CN111466339A

CN111466339A - Application of miRNA-203a-3p in promoting proliferation and migration of pancreatic cancer cells

Info

Publication number: CN111466339A
Application number: CN202010255428.6A
Authority: CN
Inventors: 章广玲; 侯晓丽; 熊亚南; 王梅梅
Original assignee: North China University of Science and Technology
Current assignee: North China University of Science and Technology
Priority date: 2020-04-02
Filing date: 2020-04-02
Publication date: 2020-07-31

Abstract

The invention provides application of miRNA-203a-3p in promoting proliferation and migration of pancreatic cancer cells, which comprises the steps of 1) preparing a product for promoting pancreatic cancer metastasis, 2) preparing a product for promoting pancreatic cancer cell migration, 3) preparing a product for improving the invasion capacity of pancreatic cancer cells, 4) preparing a product for improving the expression of genes related to pancreatic cancer, 5) preparing a model for screening drugs for treating pancreatic cancer metastasis, 6) preparing a model for promoting pancreatic cancer metastasis, wherein the nucleotide sequence of miRNA-203a-3p is shown in SEQ ID NO: 1.

Description

Application of miRNA-203a-3p in promoting proliferation and migration of pancreatic cancer cells

Technical Field

The invention relates to the technical field of biology, in particular to application of miRNA-203a-3p in promoting proliferation and migration of pancreatic cancer cells.

Background

Pancreatic Cancer (PC) is one of the most common types of cancer in digestive tract tumors, the fourth leading cause of cancer death in the united states, and one of the worst-case malignancies, with only a few patients having a chance of cure, and at the time of diagnosis, the statistics of the late stages of PDAC patients found outside of surgical resection have remained largely unchanged over the past forty years. Pancreatic cancer patients are characterized by a lack of clinical presentation until late stage, resulting in poor prognosis and high mortality. Only about 8% of pancreatic tumors are localized and resectable. The current pancreatic cancer biomarkers have relatively low sensitivity and specificity, which limits the range of clinical applications. The development of pancreatic cancer is a multigenic, multistep complex pathological and physiological process. Various factors can lead to the development of pancreatic cell carcinoma, such as: the pancreas stones mechanically stimulate the pancreatic duct mucosa epithelium to promote the hyperplasia and smoking of the pancreas ducts and people who like drinking coffee for a long time. In addition, epithelial-to-mesenchymal transition (EMT) is a process that loses the epithelial phenotype and acquires the mesenchymal phenotype, leading to dissemination and metastasis of early tumor cells. The EMT phenomenon plays a key role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer development, and researches show that EMT is used as a direct contributor of a kidney muscle fibroblast group in the development of renal fibrosis, particularly diabetic nephropathy, relates to a signal molecule and an II type EMT pathway and changes of specific miRNA expression, and inhibits EMT reversible organ fibrosis. The scientific community is exploring potential EMT markers of pancreatic cancer and mechanism action thereof at present.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide application of miRNA-203a-3p in promoting proliferation and migration of pancreatic cancer cells.

To achieve these objects and other advantages in accordance with the present invention, there is provided the use of at least one of the following miRNAs-203 a-3p in 1) -6),

1) preparing a product for promoting pancreatic cancer metastasis;

2) preparing a product for promoting pancreatic cancer cell migration;

3) preparing a product for improving the invasion capacity of pancreatic cancer cells;

4) preparing a product for improving the gene expression related to pancreatic cancer;

5) preparing a model for screening a medicine for treating pancreatic cancer metastasis;

6) preparing a model for promoting pancreatic cancer metastasis;

the nucleotide sequence of miRNA-203a-3p is shown in SEQ ID NO 1.

Preferably, the gene associated with pancreatic cancer is CERK L.

Preferably, the product is a kit or a medicament; the model is an animal model, and the animal is a mouse.

The product of any one of 1) to 6), comprising the miRNA-203a-3p,

1) preparing a product for promoting pancreatic cancer metastasis;

2) preparing a product for promoting pancreatic cancer cell migration;

6) preparing a model for promoting pancreatic cancer metastasis;

the nucleotide sequence of miRNA-203a-3p is shown in SEQ ID NO 1.

Preferably, the gene associated with pancreatic cancer is CERK L.

