CN113151464A

CN113151464A - Method for diagnosing pancreatic cancer by miRNA detection and application thereof

Info

Publication number: CN113151464A
Application number: CN202110343897.8A
Authority: CN
Inventors: 不公告发明人
Original assignee: Wuhan Boyue Zhihe Biotechnology Co ltd
Current assignee: Wuhan Boyue Zhihe Biotechnology Co ltd
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2021-07-23

Abstract

The invention discloses a miRNA (micro ribonucleic acid) marker for detecting and diagnosing pancreatic cancer and application thereof, and also discloses a detection kit containing the miRNA marker. The miRNA marker is miRNA-21-5p gene. The relative copy number of the diagnostic marker nucleotide is in positive correlation with the incidence rate of pancreatic cancer, namely, the higher the copy number of the miRNA-21-5p marker is, the easier the pancreatic cancer is. An increase or decrease in the copy number of the marker results in an increase or decrease in the expression level of miRNA-21-5 p. The invention provides a more reliable and efficient diagnosis technology for early diagnosis and/or concomitant diagnosis of pancreatic cancer, lays a solid foundation for developing miRNA-21-5p molecular inhibitors in the future, and has good application prospect.

Description

Method for diagnosing pancreatic cancer by miRNA detection and application thereof

Technical Field

The invention relates to the fields of biomedicine and gene diagnosis; in particular to the application of miRNA-21 molecules in the preparation of early diagnosis and/or concomitant diagnosis of pancreatic cancer.

Background

Recent data show that pancreatic cancer incidence is on the 9 th, mortality is on the 4 th of global tumors, and patient survival is less than 5% 5 years post-surgery [37 ]. In China, more than 9 ten thousand new cases of pancreatic cancer occur every year, and the number of death cases is nearly 90% [38 ]. At present, the treatment strategies aiming at pancreatic cancer, including surgery, chemotherapy, radiotherapy and targeted therapy, have no obvious treatment effect and cannot remarkably prolong the survival rate of pancreatic cancer after operation. Therefore, finding early pancreatic cancer specific genes and molecular markers to realize early diagnosis, treatment and prognosis of pancreatic cancer has become a hot spot of current research. In addition, miRNA is a kind of endogenous non-coding RNA with regulation and control effect, influences the generation and the progress of tumor through the regulation and control of complex gene network, and is closely related to the generation, the progress, the diagnosis and the prognosis of tumor [39-40 ]. With the explosive development of molecular biology techniques, there is increasing evidence that mirnas play an important regulatory role in the progression of pancreatic cancer [41-42 ]. Therefore, research on the function and action mechanism of miRNA is attracting more and more attention from researchers. Over the past few decades, mirnas have emerged as important molecules related to the regulation of gene expression in humans and other organisms, expanding the strategies available for diagnosing and treating a variety of diseases. To date, mirnas account for approximately 3% of the human genome [7], and under physiological conditions, mirnas play a key role in controlling tissue homeostasis and cell signaling, and are post-transcriptional mechanisms of gene expression. The coordinated function of these molecules is associated with other mechanisms that prevent the occurrence of abnormal cell proliferation, regulate cell differentiation and fine-tune mRNA in response to detected endocrine hormones and other stimuli (e.g. cytokines, chemokines, infectious or stressful conditions), and control the expression of > 60% of protein-encoding genes in humans [8 ]. Since the abnormal expression of miR-15 and miR-16 has been reported in chronic lymphocytic leukemia, there is increasing evidence that miRNA mutations or misexpression are associated with a variety of human cancers and that mirnas can act as tumor suppressors or oncogenes [9 ]. They act as regulators of gene expression and thus play a key role in regulating a wide range of cellular processes including growth, differentiation, proliferation and apoptosis.

Disclosure of Invention

The invention relates to a method for using miRNA-21-5p gene as a marker for early diagnosis and/or concomitant diagnosis of pancreatic cancer.

The invention also provides a novel miRNA for regulating pancreatic cancer, which is excavated by using the miRNA-21-5p as a marker for pancreatic cancer diagnosis and analyzing high-throughput sequencing data of tissue samples and normal tissue samples of 5 pancreatic cancer patients.

