CN108531587A - Detection methods of the microRNA -138-5p to human pancreatic cancer cell Apoptosis under hypoxemia - Google Patents

Abstract

The invention belongs to the technical field of pharmaceutical preparations containing organic active ingredients, and discloses a method for detecting the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxia. After miR-138-5p mimics are up-regulated, western blot Autophagy-related proteins in the experimental group were detected to be inhibited; autophagosomes in pancreatic tumor cells in the experimental group were observed to increase by confocal microscopy. The percentage of apoptotic cells plus the percentage of necrotic cells had significant differences among the groups, and the expression of apoptosis-related proteins in the experimental group was up-regulated by western blot. miR‑138‑5p complementarily binds to the 3'UTR region of SIRT1 and inhibits the reduction of SIRT1 expression. miR‑138‑5p plays an important role in the autophagic flow of pancreatic cancer cells under hypoxic conditions, inhibits hypoxia-induced autophagy by directly targeting and regulating the expression of SIRT1, and promotes the apoptosis of panc‑1 pancreatic cancer cells .

Description

Detection method for the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxia

technical field

The invention belongs to the technical field of pharmaceutical preparations containing organic active ingredients, and in particular relates to a method for detecting the effect of microRNA-138-5p on apoptosis of human pancreatic cancer cells under hypoxia.

Background technique

At present, the existing technologies commonly used in the industry are as follows: the degree of malignancy of pancreatic cancer is relatively high, the 5-year survival rate is less than 5%, and the prognosis of most patients is not very satisfactory. According to the 2015 Chinese cancer statistics, the death rate of pancreatic cancer ranks sixth among all cancers. The incidence of pancreatic cancer is gradually increasing every year, and it is likely to become the second most lethal disease in the near future. Therefore, it is of great significance to further elucidate the molecular mechanism of the occurrence and development of pancreatic cancer for its early diagnosis and development of new therapeutic sites. Tumor hypoxic microenvironment is one of the basic characteristics of human malignant solid tumors. Tumor hypoxia is closely related to its malignant biological behavior. Clinical studies have shown that pancreatic cancer has the appearance of no blood vessels under enhanced CT scan, and the tumor body is hypoxic. Tension, indicating that pancreatic cancer is in a hypoxic environment and has the pathophysiological characteristics of ischemia and hypoxia. Hypoxia is closely related to malignant biological characteristics such as the occurrence, development, invasion and metastasis, recurrence and chemotherapy resistance of pancreatic cancer. MicroRNA (miR) is a class of small molecules widely present in eukaryotes, with a length of about 19-23 nucleotides. It is highly conserved and does not directly encode a protein, but binds to the mRNA in the 3'-untranslated region of the target gene, thereby degrading miR and (or) inhibiting its translation. More and more evidences show that miRNAs play an important role in regulating the biological behavior of cells. However, the functions of miRNAs in pancreatic cancer are not fully understood, and their relationship with autophagy conditions is still unclear. However, miRNA and autophagy may play an important role in the occurrence and development of pancreatic cancer, so clarifying the relationship between miRNA and autophagy conditions in pancreatic cancer will help the diagnosis and treatment of pancreatic cancer.

To sum up, the problems existing in the existing technology are: little is known about the relationship between miRNA and autophagy conditions in pancreatic cancer, the function of miRNA in pancreatic cancer is not completely clear, and its relationship with autophagy conditions is not yet clear. clear.

The difficulty and significance of solving the above technical problems: the present invention explores its role in the occurrence and development of pancreatic cancer by detecting the differential expression of miRNAs in pancreatic cancer under hypoxic conditions and the detection of related biological effects.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a method for detecting the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxic conditions, by detecting the differential expression of miRNAs in pancreatic cancer under hypoxic conditions and The relevant biological effect detection explores its role in the occurrence and development of pancreatic cancer. Finally, the gene miRNA-138-5p with the most significant difference was selected as the analysis object to clarify its biological role in inhibiting the occurrence and development of pancreatic cancer.

The present invention is achieved in this way, a method for detecting the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxia, the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxia The detection method comprises the following steps:

Step 1: Differential miRNA screening of pancreatic cancer cells under normal oxygen and hypoxic conditions was performed using gene chips, and the pancreatic cancer panc-1 cell line was divided into two groups: miR-138-5p mimics transfection group and control group;

Step 2, analyzing the effects of miR-138-5p on the autophagy and apoptosis of the human pancreatic cancer cell line PANC1 under hypoxic conditions;

Step 3, the dual-luciferase reporter genotyping system verifies the complementary binding between miR-138-5p and the 3' non-coding region of SIRT1, confirming that SIRT1 is the downstream target of miR-138-5p.