The invention at least comprises the following beneficial effects:

miR-203a-3p can promote the proliferation capacity of pancreatic cancer Bxpc-3 and Panc-1 cells;

the over-expression of miR-203a-3p can promote the colony forming capability of Bxpc-3 and Panc-1 cancer cells;

the over-expression of miR-203a-3p can promote migration and invasion capacity of Bxpc-3 and Panc-1 cells;

in conclusion, the miR-203a-3p can not inhibit pancreatic cancer cell proliferation and tumor formation, but can induce the negative correlated expression of a pancreatic cancer cell metastasis related gene CERK L, improve the pancreatic cancer cell invasion capacity and promote the pancreatic cancer cell migration and metastasis in a mouse, and can be used for preparing a mouse model for pancreatic cancer metastasis, and the model can be applied to screening of drugs for treating pancreatic cancer metastasis and tools for researching the mechanism of the in-situ tumor (pancreatic cancer) to develop into the metastatic tumor (pancreatic cancer).

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows the result of conservative prediction of high expression miRNAs in pancreatic cancer tissues;

FIG. 2 is the result of the conservative prediction of low expression miRNAs species in pancreatic cancer tissues;

FIG. 3 is a graph of the correlation of differentially expressed miRNAs in pancreatic cancer tissues with clinical staging of patients;

FIG. 4 is a correlation analysis of miR-203a-3p and a target gene;

FIG. 5 shows the expression level of mRNA in HPNE and Bxpc-3, Aspc-1, Panc-1, which are normal pancreatic cells, in hsa-miR-203a-3p of high expression group; *: p<0.05, compared to the control group (n-3,

)；

FIG. 6 shows the transfection efficiency of miR-203a-3p for mRNA in pancreatic cancer Bxpc-3 cells; *: p<0.05, compared to the control group (n-3,

)；

FIG. 7 shows the transfection efficiency of mRNA of miR-203a-3p in pancreatic cancer Panc-1 cells; *: p<0.05, compared to the control group (n-3,

)；

FIG. 8 is the effect of miR-203a-3p on the proliferation of Bxpc-3 and Panc-1 cells: p<0.05, compared to the control group (n-3,

)；

FIG. 9 is a graph of the effect of miR-203a-3p on colony forming ability of various groups of Bxpc-3 cells;

figure 10 is a graph of the effect of miR-203a-3p on colony forming ability of various groups of Panc-1 cells (n-3,

)；

FIG. 11 shows the migration results of miR-203a-3p under the microscope on various groups of Bxpc-3 cells (100 ×);

fig. 12 shows the results of miR-203a-3p invasion (100 ×) on each Bxpc-3 cell group (n-3,

)；

FIG. 13 shows the results of microscopic Panc-1 cell migration (100 ×);

fig. 14 shows the results of Panc-1 cell invasion (100 ×) (n-3,

)；

FIG. 15 shows that the website predicts that miR-203a-3p is negatively correlated with CERK L.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1

1.1 screening of differentially expressed miRNAs

Logging in a Google Chrome browser, and inputting a website: https:// xenambrowser. net/datapages/, logs in TCGA database, searches for pancreatic cancer related data packets to download, analyzes using pheatmap package, extracts differential miRNAs using R language edgeR software package, picks up miRNAs with Fold difference (Fold Change) between normal pancreatic tissue (normal Mean) and pancreatic cancer patient tissue (Tumor Mean) greater than 2 Fold, i.e. log fc >2, and P <0.05 as miRNAs with statistical significance for differential expression.

1.2 analysis of conservation of differentially expressed miRNAs

And (3) carrying out conservation analysis on the screened hsa-miR-192-5p (amino acid sequence is shown as SEQ ID NO:2), hsa-miR-203a-3p and hsa-miR-451a (amino acid sequence is shown as SEQ ID NO:3), using miRBase and TargetScan websites to inquire the conservation, and finding that the gene sequences of hsa-miR-192-5p and hsa-miR-203a-3p in the high expression group have species conservation, and selecting the genes as miRNAs with differential expression. The hsa-miR-451a gene sequence in the low expression group has species conservation, and miRNAs which are differentially expressed in pancreatic cancer tissues are selected.

1.3 analysis of survival curves of patients with differentially expressed miRNAs, clinical staging

Kaplan-Meier is used for analyzing the overall survival rate of patients with low expression groups and high expression groups in pancreatic cancer tissues, and Graphpad6.0 is further adopted to prepare a Kaplan-Meier survival analysis chart. Combining gene expression data and clinical data analyzed by TCGA (Chinese character genetic Algorithm) to find out tumor stage diagnosis information, and making a miRNA box diagram of differential expression and TNM stage scatter diagrams (stage i, stage ii, stage iii and stage iv). miRNAs with statistically significant patient survival curves (P <0.05) were further analyzed for their differential expression and clinical staging in pancreatic cancer tissues.

1.4 prediction of related target genes

And (2) predicting the target genes of hsa-miR-192-5p and hsa-miR-203a-3p with remarkable difference in a high expression group and hsa-miR-451a with remarkable difference in a low expression group by using a DIANA TOO L S v 5.0.0 biological information website, and screening the target genes with higher grade ranking according to the grade of the combination degree of the target genes, wherein each miRNA predicts at least 10 target genes.