The invention also discovers the difference of miRNA-21-5p in clinical sample sequencing data, including that the expression level of miRNA-21-5p in tumor tissues is 30-100 times higher than that in normal tissues.

The invention also discloses a mimic sequence of miRNA-21-5p as shown in SEQ ID NO: 2, the inhibitor sequence of miRNA-21-5p is shown in SEQ ID NO: 4

The invention also discovers the function of the miRNA-21-5p at the cellular level, and the function comprises that after the miRNA-21-5p mimics are transfected into cells, the proliferation rate of the cells is accelerated, the apoptosis rate is slowed, and the cell division is promoted; the miRNA-21-5p inhibitor is transfected into cells, the proliferation rate of the cells is reduced, the apoptosis rate is increased, and cell division is inhibited.

The invention also relates to application of the miRNA-21-5p in preparation of a kit for early diagnosis and/or concomitant diagnosis of pancreatic cancer. The pancreatic cancer diagnostic kit comprises a miRNA-21-5p detection primer pair, a reference gene U6 detection primer pair, a buffer reagent and a reverse transcription cDNA primer. The method for detecting miRNA-21-5p by the kit comprises the steps of RNA extraction, cDNA reverse transcription synthesis, PCR amplification, calculation and judgment of amplification product results and the like. Wherein the miRNA-21-5p primer pair has a sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10, the primer pair sequence of the internal reference U6 gene is shown as SEQ ID NO: 6 and SEQ ID NO: 7.

description of the drawings:

FIG. 1 shows that miRNA-21-5p is screened as an important diagnostic marker by high-throughput sequencing of a tumor sample;

FIG. 2 is a graph showing the effect of miRNA-21-5p on the proliferation potency of CFPAC-1 cells;

FIGS. 3-7 are graphs showing the effect of miRNA-21-5p on CFPAC-1 cell cycle division;

FIGS. 8-12 show the effect of miRNA-21-5p on CFPAC-1 apoptosis;

FIG. 13 is a graph of the effect of EdU fluorescence analysis of miRNA-21-5p on CFPAC-1 cell proliferation.

The specific implementation mode is as follows:

example 1: pancreatic cancer detection early diagnosis and/or detection of marker miRNA-21-5p accompanying diagnosis

Tumor tissues and normal tissues of 5 patients are collected for pancreatic cancer selection, high-throughput sequencing is carried out, and 171 miRNAs are screened to be up-regulated and 188 miRNAs are screened to be down-regulated. Wherein the expression level of miRNA-21-5p is 30-100 times higher than that of normal tissues in the tumor.

The following experimental methods and contents are used in this embodiment:

1. extracting total RNA of tissues and detecting the quality of the total RNA. And (3) extracting total RNA by using a TRIzol method, and after the total RNA is extracted, carrying out RNA purity detection by using Agilent2100 and Nanodrop, and detecting whether the RNA is degraded or not and whether a mixed peak exists or not. After the total RNA is finished, firstly, Qubit2.0 and Nanodrop are used for preliminary quantification, then, 1% agarose gel electrophoresis is used for detecting the integrity of the extracted RNA, and then Agilent2100 is used for accurately detecting the RNA (RIN/RQS >7), so that the quality of the RNA is ensured after the requirement is met. And after the RNA quality meets the requirement, constructing the library.

2. And (5) constructing a sequencing library. Construction of a miRNA Library Using NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) kits A DNA high sensitivity chip was used to evaluate Library quality in Agilent 2100. And (3) sequencing the miRNA by adopting an Illumina Hiseq sequencing platform and a SE50 sequencing strategy.

3. High throughput sequencing. Samples encoded by index were resolved on a cBot Cluster generation system according to the Kit description method using TruSeq PE Cluster Kit v3-cBot-hs (illumina). After cluster generation, the library was sequenced on the Illumina HiSeq 3000 platform, yielding 50 bp single-ended reads. The number of reads of miRNA for each sample was 14658590-18771808, respectively. The clean bases of the miRNA of each sample are respectively 5-62M reads. The error rate of miRNA for each sample was < 0.01%, respectively.