Further, in the second step, the effects of miR-138-5p on the autophagy and apoptosis of the human pancreatic cancer cell line PANC1 under hypoxic conditions were analyzed using flow cytometry, confocal microscopy and western blot techniques.

Another object of the present invention is to provide a drug for inhibiting the proliferation of pancreatic cancer cells obtained from the method for detecting the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxia.

Another object of the present invention is to provide a pancreatic cancer cell line with high liver metastasis obtained from the method for detecting the effect of microRNA-138-5p on apoptosis of human pancreatic cancer cells under hypoxia.

In summary, the advantages and positive effects of the present invention are: the miRNA chip shows that, compared with pancreatic cancer cell lines under normoxia, 83 differentially expressed miRNAs were screened in pancreatic cancer tissues, of which 73 were highly expressed, and 10 The expression of the bar was low, and the difference was statistically significant (P<0.05). After miR-138-5p mimics were up-regulated, autophagy-related proteins in the experimental group were inhibited by western blot detection; autophagosomes in pancreatic tumor cells in the experimental group increased by confocal microscopy. The percentage of apoptotic cells plus the percentage of necrotic cells had significant differences among the groups (P<0.05), and the expression of apoptosis-related proteins in the experimental group was up-regulated by western blot. miR-138-5p complementarily binds to the 3'UTR region of SIRT1 and inhibits the reduction of SIRT1 expression. miR-138-5p plays an important role in the autophagy flow of pancreatic cancer cells under hypoxic conditions, inhibits hypoxia-induced autophagy by directly targeting and regulating the expression of SIRT1, and promotes the apoptosis of panc-1 pancreatic cancer cells . This may be one of the mechanisms by which it suppresses the survival of pancreatic cancer cells.

Description of drawings

Fig. 1 is a flow chart of the method for detecting the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxia provided by the embodiment of the present invention.

Fig. 2 is a clustering diagram of differential expression of microRNAs in pancreatic cancer cell lines under normoxic conditions (N) and hypoxic conditions (H) provided in an embodiment of the present invention.

Fig. 3 is a schematic diagram of overexpression of miR-138-5p inhibiting the dephosphorylation of the autophagy initiation complex mTOR under hypoxic conditions provided by the embodiment of the present invention.

Fig. 4 is a schematic diagram showing the inhibitory effect of overexpressed miR-138-5p on autophagy of pancreatic tumor cells under the hypoxic culture condition of A provided in the embodiment of the present invention, which is gradually obvious with the increase of transfection concentration.

Fig. 5 is the confocal laser provided by the embodiment of the present invention showing that the autophagy-lysosome generation of pancreatic cancer cells transfected with Pre-miR-138-5p is inhibited. Cells expressing miR-138-5p have reduced red free spots (autophagy lysosomes) and increased yellow spots (autophagosomes) B. Statistical histograms of the two spots in each group, and a schematic diagram of the specific statistical differences between the groups.

Fig. 6 is a histogram of the apoptosis ratio of experimental group A and the control group after overexpression of miR-138-5p under the hypoxic culture condition provided by the embodiment of the present invention. It can be seen from the figure that the expression of miR-138-5p is up-regulated , the number of apoptotic cells in the experimental group was significantly increased; B the histogram of the apoptotic ratio of each group, comparing the specific statistical significance of the differences among the groups; C after overexpressing miR-138-5p, the apoptosis-related proteins cytochrome C and splicing body-caspase3 Expression is upregulated.

Figure 7 is the miR-138-5p target prediction and verification provided by the embodiment of the present invention A: Targetscan software predicts the miR-138-5p binding site in the 3'UTR region of SIRT1; B: double fluorescein plum experiment; C: Western The blot experiment showed that the expression of miR-138-5p in up-regulated PANC1 cells decreased the expression of SIRT1 protein.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The invention detects the expression of differential microRNA (miR) in human pancreatic cancer under hypoxic conditions, and explores its influence on pancreatic cancer in hypoxia-induced autophagy.

As shown in Figure 1, the method for detecting the effect of microRNA-138-5p on the apoptosis of human pancreatic cancer cells under hypoxia provided by the embodiments of the present invention comprises the following steps:

S101: Differential miRNA screening of pancreatic cancer cells under normoxic and hypoxic conditions using gene chips, and the pancreatic cancer panc-1 cell line was divided into two groups: miR-138-5p mimics transfection group and control group;

S102: Analyze the effects of miR-138-5p on the autophagy and apoptosis of the human pancreatic cancer cell line PANC1 under hypoxic conditions by using techniques such as flow cytometry, confocal microscopy and western blot;

S103: The dual-luciferase reporter genotyping system verified the complementary binding of miR-138-5p to the 3' non-coding region (3'UTR) of SIRT1 to confirm that SIRT1 is the downstream target of miR-138-5p.