1.5 analysis of target Gene survival curves and their correlation with miRNAs

Next, the survival curves of the target genes in pancreatic cancer were analyzed using a database, excluding validated target genes and target genes without raw data, and statistical significance of the correlations between them and significantly expressed miRNAs (P <0.05) was continued for target genes.

1.6 real-time fluorescent quantitative PCR is used for verifying the expression quantity of mRNA in normal pancreatic epithelial HPNE cells and pancreatic cancer Bxpc-3 cells, Aspc-1 cells and Panc-1 cells

The expression level of mRNA of miR-192-5p, miR-203a-3p and hsa-miR-451a is detected in pancreatic cancer cell lines Bxpc-3, Aspc-1 and Panc-1 by using normal pancreatic epithelial HPNE cells as a control, the cells are inoculated in a 6-well plate, after 48 hours, the total RNA of 4 cell strains is extracted by using a kit according to the Trizol RNA standard, and the whole operation is carried out on ice.

The specific operation steps are as follows:

1) two days before transfection, the cell line in logarithmic growth phase is taken, washed twice with normal saline, digested in a 1ml culture flask with 0.25% trypsin (containing 0.02% EDTA), blown into single suspension cells, counted on a Bowden counting plate according to 5 × 10⁵One well in a volume of 2ml was inoculated in a 6-well plate and placed in a 5% CO2 incubator at 37 ℃ overnight. The cell density before transfection reaches 70% -90% of the plate bottom area. The miRNA expression in the pancreatic cancer cells is detected without transfecting a cell line, and the RNA of the cells in the logarithmic growth phase is directly extracted.

2) Cell transfection: is used for detecting miR-203a-3p overexpression and inhibiting preparation of miR-203a-3p expression amount transfection solution in pancreatic cancer cells after endogenous miR-203a-3p of the cells is inhibited.

(1) Solutions A and B (the following doses used to transfect 1 well cells) were prepared separately in two sterile 1.5ml EP tubes:

solution A: and diluting 4 mu l of each of the miR-203a-3p micid and the NC-micid or 10 mu l of each of the miR-203a-3p inhibitor and the NC-inhibitor by using a serum-free culture medium Opti-MEM, slightly blowing, uniformly mixing and instantly centrifuging for 30s, wherein the final amount is 250 mu l.

Solution B, the liposome L ipofectamine2000 was mixed gently before preparation, and then 4. mu.l of L ipofectamine2000 was diluted with Opti-MEM, mixed gently, centrifuged instantaneously for 30s to a final volume of 250. mu.l, and allowed to stand at room temperature for 5 min.

Adding the solution B into the solution A, lightly blowing and beating with a gun head for 4-5 times, centrifuging for 15s instantly, and standing at room temperature for 15-20min (no more than 20 min).

(2) Preparation of transfection: taking out the culture plate when the time is close to 15-17min, completely sucking the original culture solution, washing with 0.9% physiological saline for 2 times, slowly dripping the mixed transfection solution into corresponding wells at a rate of 500 μ l/well, supplementing 1.5ml of serum-free culture medium Opti-MEM into each well, shaking the culture plate to fully mix the reagents, placing at 37 deg.C and 5% CO₂

Culturing in a constant temperature incubator for 4-6 h.

(3) Liquid changing: after 4-6h of transfection, the liquid is changed, the transfection liquid is completely sucked, 10% FBS RPM-1640 culture medium without antibiotics is added into each hole, and the culture is continued to be placed in an incubator for 48 h.

3) Extracting cell RNA by a Trizol method:

(1) completely sucking the original culture solution, cleaning twice with normal saline, adding 500 mul Trizol into each well, shaking the culture plate lightly to make Trizol contact with cells fully, carrying out ice bath for 10min to make the cells fully cracked, blowing to make the cells completely fall off, then transferring into 1.5ml enzyme-free EP tube, adding chloroform (precooled at 4 ℃) with one fifth of the total volume of the liquid, shaking up and down and mixing uniformly for 15s, and standing on ice for 5 min.

(2) Centrifuging at 12000rpm for 15min at 4 deg.C, separating the solution into three layers, the upper water sample layer is RNA, and sucking 100-.

(3) Adding pre-cooled isopropanol according to the ratio of 1:1, mixing uniformly for 10s from top to bottom, and carrying out ice bath for 10 min.

(4) Centrifugation was carried out at 12000rpm for 10min at 4 ℃ to obtain a white RNA precipitate, and the supernatant was carefully discarded.

(5) Washing: adding 500 μ l ethanol solution prepared from 75% chilled ethanol (prepared from anhydrous ethanol and DEPC water at a ratio of 3: 1) DEPC water, gently flicking to make precipitate, centrifuging at 7500rpm at 4 deg.C for 5 min.