4. And (4) bioinformatics analysis. And finishing data preliminary filtration by removing joints at two ends of reads, removing low-quality reads, removing pollution and the like, screening out miRNA with the length of 18-25 nt from clean readings, and positioning to a reference sequence. And performing quality control analysis on raw reads by using FastQC. Used in conjunction with mirefo software and mirdrep 2 software to analyze the function of the new mirnas. Differential expression of mirnas was analyzed using negative binomial distribution of DESeq 2. All sequencing procedures were performed by Novogene Company. Then, the differential expression analysis is carried out on the known miRNA, and miRNA-21-5p is screened out as an important diagnostic marker (figure 1).

Example 2: pancreatic cancer early diagnosis and/or verification of concomitant diagnosis marker miRNA-21-5p

The following experimental methods and experimental conclusions are disclosed in this embodiment.

1. Instrumentation and emphasis reagents. Human pancreatic cancer cell line CFPAC-1: purchased from the national laboratory cell resource sharing platform (Beijing collaborating with cell Bank). SW-CJ-1D biological clean bench, Suzhou decontamination, Inc.; MCO-18AC CO2 cell culture incubator (Panasonic, Japan); XZ-10 inverted microscope (Guangzhou Mingmei opto-electro technology, Inc.); SMR60047 enzyme-labeled detector (Wuhan Youlsheng bioscience Equipment, Inc.); 80-2 centrifuge (Zhengrong instruments, Inc., Changzhou); BS110 electronic balance (Sartorius, usa); Milli-Q ultrapure water system (Millpore, USA); MDF-U32V Low temperature refrigerator (SANYO, Japan); gilson Gilso pipettor (Gilson corporation, france); vertical automatic steam sterilizer model LDZX-40B (Systec, Germany); k20 Metal bath (Hangzhou blue flame science, Inc.); PHS-10A pH meter (Hangzhou Vanda instrument and meter factory); a quantitative pipettor (Thermo corporation, usa); disposable needle filters (Pall Life Sciences, USA); MF53 inverted fluorescence microscope (guangzhou mingmei photoelectric technology, ltd); an electric heating constant temperature air blast drying oven (Changzhou ordinary instrument manufacturing Co., Ltd.); NanoDrop @ 2000c spectrophotometer (Roche, Switzerland); flow cytometry (BD corporation, usa); PCR amplificators (Roche, Switzerland); PP-1105 electrophoresis apparatus (Beijing Kaiyuan Xinrui apparatus Co., Ltd.); MP-8120 transfer electrophoresis apparatus tank (Beijing Kaiyuan Xinrui apparatus Co., Ltd.); MP-3030 vertical electrophoresis tank (Beijing Kaiyuan Xinrui instruments Co., Ltd.); TGL-16c refrigerated centrifuge (Shanghai' an Tint scientific Instrument factory); an IMS-20 ice maker (snow appliances, Inc., Utility); WD-9405A decoloring shaking table (six instruments factories in Beijing); Annexin-V-FITC apoptosis detection kit (Nanjing Kai Biotechnology Co., Ltd.); cell cycle detection kits (Nanjing Kaikyi Biotechnology Co., Ltd.); gene primers (Shanghai Biotechnology engineering Co., Ltd.); a reverse transcription kit (Takara, Japan); trizol solution (Solarbio, Beijing); SDS-PAGE gel preparation kit (Wuhan Bai Qian degree Biotechnology Co., Ltd.); RIPA Total protein lysate (Wuhan Bai Qian degree Biotech Co., Ltd.); BCA protein concentration assay kit (Wuhan Bai Qian degree Biotech Co., Ltd.); ECL chemiluminescence assay kit (Wuhan Bai Qian degree Biotechnology Co., Ltd.); protease Inhibitor Cocktail (ROCHE, switzerland).

