of an inhibitor of PIM1 protein, which is SGI-1776, a pharmaceutically acceptable salt thereof, or PIM 1-specific interfering RNA, in the preparation of a medicament for treating gallbladder cancer. Further, the medicament is for alleviating or treating gallbladder cancer, or for improving the prognosis of a patient with gallbladder cancer.

It is noted that the stage of the gallbladder cancer TNM according to any of aspects 1-3 may be stages III-IV (preferred) or stages I-II.

The invention shows that the PIM1 has very important significance in clinical practice, and can become a new generation of tumor biological diagnosis marker and a molecular target for tumor treatment; especially, the research on the brand-new target of gallbladder cancer mainly based on PIM1 is developed, and a new way for developing new anti-tumor drugs is opened up.

Drawings

FIG. 1.1 is a Western blot in which PIM1 corresponds to expression levels of PIM1 protein in gallbladder cancer tissue;

FIG. 1.2 is a Western blot in which PIM1 corresponds to expression levels of PIM1 protein in four gallbladder cancer cell lines.

FIG. 2.1 shows statistical analysis of the differences in expression of PIM1 protein in gallbladder and paracarcinoma tissues by tissue chip technique and immunohistochemical technique, which we found: PIM1 was expressed at significantly higher levels in gallbladder cancer samples than in paracancerous samples;

fig. 2.2A is a correlation analysis of expression levels of PIM1 with TNM staging, and we found that: PIM1 is expressed in gallbladder cancer samples with TNM stages III-IV and is significantly higher than the gallbladder cancer samples with I-II;

fig. 2.2B shows a statistical analysis of the correlation of PIM1 expression levels with distant metastasis of gallbladder cancer by histochip and immunohistochemical techniques, which we found: PIM1 was expressed at significantly higher levels in the pooled metastatic gallbladder cancer samples than in the non-pooled and metastatic gallbladder cancer samples;

fig. 2.3 is a correlation analysis between PIM1 expression level and overall patient survival, and Kaplan-Meier survival analysis results show that the overall survival time of patients with high PIM1 expression group is significantly shorter than that of patients with low PIM1 expression group, i.e. high PIM1 expression suggests a poorer clinical prognosis.

FIG. 3.1A is a graph showing the comparison of proliferation of gallbladder cancer cells in experiment 3.1GBC-SD, and FIG. 3.1B is a graph showing the comparison of proliferation of gallbladder cancer cells in experiment 3.1 NOZ; FIG. 3.2 is a graph comparing the migration of gallbladder cancer cells in experiment 3.2; FIG. 3.3 is a comparison of the invasion of gallbladder cancer cells in experiment 3.3; in each figure, the experimental group uses interfering RNA to inhibit the expression of PIM1, and the control group uses gallbladder cancer cells without interfering RNA, and the results show that: after the expression of the PIM1 is down-regulated, the proliferation, migration and invasion capacities of the cells are obviously weakened.

Fig. 4 is a graph of the variation of the normalized photon flux of the experimental and control groups of PIM1, which studies the effect of PIM1 on the tumorigenic capacity of the nude mice after being down-regulated, and shows that: the experimental group with the PIM1 knockout had significantly lower tumor volume than the control group.

Detailed Description

The invention is explained below by means of a few exemplary experiments, to which the invention is not restricted.

Western blot detection of expression of PIM1 protein in gallbladder cancer tissues and cells

Experiment 1.1:

adding tissue protein lysate into 9 cases of gallbladder cancer tissues preserved by liquid nitrogen, fully grinding on ice by using an electric homogenizer, placing on ice for shaking table reaction for 1h, centrifuging at 4 ℃ at 10000r/min for 15min, and taking supernatant. And detecting the concentration of the protein by using a BCA method microplate reader, adjusting the protein to be consistent, adding the buffer solution, and boiling for 5min at 100 ℃. Protein samples were each added to a preformed 12% polyacrylamide gel lane and electrophoresed at 110V for 90 min. After protein electrophoresis is finished, the membrane is electrically transferred to a nitrocellulose membrane, the membrane is placed in PBST solution containing 5% BSA for sealing for 1h, primary antibody is added in an environment at 4 ℃ for incubation overnight, after rewarming, fluorescent secondary antibody is incubated for 2h, then the optical density of an antibody specific strip is analyzed by using a luminescence image analyzer, and the relative protein expression level is subjected to normalized analysis by comparing the optical density of PIM1 protein/beta-actin.