(6) The supernatant was discarded, the tube was left open for 10min, and 10-20. mu.l of DEPC was added to each tube to dissolve the pellet.

(7) mRNA purity detection: respectively reserving 1 mul for quantification, and detecting the absorbance value and mRNA concentration of the extracted RNA at 260-280 nm by using an ultramicro spectrophotometer, wherein OD260/OD280 is 1.8-2.0. The remaining RNA was stored in a freezer at-80 ℃ for further use.

4) Preparation of cDNA: and performing reverse transcription by using the extracted total RNA as a template and a miRNA first strand cDNA synthesis kit (dye method). The PCR reaction system is as follows:

setting the reaction conditions of a PCR instrument after instantaneous centrifugation: 60 minutes at 37 ℃, 5 minutes at 85 ℃ and 4 ℃. And (3) carrying out real-time fluorescence quantitative PCR on the synthesized cDNA product or storing the cDNA product in a refrigerator at the temperature of minus 20 ℃.

5) Real-time fluorescent quantitative PCR detection of mRNA expression level of miRNA

The qRT-PCR primer is designed and synthesized by Shanghai Jima Gene biology Limited company through an email connection mode, and the sequences of the U6 upper and lower primers and the miRNA upper and lower primers (SEQ ID NO: 4-SEQ ID NO:11) are as follows:

miR-192-5p-FO：5’-CTGCTGCTGCTGACCTATGAAT-3’

miR-192-5p-RE：5’-CAGTTCCTCAGCAGATGTTGGTAT-3’

miR-203a-3p-FO：5’-CCGCTCGTGAAATGTTTAGG-3’

miR-203a-3p-RE：5’-CAGAGCAGGGTCCGAGGTA-3’

miR-451a-FO：5’-CAGAGCAGGGTCCGAGGTA-3’

miR-451a-RE：5’-CAGAGCAGGGTCCGAGGTA-3’

U6 snRNA-FO：5’-CGCTTCGGCAGCACATATAC-3’

U6 snRNA-RE：5’-TTCACGAATTTGCGTGTCATC-3’

when in use, the obtained reaction solution of the reverse transcription product is diluted by 50 times and used as a template to establish a 20ul reverse transcription reaction system, and the reaction system is as follows:

after the sample loading is finished, the sample is instantaneously centrifuged for 15s and placed in a qRT-PCR instrument. Setting a reaction system: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 12 s; annealing at 60 ℃ for 40 s; extension at 72 ℃ for 30s, 40 cycles, each set of 2 duplicate wells.

Real-time fluorescent quantitative PCR (polymerase chain reaction) is used for detecting the expression levels of mRNA (messenger ribonucleic acid) of miR-192-5p, miR-203a-3p and hsa-miR-451a, U6 is used as a positive control gene to correct the copy number of a PCR template, and the relative expression quantity of the gene adopts 2^-ΔΔCtAnd (3) calculating by the following formula: the relative expression quantity of the gene is 2^–ΔΔCt。

1.7 qRT-PCR respectively detects the transfection efficiency of mRNA of NC-imic, miR-203a-3p imic, NC-inhibitor and miR-203a-3p inhibitor;

respectively transfecting NC-imic, miR-203a-3 p-imic, NC-inhibitor and miR-203a-3p inhibitor in Bxpc-3 cells and Panc-1 cells, and detecting the expression level of each group of mRNA (messenger ribonucleic acid) by using the cells according to the specification of 5 × 10⁵One well, 2ml volume suspension was seeded in 6 well cell culture plates and placed in a 5% CO2 incubator at 37 ℃ overnight. Extracting total RNA by using a Trizol method 48 hours after transfection; and (3) preparing cDNA (complementary deoxyribonucleic acid) by taking the extracted total RNA as a template to perform a real-time fluorescence quantitative PCR (polymerase chain reaction) experiment, wherein the specific operation is performed according to the step 1.6. Data analysis according to the Comparative Delta Ct relative quantification method, U6 was used as an endogenous control to normalize gene levels, calculating the formula: the relative expression quantity of the gene is 2^–ΔΔCt。

1.8 CCK-8 determination of Effect of miR-203a-3p on the proliferation capacities of Bxpc-3 and Panc-1 cells

1) Cell transfection: transfection was performed according to procedure 1.6.