2. Experimental methods

Cell transfection experiments: (1) one day prior to transfection, 1X 106/mL CFPAC-1 cells were seeded in 6-well plates and 2mL serum-containing IMDM medium was added. Culturing in an incubator with CO2 concentration of 5% at 37 deg.C until cell confluence reaches 70-80%.

(2) And adding mimic mimics of miRNA-21-5p and inhibitor to 100 mu L of Opti-MEM serum-free medium, and mixing the mixture softly. Another 10. mu.L of Lipofectamin reagent was added to 100. mu.L of serum-free Opti-MEM, gently mixed, and left at room temperature for 5 min.

(3) The DNA and the Lipofectamin 2000 solution are mixed, gently mixed, and placed at room temperature for 20min, so that the miRNA and the Lipofectamin form a complex. 200 μ L of DNA/Lipofectamin complex was added to the wells of the plate containing cells and 1.8 mL of serum-free medium, and the cell culture plate was gently shaken back and forth.

(4) After incubating the cells in a CO2 incubator at 37 ℃ for 6h, the fresh serum-containing medium was replaced and the plates were shaken and mixed well. After a further 48h incubation, cells were collected for subsequent experiments.

Cell proliferation assay: the log phase CFPAC-1 cells were harvested and plated in 96 well plates and cultured at a cell density of 6000 cells/well with 100 μ L/well to a cell confluency of 80%. According to the transfection reagent step, the miRNA is added into opti-MEM serum-free medium, mixed evenly and softly, mixed with a proper amount of Lipofectamin 2000, and kept standing for 20min at room temperature so as to form a compound of the miRNA and the Lipofectamin. The miRNA-Lipofectamin complex was added to a 96-well plate containing cells and 100 μ L of culture medium, and after 6 hours, culture was continued for 48 hours after changing to fresh complete medium. At the end of the incubation, 10 μ L of CCK8 solution (to avoid air bubble formation) was added to each well of the 96-well plate using a pipette gun and the plate was incubated in the incubator for an additional 2 hours. The 96-well plate was placed on an orbital shaker and mixed gently for 5 minutes before measurement on the microplate reader to ensure uniform color distribution. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader, and 3 wells per group were repeated. Proliferation of each group of cells was calculated from the standard curve.

Apoptosis assay: CFPAC-1 cells in log phase were harvested and plated in 6-well plates and incubated until the cell confluence was 80% with 2mL of CFPAC-1 cells at 1X 106 cells per well. And adding the miRNA/siRNA into opti-MEM serum-free medium, mixing the miRNA/siRNA and the opti-MEM serum-free medium softly, mixing the mixture with a proper amount of Lipofectamin 2000, and standing the mixture for 20min at room temperature so as to form a compound of the miRNA and the Lipofectamin. The miRNA-Lipofectamin complex was added to a 6-well plate containing 2mL of culture medium, and after 6 hours, the culture was continued up to 48 hours after changing to fresh complete medium. After the culture is finished, digesting and collecting the CFPAC-1 cell suspension in a 2mL centrifuge tube, placing the centrifuge tube in a 1500 rpm condition for 5min, and removing the supernatant to leave a precipitate. After washing with 1mL of precooled PBS, cell debris was removed from the suspension after centrifugation at 1500 rpm for 5min, and the procedure was repeated 2 times. And sucking 300 mu L of Binding Buffer for resuspending the sediment by using a pipette gun. Then 5 muL of Annexin V-FITC and 5 muL of Propidium Iodide staining solution are respectively added into the suspension and mixed evenly and gently again. The suspended cell fluid was incubated at room temperature for 5-15 min and then placed in an ice bath for detection. And (3) carrying out apoptosis detection on the prepared sample by a flow cytometer in sequence, and analyzing the result by Flomax software.