As a result, it was found that: in 6 cases of gallbladder cancer tissues, significant expression of PIM1 was detected, as shown in FIG. 1.1.

Experiment 1.2:

referring to experiment 1.1, the expression level of PIM1 protein in gallbladder cancer cells from cell lines GBC-SD, NOZ, OCGU1 and SGC-996 was analyzed by Westernblot, and the results suggest that PIM1 is highly expressed in all of the four cell lines, and the expression in GBC-SD and NOZ of the gallbladder cancer cells is higher than that in SGC-996 and OCGU1 cells, as shown in FIG. 1.2.

Second, chip experiment

Immunohistochemical analysis of PIM1 protein expression in gallbladder cancer tissue and paracarcinoma tissue

Constructing a gallbladder cancer tumor tissue chip, wherein the tissue sample source is the first affiliated hospital of Zhengzhou university, the gallbladder cancer tumor tissue chip comprises 52 gallbladder cancer tissues and 27 adjacent cancer tissues, and clinical information comprises sex, age, TNM stage, tumor size, grade, follow-up information and the like; the diameter of the chip sample point is 1.5mm, and the fixing mode is formalin fixation. Secondly, a commercialized tissue chip (Shanghai core Biotechnology company) is used at the same time, wherein the commercialized tissue chip comprises 79 gallbladder cancer tissues and 20 cancer collateral tissues, and clinical information comprises sex, age, TNM stage, tumor size, grade, follow-up information and the like; the diameter of the chip sample point is 1.5mm, and the fixing mode is formalin fixation.

Immunohistochemistry main operation steps:

baking the tissue chip, dewaxing, hydrating, repairing antigen, sealing, adding rabbit anti-human PIM1 polyclonal antibody (diluted 1: 100), and incubating at 4 deg.C overnight; adding HRP-labeled goat anti-rabbit IgG working solution dropwise, incubating at 37 ℃ for 60min, incubating at 37 ℃ for 30min with SABC reagent, and then developing with DAB; performing hematoxylin counterstaining; xylene transparent, neutral gum mounting. Panoramic scanning is carried out by using a tissue chip scanner, 2 senior pathological doctors respectively and independently score, and then the scoring result is unified through discussion. Five grades were assigned according to the degree of staining, with 1 score indicating almost no staining; score 2 indicates weak staining; score 3 indicates moderate staining; score 4 indicates strong dyeing and score 5 very strong dyeing. Meanwhile,

scores

1, 2 and 3 were defined as low expression, and scores 4 and 5 were regarded as high expression for statistical analysis.

Statistical treatment:

the mapping software used Graph pad Prism5, the photo finishing used Adobe Illustrator CS6, and the statistical analysis used SPSS 23.0. Differences in clinical characteristics from PIM1 expression levels were tested using the chi-square test; single-factor regression analysis and multi-factor regression analysis are adopted to explore independent risk factors of the gallbladder cancer; correlation of PIM1 expression levels with overall survival time of gallbladder cancer patients Kaplan-Meier survival analysis was used. The statistical analysis result P <0.05, the difference was considered statistically significant.

2.1: the staining result shows that the PIM1 subcellular localization is mainly distributed in cytoplasm and cell membrane, positive cells are obviously brownish yellow or brownish brown particles, and negative cells are light-colored particles or colorless; the expression level of PIM1 protein in gallbladder cancer tissue is high (positive rate is 75.8%), while the expression level in the majority of paracarcinoma tissues is low (positive rate is 53.2%), and the difference has statistical significance (P <0.05) compared between two groups, as shown in figure 2.1.