2) The Cell proliferation experiment utilizes Cell Counting Kit-8(CCK8) reagent to determine the proliferation rate, 1000 cells are inoculated into a 96-well plate at the density of each well, the cells of Bxpc-3 and Panc-1 are transfected by using Opti-MEM and L ipofectamine2000 respectively by using miR-203a-3p mimic, NC-mimic, miR-203a-3p inhibitor and NC-inhibitor, after 0h, 24h, 48h and 72h of incubation in a Cell incubator, the cells are labeled by using CCK-8, after 4h, the OD value of the cells at the wavelength of 450nm is determined by using an enzyme labeling instrument, and each group is repeated three times.

3) Data processing: the absorbance values of each group of cells were analyzed by mean and standard deviation calculation, and a line graph was created using Prism 7 software.

1.9 cell colony formation assay to test the Effect of miR-203a-3p on pancreatic cancer cell colony formation ability

1) The Bxpc-3 and Panc-1 cells are inoculated on a 6-hole culture plate, cultured for 48 hours at the constant temperature of 37 ℃ and 5% CO2, and transfected with an overexpression plasmid miR-203a-3p and a control NC-mimic thereof; the miR-203a-3p inhibitor and a control NC-inhibitor thereof are applied to pancreatic cancer cells Bxpc-3 and Panc-1, the transfection method is similar to the step of section 1.6, and the cell inoculation quantity and the transfection reagent dosage are calculated according to the bottom area of the culture plate.

2) After the cells are transfected for 48 hours, sucking out the original culture solution, washing for 2 times by using normal saline, adding 300 mu l of pancreatin into each hole for digestion, adding 2 ml/hole of RPM-1640 culture medium, and blowing and beating the cells to form uniform single cell suspension. Counting by using a cell counting plate, transferring the cells into a 6-well plate according to 1000 cells/well, setting 2 multiple wells in each group, and placing the cells in an incubator for continuous culture.

3) Replacing fresh culture solution every 2-3 days during the culture period, counting from the 14 th day of inoculation, observing the cell clone formation condition under a microscope, selecting colonies with the cell number being more than or equal to 50 as counting objects, completely sucking culture solution in each hole, washing twice with normal saline, dyeing the clones by a crystal violet dyeing method for 5min, washing 2-3 times with normal saline, and photographing to store experimental results.

4) Data processing, mean and standard deviation of the number of colonies formed by each group of cells were calculated, and the cell colony formation rate (number of cell colonies/number of initially seeded cells) was calculated as × 100%.

1.10Transwell experiment for detecting the influence of miR-203a-3p on migration and invasion capacity of pancreatic cancer cells

1) Cells are inoculated on a 6-hole cell culture plate, after 24 hours of culture, an overexpression plasmid group miR-203a-3p and a control group NC-mici or miR-203a-3p inhibitor group and a control group NC inhibitor group thereof are transfected on pancreatic cancer cells Bxpc-3 and Panc-1, the transfection method refers to the step of 1.6, and the inoculation number of the cells and the dosage of a transfection reagent are calculated according to the bottom area ratio of the culture plate.

2) Migration experiment, after 48h of cell transfection, washing the transfected cells with physiological saline for 2 times, digesting the transfected cells with 300. mu.l/well pancreatin, blowing the cells into suspension state with RPM-1640 medium without fetal bovine serum, and adjusting the cell density to 2 × 10⁴200. mu.l of cell suspension was added to the upper chamber of the Transwell, 800. mu.l of RPM-1640 medium containing 20% fetal bovine serum was added to the lower chamber, and 2 multiple wells were set for each group, and the cells were cultured in an incubator for 24 hours continuously.

3) Invasion test: 48h after transfection of the cells, matrigel (Metrigel gel) was prepared at a concentration of 1mg/ml throughout the ice, 50. mu.l of the gel was added to each well of the upper chamber, and the gel was spread on the bottom of the upper chamber by gently tapping the air. Then it was placed at 37 ℃ in 5% CO₂Preparing RPM-1640 culture medium without fetal calf serum and RPM-1640 culture medium containing 20% fetal calf serum during the 15min of solidification in a thermostat, cleaning the culture plate with normal saline for 2 times, digesting and transfecting cells with 300 mul/hole pancreatin, blowing and beating the cells to be in a suspension state by using the RPM-1640 culture medium without fetal calf serum, and adjusting the cell density to be 2 × 10⁴A hole. The cell suspension of 200. mu.l was pipetted into the upper chamber of a Transwell in which the gel was spread, and 800. mu.l of an RPM-1640 medium containing 20% fetal bovine serum was added to the lower chamber, and 2 multiple wells were set for each group, and continuously cultured in an incubator for 24 hours.