Cell cycle analysis: CFPAC-1 cells in log phase were harvested and plated in 6-well plates and incubated until the cell confluence was 80% with 2mL of CFPAC-1 cells at 1X 106 cells per well. And adding the miRNA into the opti-MEM serum-free medium, mixing the miRNA and the opti-MEM serum-free medium gently, mixing the miRNA with an appropriate amount of Lipofectamin 2000, and standing the mixture at room temperature for 20min to form a compound of the miRNA and the Lipofectamin. The miRNA and Lipofectamin complex is added into a 6-well plate containing 2mL of culture solution, and after 6 hours, the culture is replaced by fresh complete culture medium, and then the culture is continued for 48 hours. After the culture, the stock culture solution, the PBS after washing and the CFPAC-1 cell suspension after digestion are collected in a 2mL centrifuge tube, and are centrifuged for 5min under the condition of 1500 rpm, and the supernatant is discarded to leave a precipitate. After washing with 1mL of precooled PBS, cell debris was removed from the suspension after centrifugation at 1500 rpm for 5min, and the procedure was repeated 2 times. Adding appropriate amount of 75% ethanol, fixing at 4 deg.C for 2 hr, centrifuging at 1500 rpm for 5min, discarding the supernatant to obtain precipitate, and washing with precooled PBS for 2 times to remove residual anhydrous ethanol. 400 uL of ethidium bromide (PI, 50 ug/mL) and 100 uL of RNase A (100 ug/mL) were added to the cell pellet in this order, and the pellet was incubated at 4 ℃ for 30min in the absence of light. This was then placed in an ice bath for detection, and detection was performed under a flow cytometer using standard procedures and periodic analysis using the software ModFit.

EdU fluorescence analysis: the CFPAC-1 cells in log phase were harvested and plated in 96-well plates and 100. mu.L of cells containing 103-104 cells per well were cultured until the cell confluence was 80%. And adding the miRNA into the opti-MEM serum-free medium, mixing the miRNA and the opti-MEM serum-free medium gently, mixing the miRNA with an appropriate amount of Lipofectamin 2000, and standing the mixture at room temperature for 20min to form a compound of the miRNA and the Lipofectamin. The miRNA and Lipofectamin complex is added to the cells containing the culture solution, and after 6 hours, the cells are replaced by fresh complete medium and then cultured for 48 hours. The EdU reagent was diluted with the cell culture medium at a ratio of 1000:1 to prepare a 50. mu.M EdU medium, and 100. mu.L of 50. mu.M EdU medium was added to each well and incubated at 37 ℃ for 2 hours. After incubation, the medium was discarded and the cells were washed 2 times with pre-cooled PBS for 5 min/time. 100 mu L of 1 XApollo dyeing reaction liquid is added into each hole, and after incubation for 30min under the conditions of dark, room temperature and a decoloring shaking table, the reaction liquid is discarded. Adding 100 μ L of penetrating agent containing 0.5% TritonX-100, and washing with decolorizing shaker for 2-3 times (10 min/time). Then, 100 μ L of methanol was added to each well in sequence to wash the cells for 2 times and 5 min/time, and precooled PBS was added to each well to wash the cells for 2 times and 5 min/time. And (4) observing under a fluorescence microscope and imaging.

3. Results of the experiment

(1) The miRNA-21-5p can promote cell proliferation by high expression and inhibit cell proliferation by low expression. The miRNA-21-5p mimic promotes the expression of miRNA-21-5p genes, thereby improving the proliferation capacity of CFPAC-1 cells. However, when the gene expression of miRNA-21-5p is inhibited, the proliferation capability of CFPAC-1 cells is obviously inhibited. The results prove that miRNA-21-5p can regulate the proliferation capacity of CFPAC-1 of pancreatic cancer cells, and the proliferation activity of CFPAC-1 cells is in positive correlation with the expression of miRNA-21-5p (figure 2).

(2) Compared with the mimic NC group, the miRNA-21-5p mimic can obviously increase the proportion of cells in the G0/G1 phase and promote the cell division of CFPAC-1. In contrast, miRNA-21-5p is effective in reducing the proportion of cells in G0/G1 phase and blocking the CFPAC-1 cell cycle in S phase, thereby inhibiting the proliferation of cells, compared with the inhibitor NC group. The above results reveal that miRNA-21-5p regulates the cycle division of CFPAC-1 cells, and high expression of the miRNA-21-5p can promote cell division; low expression can cause cell cycle arrest (fig. 3-7).