The application principle of the present invention will be further described in conjunction with experiments below.

1 Materials and methods

1.1: Source of materials: The human pancreatic cancer cell line PANC1 was donated by Professor Qin Renyi from Tongji Medical College, Huazhong University of Science and Technology. High-glucose DMEM culture medium, special grade fetal bovine serum, double antibody solution and OPTI-MEM were purchased from Gibco, USA. miR-138-5pmimics, non-specific NC was designed and synthesized by Guangzhou Ribobiological Company. The GAPDH primary antibody was purchased from Wuhan Sanying Biotechnology Company, and mTOR, LC3II, P62, and cleavage casepase 3 were purchased from CST Company in the United States. The secondary antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. Lip02000 reagent was purchased from Invitrogen, USA. Apoptosis detection kit was purchased from KGI Corporation. The mRFP-GFP-LC3 dual fluorescent autophagy indicator virus was purchased from Shanghai Hanbio Company. miR-138-5p adenovirus was purchased from Shandong Weizhen Biological Company.

1.2 Cell culture: PANC1 cells were cultured in high-glucose DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, and streptomycin in a nuclei humidity incubator at 37°C and 5% CO ₂ under normal oxygen. Culture in a nuclei humidity incubator at 37°C, 5% CO ₂ , 1% O ₂ under hypoxia. 0.25% trypsin solution was used for nitrification, subculture, and the cells in the logarithmic growth phase were selected for experiments.

1.3 miR chip data analysis: clustering method was used to analyze the differential miRNAs of three pairs of pancreatic tumor cells under normoxia and hypoxia. It is meaningful that each signal distribution curve basically coincides. The ratio of the two normalized signals to be compared was taken as the comparison data (FoldChange=SignalA/SignalB, FC), and the significant P value was calculated. P<0.05 was considered as a statistically significant difference.

1.4 Western Blotting to detect protein expression: extract protein from pancreatic tumor cells with logarithmic growth, take 20ug of total cell protein for polyacrylamide gel electrophoresis, transfer to polyethylene double oxide (PVDF) membrane, shake and seal, Add primary antibody (1:1000), incubate overnight at 4°C, add horseradish peroxidase-labeled secondary antibody (1:2000), incubate at 37°C for 2 hours, add luminescence reagent after washing the membrane, after the electrochemiluminescence kit emits light Exposure, with GAPDH as internal reference.

1.5 Annexin V-FITC/PI double-staining flow cytometry to detect cell apoptosis: Collect cells from different treatment groups in centrifuge tubes, add 4°C pre-cooled PBS to wash the cells three times after centrifugation, centrifuge at 800r/min for 5min, and discard the supernatant. Suspend the cells with 200 μl BindingBuffer. Add 3 μl Annexin V-FITC and 5 μl PI to each tube in turn, mix well; react at room temperature in the dark for 20 minutes; run the flow cytometer to detect and record the results. luc-SIRT1 and luc-SIRT1 mutants were identified by DNA sequencing. Use lipofectamine 3000 to transfect PANC1 cells with SIRT1 3'UTR wild-type and mutant recombinant vectors, each vector set up a blank group, transfect miR-138-5p mimic, mimic negative control, miR-138-5p inhibitor, inhibitor The negative control group was cultured for 24 hours, and operated according to the instructions of Prgmega’s double fluorescein detection kit. Each group had 3 replicate wells, and the fluorescence intensity of the cells in each well of the 24-well plate was detected. The fluorescence measurement ratio of each hole is firefly luciferase activity (F)/renilla luciferase activity value (R), and the double fluorescence measurement ratio of each group is analyzed by △ activity endorsement, △ activity multiple = (R/F) sample /(R/F) control.

1.6 Construction of dual luciferase reporter vector PCR amplified the region of this site, respectively constructed SIRT1 wild-type and mutant vectors inserted into the luciferase reporter gene vector (pmiR-Report, Riobo, China), and detection: TargetScan prediction The binding site of miR-138-5p in the 3' UTR of human SIRT1 gene,

1.7 Statistical processing: SPSS 22.0 statistical software was used for data analysis. The t test was used for the measurement data, and the x2 test was used for the count data, and P<0.05 was considered statistically significant.

2 results

2.1 Pancreatic cancer cell miRNA chip screening under normal oxygen and hypoxic conditions: using miR chip (agilent V13.0) to detect 3 pairs of pancreatic cancer cells cultured under different conditions. Results A total of 83 differential miRs were screened out, including 10 low-expression miRNAs including miR-138-5p. Cluster analysis of these differential genes preliminarily confirmed that miR-138-5p may play an important role in the malignant biological behavior of pancreatic cancer cells (Figure 2).