4) The next day, the upper chamber is taken out by a pair of tweezers, the culture solution in the upper chamber is completely sucked, the residual cells in the upper chamber are slightly washed away by normal saline, the cotton swab is lightly wiped on the polycarbonate micro-membrane in the upper chamber, the ester membrane cells are fixed by 1% paraformaldehyde solution for 30min, the ester membrane cells are stained by 0.2% crystal violet solution for 5min, the normal saline is decolorized for 3-4 times, 5 visual fields are randomly selected under a 100-fold microscope for photographing, the average value and the standard deviation of the number of the cells penetrating the membrane are calculated, and the cell mobility and the invasion rate are calculated, wherein the cell mobility is [ (the number of the migrated cells in the experimental group-the number of the migrated cells in the control group)/the number of the migrated cells in the control group ] ×, and the cell invasion rate is [ (the number of the invaded cells in the experimental group-the number of the control group.

1.11 fluorescent report vector experiment verifies the targeting relationship between the two

To identify the direct target relationship between miR-203a-3p and the CERK L mRNA 3' UTR, fluorescence intensity was determined using a dual luciferase reporter assay system (Promega, Madison, WI). Bxpc-3 and Panc-1 cells (pcDNA3/EGFP, pcDNA3/EGFP-3 ' UTR and pcDNA3/EGFP-3 ' UTR-mut each 1ug) were co-transfected with pcDNA3/pri-miR-203a-3p and CERK L wildlife 3' UTR or mutant 3' UTR. cells were lysed 48h after transfection by comparison with a control group (EGFP reporter group not containing miR-203a-3p action site, i.e. blank control group, containing specific mutant EGFP reporter vector, i.e. negative control group), and the intensity of red fluorescent protein RFP expression (EGFP, 558nm, green fluorescent protein emission, 583nm) and excitation light intensity of emission (507 nm) was determined using a fluorescence emission chemiluminescence apparatus spectramuci EM.

Results of the experiment

Screening of TCGA database differentially expressed miRNAs miRNA differential analysis the pheatmap R language package was usedhttps://cran.r-project.org/web/packages/pheatmap/)。

The miRNAs were chosen as statistically significant if normal pancreatic tissue (normal Mean) differed from pancreatic cancer patient tissue (Tumor Mean) by a factor (Fold Change) greater than 2, i.e., logFC >2, and P < 0.05.

Compared with normal pancreatic tissues, 9 miRNAs in the high expression group and 9 miRNAs in the low expression group in the pancreatic cancer patient tissues have statistical significance (P <0.05) in differential expression (shown in tables 1-2).

TABLE 1 MiRNAs with major deregulation in the tissue high expression group of pancreatic cancer patients (9)

TABLE 2 MiRNAs with major deregulation in the tissue underexpression group of pancreatic cancer patients (9)

Species conservation prediction results for differentially expressed miRNAs

MiRBase and TargetScan websites are used for inquiring the conservation of miRNA, and hsa-miR-192-5p, hsa-miR-194-5p, hsa-miR-203a-3p, hsa-miR-203a-5p and hsa-miR-592 gene sequences in high expression groups are found to have species conservation (figure 1), and are selected for analyzing miRNAs with differential expression in pancreatic cancer tissues. In the high expression group, species conservation does not exist in hsa-miR-192-3p, hsa-miR-215, hsa-miR-4724 and hsa-miR-6514, and the hsa-miR-196a-1, hsa-miR-196a-2 and hsa-miR-203a-5p have no original data in TCGA and are not used as research objects, and the survival curves of miRNAs with wide conservation are further analyzed.

Similarly, miRBase and TargetScan websites are applied to inquire that the gene sequences of the low expression groups hsa-miR-142-5p, hsa-miR-150-5p, hsa-miR-206 and hsa-miR-451a have species conservation (figure 2), and the miRNAs are selected for analyzing differential expression in pancreatic cancer tissues. In the low expression group, the species conservation of hsa-miR-142-3p, hsa-miR-150-3p, hsa-miR-1537-5p, hsa-miR-3191-3p, hsa-miR-3191-5p, hsa-miR-4665-3p, hsa-miR-4722-5p and hsa-miR-6502-3p is not taken as a research object, and the survival curves of miRNAs with wide conservation are further analyzed.

Example 2

Species-conservative differential expression miRNAs survival curves and clinical staging

The results of analyzing miRNAs which are differentially expressed and have species conservation in pancreatic cancer tissues and the expression levels of the miRNAs are related to the survival of patients indicate that the expression levels of hsa-miR-192-5P, hsa-miR-203a-3P and hsa-miR-451a in high expression groups and low expression groups in the pancreatic cancer tissues are related to the survival rate of the patients, and the results have statistical significance (P < 0.05).

miRNAs with statistical significance (P <0.05) in the survival rate of a high expression group of pancreatic cancer patients comprise hsa-miR-192-5P and hsa-miR-203 a-3P. miRNAs with statistically significant survival (P <0.05) in patients with low expression groups have hsa-miR-451a for further analysis of their clinical staging in pancreatic cancer tissues (FIG. 3).