(3) The high expression of miRNA-21-5p can reduce apoptosis, and the low expression can promote apoptosis. Compared with the mimic NC group, the miRNA-21-5p with high expression can reduce the apoptosis ratio of CFPAC-1. On the contrary, when the expression of miRNA-21-5p is inhibited, the CFPAC-1 cell can be remarkably promoted to generate apoptosis. In conclusion, miRNA-21-5p is in negative correlation regulation to regulate the apoptosis of CFPAC-1 cells, and the regulation effect of miRNA-21-5p on the proliferation and cycle division of CFPAC-1 cells is proved (figure 8-figure 12).

(4) The high expression of miRNA-21-5p can promote cell proliferation, and the low expression can inhibit cell proliferation. Compared with a mimic NC group, the miRNA-21-5p mimic obviously activates the proliferation activity of the CFPAC-1 cells; on the contrary, compared with the inhibitor NC group, the miRNA-21-5p inhibitor effectively reduces the proliferation activity of the CFPAC-1 cells. The above results demonstrate from a molecular level that miRNA-21-5p enhances the proliferative activity of CFPAC-1 (FIG. 13).

Sequence listing

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Claims

1. The miRNA marker for clinical diagnosis of pancreatic cancer is characterized in that the biomarker is miRNA-21-5p gene, and the nucleotide sequence of the biomarker is shown in SEQ ID NO: 1 is shown.

The application of miRNA-21-5p gene as a biomarker for clinical diagnosis of pancreatic cancer is characterized in that the nucleotide sequence of miRNA-21-5p gene is shown as SEQ ID NO: 1 is shown.

3. The use of claim 2, wherein a significant increase in the relative copy number of nucleotides of the miRNA-21-5p gene positively correlates with pancreatic carcinogenesis.

4. The use of claim 3, wherein the increase in the relative copy number of nucleotides of the miRNA-21-5p gene, which is a biomarker, promotes the proliferation of tumor cells, decreases the apoptosis rate of tumor cells, and promotes the cell cycle; conversely, a decrease in copy number will inhibit the proliferation of tumor cells, increase the rate of apoptosis of tumor cells, and inhibit the cell cycle.

5. A mimic of miRNA-21-5p gene, wherein the nucleotide sequence of the mimic of miRNA-21-5p gene is as shown in SEQ ID NO: 2 is shown in the specification; the control nucleotide sequence of the miRNA-21-5p gene mimic is shown in SEQ ID NO 3: as shown.

6. The use of claim 5, wherein the expression level of miRNA-21-5p in the cell is significantly increased by transfecting the miRNA-21-5p gene mimic into a cell line.

7. An inhibitor of miRNA-21-5p gene, wherein the nucleotide sequence of the inhibitor of miRNA-21-5p gene is shown in SEQ ID NO: 4 is shown in the specification; the reference nucleotide sequence of the miRNA-21-5p gene inhibitor is shown as SEQ ID NO: 5, respectively.

8. The use of claim 7, wherein the miRNA-21-5p gene inhibitor is transfected into a cell line, so that the expression level of miRNA-21-5p in the cell can be significantly reduced.

Application of miRNA-21-5p gene in preparation of kit for diagnosing pancreatic cancer.

10. A kit for diagnosing pancreatic cancer is characterized by comprising an RNA extraction reagent, a miRNA-21-5p gene detection primer pair, an internal reference U6 gene primer pair, a buffer reagent, a reverse transcription cDNA primer and an instruction book;

wherein, the sequence of the forward primer of the internal reference RNA U6 is shown as SEQ ID NO: 6, the reverse primer sequence is shown as SEQ ID NO: 7 is shown in the specification;

the reverse transcription cDNA primer sequence is shown as SEQ ID NO: 8 is shown in the specification;

the sequence of the fluorescent quantitative PCR forward primer is shown as SEQ ID NO: 9 is shown in the figure;

the fluorescent quantitative PCR reverse primer sequence is shown as SEQ ID NO: shown at 10.