2.2 expression levels of PIM1 in relation to clinical characteristics

Clinical characteristics of follow-up patients and expression levels of PIM1 are analyzed by chi-square test, and the results show that the age, sex and tumor size of the patients have no statistical significance with the expression difference of PIM1, and TNM stage and distant metastasis of tumors have statistical significance with the expression difference of PIM 1.

2.2.1 correlation of expression levels of PIM1 with TNM staging

The expression level of PIM1 was significantly higher in patients in stages III to IV of TNM than in patients in stages I to II, the difference was statistically significant (P <0.05), and was plotted using Graph pad Prism5, see FIG. 2.2A.

Fig. 2.2A is a correlation analysis of expression levels of PIM1 with TNM staging.

2.2.2 correlation of expression levels of PIM1 with the occurrence of distant metastasis

The level of expression of PIM1 was significantly higher in patients with distant metastasis than in patients without distant metastasis, and the difference was statistically significant (P <0.05), see fig. 2.2B.

2.3 correlation between clinical characteristics and patient Overall survival

The single-factor regression analysis of 66 patients with complete follow-up data shows that the age, the sex and the tumor size have no significant correlation with the overall survival rate of the patients (P >0.05), the TNM stage and the distant metastasis of the tumor and the expression of PIM1 have significant correlation with the overall survival rate of the patients (P <0.05), and the single-factor regression analysis is a potential risk factor; further, multifactorial regression analysis proves that the high-level expression of PIM1 has a significant correlation with the overall survival rate of the patient and is an independent risk factor of the overall survival rate of the gallbladder cancer.

The correlation between PIM1 expression levels and overall patient survival is shown in figure 2.3: the overall survival of patients with high expression of PIM1 was significantly lower than that of patients with low expression of PIM1, with the difference statistically significant (P < 0.05).

Third, in vitro cell experiment

Taking cancer cells from two cell lines of gallbladder cancer GBC-SD and NOZ as an example, an experimental group uses PIM1 specific interfering RNA to inhibit the expression of PIM1 in the gallbladder cancer cells, the sense strand sequence of the used interfering RNA is GAUAUGGUGUGUGGAGAUAtt from 5 'to 3', the antisense strand is UAUCUCCACACACCAUAUCtt (shown as a sequence 1 after tt is removed), a control group corresponds to normal cancer cells which are not treated by the interfering RNA, and the influence on the proliferation, migration and invasion of the gallbladder cancer cells is analyzed; the result shows that after the expression of the PIM1 is down-regulated, the proliferation, migration and invasion capacities of the cells are obviously weakened, and the details are as follows:

experiment 3.1: CCK-8 measures cell proliferation.

After transfection screening, cells in the logarithmic growth phase were digested, and cell density was adjusted to the appropriate concentration after cell counting. Inoculating the cell suspension into a 96-well plate at a volume of 200 ul/well, and culturing at 37 ℃ in a 5% CO2 incubator; under the condition of keeping out of the light, adding the CCK8 solution into the 96-well plate at the volume of 10 ul/well, incubating for 2h in an incubator, setting the wavelength on an enzyme-linked immunosorbent assay (ELISA) detector to be 450nm, and detecting the absorbance (A450) value of each well. Set zero setting hole and contrast hole simultaneously, every group sets for 3 compound holes. The mean and standard deviation of the two groups were analyzed separately, and the differences were statistically significant (P < 0.05).

The results of the absorbance experiments are shown in FIG. 3.1A/3.1B.

Experiment 3.2: cell scratch detects cell migration.

The cell processing method is shown in the experiment, 2 ml/hole of cell suspension is added into a 6-hole plate, when the confluence degree of cells of each hole reaches 80-90%, a Tip gun head is used for drawing a straight line in the hole to form scratches among single-layer cells; the cells exfoliated during streaking were then washed with sterile PBS until no exfoliated cells were present. According to the time point designed in advance, the 6-well plate is placed under a microscope for observation and photographing, the migration distance between scratches of the cells is measured, and the experiment is repeated three times. The mean and standard deviation of the two groups were analyzed separately, and the differences were statistically significant (P < 0.05). The results are shown in FIG. 3.2.