Example 3

Prediction of target gene outcome

And (3) predicting the target genes of hsa-miR-192-5p and hsa-miR-203a-3p with obvious difference in the high expression group and hsa-miR-451a with obvious difference in the low expression group by using a DIANA TOO L S v 5.0.0 biological information website, and screening the target genes with higher scores according to the scores of the combination degree of the target genes.

Next, the survival relevance of the miR-203a-3P candidate target gene and pancreatic cancer patients is analyzed, and the results indicate that the candidate target genes with expression levels relevant to the survival of the patients and statistical significance (P <0.05) are ZNF148, TMEM69, ABCE1, CERK L, TSHR, MBN L2, HNRNP L and CCDC50, and the relevance of the candidate target genes and miR-203a-3P is analyzed, and after the analysis, ZNF148, TMEM69, ABCE1, MBN L2, HNRNP L, CCDC50 and miR-203a-3P are found to be positively correlated (r values are 0.07115, 0.1145, 0.06799, 0.04999, 0.2795 and 0.2765 respectively), and CERK L, TSHR and miR-203a-3P are negatively correlated (r values are-0.4603 and-0.2229 respectively) (FIG. 4).

Example 3

Results of verifying mRNA expression levels of mRNAs in HPNE cells, Bxpc-3 cells, Aspc-1 cells and Panc-1 cells by qRT-PCR

And (3) detecting the expression levels of hsa-miR-192-5p, hsa-miR-203a-3p and hsa-miR-451a in normal pancreatic epithelial cells HPNE and pancreatic cancer cells Bxpc-3, Aspc-1 and Panc-1 by using qRT-PCR.

① the mRNA expression level of the high expression group hsa-miR-203a-3p in normal pancreatic epithelial cells HPNE and pancreatic cancer cells Bxpc-3, Aspc-1 and Panc-1 is verified by a qRT-PCR method.

The qRT-PCR result shows that in the HPNE of normal pancreatic epithelial cells and Bxpc-3, Aspc-1 and Panc-1 of pancreatic cancer cells, the mRNA expression level of hsa-miR-203a-3P is increased, the relative expression amounts are 7.115 times, 7.714 times and 6.154 times of those of a control group respectively (figure 7), and the difference has statistical significance (P < 0.05).

Example 4

Results of detecting transfection efficiency of NC-mimic, miR-203a-3p mimic, NC-inhibitor and miR-203a-3 inhibitor by qRT-PCR

The experiment is divided into a miR-203a-3p mimic group, a control NC-mimic group, a miR-203a-3p inhibitor group and an NC-inhibitor control group, and 4 groups are transfected in Bxpc-3 and Panc-1 cells respectively. And (3) detecting the expression level of miR-203a-3p mRNA in each group of cells by qRT-PCR. The results show (FIG. 6a, FIG. 7a) that the mRNA expression level of the miR-203a-3P group is high after miR-203a-3P is over-expressed compared with the control group, the relative expression amounts are respectively 26.234 times and 27.947 times of the control group, the difference is statistically significant (P <0.05), and the miR-203a-3P micic transfection can effectively increase the expression of miR-203 a-3P. And after the function of the endogenous miR-203a-3P is sealed, the mRNA expression level of the miR-203a-3P is lower than that of a control group (figure 6b and figure 7b), the relative expression amounts are respectively 0.414 times and 0.534 times of those of the control group, the difference has statistical significance (P <0.05), and the miR-203a-3P inhibitor is prompted to be transfected to effectively reduce the expression of the miR-203a-3P, and the difference has statistical significance (P < 0.05).

Example 5

miR-203a-3p promotes proliferation capacity of pancreatic cancer Bxpc-3 and Panc-1 cells

The experiment is divided into a miR-203a-3p mimic group, a control NC-mimic group, a miR-203a-3p inhibitor group and an NC-inhibitor control group. The CCK-8 experiment is adopted to detect the proliferation of Bxpc-3 and Panc-1 cells in each group, and the result shows that miR-203a-3P imic promotes the proliferation of Bxpc-3 and Panc-1 cells (P <0.05), and miR-203a-3pinhibitor inhibits the proliferation of Bxpc-3 and Panc-1 cells, and the difference has statistical significance (P <0.05) (as shown in FIGS. 8a and 8b, and as shown in tables 3-4).