2.2 Overexpression of miR-138-5p inhibited the transformation of pancreatic cancer cell line PANC1 mTOR to pmTOR, and at the same time inhibited the expression of LC3II, and the difference in protein expression was statistically significant (P<0.05, Figure 3). With the increase of miR-138-5p transfection concentration in the pancreatic cancer cell line PANC1, the expression inhibition of LC3II was gradually obvious and reached the peak at 100nM. (Figure 4)

2.3 Laser confocal microscope detection: use a 40 times oil lens to observe the sample, and output the image after processing. The result is shown in Figure (6). The aggregation of LC3 into plaques indicates the formation of autophagosomes (green), and the red free spots represent the formation of autolysosomes. The results are shown in the figure. Compared with the control group, the number of red spot-positive cells in the pre-miR-138-5p treatment group was significantly reduced, and the number of yellow spots was significantly increased. Statistical analysis showed that the difference between the two groups was statistically significant (P<0.05, Figure 5).

2.4 Overexpression of miR-138-5p promotes apoptosis of pancreatic cancer cells under hypoxic conditions: flow cytometry analysis showed that PANC1 cells transfected with miR-138-5p had increased apoptosis under hypoxic conditions. Western Blot detection of apoptotic protein splicing body casepase3 and cytochrome C found that the experimental group was significantly higher than the control group, and the difference was statistically significant (P<0.05, Figure 6).

2.5 SIRT1 is the direct target of miR-138-5p: Bioinformatics software is used to predict the possible targets of miR-138-5p. It can be seen that both TargetScan and miRANDa bioinformatics software can predict that SIRT1 is miR-138-5p Direct target ( FIG. 7A ), the direct target of miR-138-5p in SIRT1 in pancreatic cancer cells was verified by double luciferase assay ( FIG. 7B ). At the same time, Wstern blot also verified that up-regulating the expression of miR-138-5p can inhibit the expression of SIRT1 (Figure 7C), showing that SIRT1 is the direct target of miR-138-5p in pancreatic cancer.

The results of the present invention show that when the expression of miR138-5p in pancreatic cancer cells is up-regulated, Western blot detection shows that it significantly inhibits the autophagy of pancreatic cells under hypoxic conditions. -5p can inhibit the formation of autophagy lysosomes in tumor cells and cause the swollen and ruptured mitochondria in cells to release apoptotic proteins, thereby causing pancreatic tumor cell apoptosis. While up-regulating miR-138-5p, the expression of SIRT1 was found to be significantly reduced. Further, the dual-luciferase reporter gene analysis system confirmed that SIRT1 is the downstream molecular target of miR-138-5p, which can at least partially inhibit tumors by regulating the expression of SIRT1 The autophagy state of cells plays an important role in the occurrence and development of tumors, especially the low expression of miR-138-5p in the survival of pancreatic cancer. miR-138-5p may serve as an important marker of pancreatic cancer malignant behavior, and may provide an experimental basis for biological targeted therapy in terms of pancreatic cancer chemosensitivity.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (4)

1. detection methods of the microRNA -138-5p to human pancreatic cancer cell Apoptosis under a kind of hypoxemia, which is characterized in that described Detection method includes the following steps to human pancreatic cancer cell Apoptosis by microRNA -138-5p under hypoxemia：

Step 1 carries out difference miRNA screenings, by cancer of pancreas using genetic chip to pancreatic cancer cell under normal oxygen and hypoxia Panc-1 cell lines are divided into two groups：MiR-138-5p mimics transfection groups, control group；

Step 2 analyzes the miR-138-5p shadows of the autophagy to human pancreatic cancer cell PANC1 and apoptosis under low oxygen conditions It rings；

Step 3, luciferase reporter gene classification system verify the mutual of the ends miR-138-5p and SIRT13' non-coding region It mends and combines, it was demonstrated that SIRT1 is miR-138-5p downstream effects target spots.

2. microRNA -138-5p is to the detection method of human pancreatic cancer cell Apoptosis under hypoxemia as described in claim 1, It is characterized in that, flow cytometer, Laser Scanning Confocal Microscope and western blot technologies is utilized in the step 2, analyze miR- The influence of the 138-5p autophagy to human pancreatic cancer cell PANC1 and apoptosis under low oxygen conditions.

3. microRNA -138-5p is to human pancreatic cancer cell Apoptosis under a kind of hypoxemia described in claim 1~2 any one The obtained inhibition Cell Proliferation of Pancreatic Cancer Cell drug of detection method.

4. microRNA -138-5p is to human pancreatic cancer cell Apoptosis under a kind of hypoxemia described in claim 1~2 any one The obtained cancer of pancreas highly metastatic potential in the liver cell line of detection method.