Experiment 3.3: the Transwell experiment detects the invasion condition of the cells.

Putting the Matrigel gel stored at the temperature of minus 20 ℃ in a refrigerator at the temperature of 4 ℃ overnight, diluting the Matrigel in a culture solution without serum according to a proportion, uniformly spreading the Matrigel on a Transwell chamber membrane, and standing and solidifying the Matrigel; removing residual culture medium from the culture plate, adding 50ul of serum-free culture medium containing 10g/L BSA into each well, and incubating at 37 deg.C for 30 min; after the cells of the experimental group and the control group are subjected to conventional digestion and centrifugation, the cells are resuspended by BSA serum-free culture solution containing 10g/L, and the cell concentration is adjusted; 200ul of cell suspension was inoculated into the upper chamber of the Transwell chamber, then the Transwell chamber was placed in a 24-well plate well, and 500ul of a culture solution containing 10% FBS was added to the lower chamber of the 24-well plate; placing at 37 ℃ and 5% CO₂Conventionally culturing for 36h in a cell culture box; taking out the chamber, washing with PBS, carefully wiping off the cells on the inner layer of the microporous membrane with a wet cotton swab, fixing with 95% ethanol, and staining; the number of cells passing through the lower layer of the microporous membrane was counted by randomly selecting 5 fields under the microscope, and the average was taken, 3 cells per group, and the experiment was repeated 3 times. The mean and standard deviation of the two groups were analyzed separately, and the differences were statistically significant (P)<0.05). The results are shown in FIG. 3.3.

Experimental groups changed to the PIM1 protein inhibitor SGI-1776 to inhibit PIM1 expression, and the above 3 experiments were repeated, and as a result, it was also found that: after the expression of the PIM1 is down-regulated, the cell proliferation, migration and invasion capacities are obviously weakened.

Fourthly, nude mouse tumorigenesis experiment:

and (3) knocking out PIM1 by lentivirus in an experimental group of cancer cells from the gallbladder cancer cell line GBC-SD, carrying out amplification culture after screening, not knocking out lentivirus in a control group, preparing cell suspensions of cells in a plurality of groups of growth phases, inoculating the cell suspensions to the right side axilla of the nude mouse, and establishing a nude mouse subcutaneous transplantation tumor model. The standardized photon flux of each group of nude mice is measured and recorded periodically (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks) by using a small animal living body imager, and is used for indirectly reflecting the tumor size of the nude mice to draw a curve.

The results show that the PIM1 knockout group had significantly lower tumor normalized photon flux than the control group, and the results of the experiment are shown in fig. 4.

<110> first subsidiary Hospital of Zhengzhou university

<120> kit and medicine based on gallbladder cancer marker

<160> 1

<210> 1

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gauaugguguguggagaua 19

Claims

Use of SGI-1776 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of gallbladder cancer.
2. The use according to claim 1, wherein the anti-gallbladder cancer medicament is for ameliorating or treating gallbladder cancer, or for improving the prognosis of a gallbladder cancer patient.
3. A pharmaceutical composition comprising PIM 1-specific interfering RNA and SGI-1776, or a pharmaceutically acceptable salt thereof.
4. The pharmaceutical composition of claim 3, wherein the sense strand sequence of said RNA is GAUAUGGUGUGUGGAGAUAtt from 5 'to 3' and the antisense strand sequence of said RNA is UAUCUCCACACACCAUAUCtt from 5 'to 3'.
5. A kit comprising an antibody that binds to a marker for gallbladder cancer diagnosis and/or prognosis, which is an antibody against human PIM1 protein.
6. Kit according to the preceding claim, characterized in that it does not comprise other antibodies that bind to markers for the diagnosis and/or prognosis of gallbladder cancer.
7. The kit of any preceding claim, wherein the kit is for diagnosing, predicting, detecting or screening human gallbladder cancer cell spreading, clinical stage of gallbladder cancer or gallbladder cancer patient prognosis.
8. The kit or use according to any preceding claim, wherein the gallbladder cancer is stage III-IV or stage I-II TNM.