Table 3 effect of miR-203a-3p on the proliferative capacity of Bxpc-3 cells (n-3,

)

note: *: p <0.05, x: p <0.01, compared to NC group;

table 4 effect of miR-203a-3p on Panc-1 cell proliferation potency (n-3,

)

note: *: p <0.05, x: p <0.01, compared to NC group;

example 6

Ability of miR-203a-3p to promote pancreatic cancer cell colony formation

Colony formation conditions of cells in each group are observed through pancreatic cancer cell Bxpc-3 and Panc-1 colony formation experiments, and the experiments are divided into a miR-203a-3p micic group, a control group, a miR-203a-3p inhibitor group and an NC inhibitor group. As shown in FIG. 9 and FIG. 10, the results show that in Bxpc-3 and Panc-1, compared with the NC mimic control group, the cell colony number of the miR-203a-3P overexpression group is obviously increased, and the difference has statistical significance (P < 0.05); compared with an NC inhibitor control group, after the function of endogenous miR-203a-3P of the cells is inhibited in Bxpc-3 and Panc-1, the cell colony formation number is obviously reduced, the difference has statistical significance (P <0.05), and the results show that the over-expression of miR-203a-3P can promote the colony formation capacity of Bxpc-3 and Panc-1 cancer cells.

Example 7

Ability of miR-203a-3p to promote migration and invasion of pancreatic cancer cells

To demonstrate that miR-203a-3p affects the change in pancreatic cancer cell migration and invasive capacity, experiments were performed in Bxpc-3 and Panc-1 cells in groups as follows: and (3) adopting a Transwell experiment to observe migration invasion and attack conditions of pancreatic cancer cells in four groups of NC-mic, miR-203a-3p mic, NC-inhibitor and miR-203a-3p inhibitor. The results show (FIG. 11, FIG. 12, FIG. 13 and FIG. 14) that over-expression of miR-203a-3P can promote the migration and invasion abilities of Bxpc-3 and Panc-1 cells compared with the control promotion group, and the difference has statistical significance (P < 0.05); compared with a control inhibition group, after the expression of miR-203a-3P is inhibited, the cell migration and invasion number is obviously reduced, and the difference has statistical significance (P is less than 0.05). These results indicate that the miR-203a-3p overexpression can promote the migration and invasion capacity of pancreatic cancer cells Bxpc-3 and Panc-1.

Example 8

CERK L is related target gene of miRNA-203a-3p

The possibility target gene of miR-203a-3p is predicted through bioinformatics websites (DIANA TOO L S, TargetScan, StarBase), three websites predict that CERK L is the related target gene of miR-203a-3p, and a highly conserved complementary sequence exists between miR-203a-3p and CERK L, and the search finds that the CERK L3' UTR has a complementary sequence with miRNA-203a-3p at position 1499-1506, the combination score of the two is 98, and the CERK L is the downstream target gene of miR-203a-3p (FIG. 15) which is in negative correlation (r value is-0.4603).

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

<110> North China university of Engineers

Application of <120> CN20BD 007A-miRNA-203 a-3p in promoting proliferation and migration of pancreatic cancer cells

<160>3

<210>1

<211>21

<212>RNA

<213> Artificial sequence

<220>

<400>1

cugaccuaug aauugacagc c 21

<210>2

<211>22

<212>RNA

<213> Artificial sequence

<220>

<400>2

gugaaauguu uaggaccacu ag 22

<210>3

<211>22

<212>RNA

<213> Artificial sequence

<220>

<400>3

aaaccguuac cauuacugag uu 22

Claims

The application of miRNA-203a-3p in at least one of the following 1) to 6),

1) preparing a product for promoting pancreatic cancer metastasis;

2) preparing a product for promoting pancreatic cancer cell migration;

3) preparing a product for improving the invasion capacity of pancreatic cancer cells;

4) preparing a product for improving the gene expression related to pancreatic cancer;

5) preparing a model for screening a medicine for treating pancreatic cancer metastasis;

6) preparing a model for promoting pancreatic cancer metastasis;

the nucleotide sequence of miRNA-203a-3p is shown in SEQ ID NO 1.
2. The use of claim 1, wherein the gene associated with pancreatic cancer is CERK L.
3. The use of claim 1, wherein the product is a kit or a medicament; the model is an animal model, and the animal is a mouse.
4. The product of any one of claims 1) to 6), comprising the miRNA-203a-3p of claim 1,

1) preparing a product for promoting pancreatic cancer metastasis;

2) preparing a product for promoting pancreatic cancer cell migration;

3) preparing a product for improving the invasion capacity of pancreatic cancer cells;

4) preparing a product for improving the gene expression related to pancreatic cancer;

5) preparing a model for screening a medicine for treating pancreatic cancer metastasis;

6) preparing a model for promoting pancreatic cancer metastasis;

the nucleotide sequence of miRNA-203a-3p is shown in SEQ ID NO 1.
5. The product of claim 4, wherein the gene associated with pancreatic cancer is CERK L.
6. The product of claim 4, wherein the product is a kit or a medicament; the model is an animal model, and the animal is a mouse.