CN117867125A - Colorectal cancer molecular marker miR-16-2-3P and application thereof - Google Patents
Colorectal cancer molecular marker miR-16-2-3P and application thereof Download PDFInfo
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Abstract
The invention discloses colorectal cancer molecular marker miR-16-2-3P and application thereof. The miR-16-2-3P is found to be highly expressed in colorectal cancer cells and tissues for the first time, can be used as a molecular marker for diagnosing early colorectal cancer, is closely related to increase of tumor angiogenesis, and is expected to be developed into a therapeutic drug for colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a colorectal cancer molecular marker miR-16-2-3P and application thereof.
Background
Colorectal cancer is a common malignant tumor of the digestive tract, and the new incidence and the death rate of colorectal cancer are respectively in the front of cancer, and are fourth malignant tumors worldwide. Colorectal cancer cell proliferation is an important biological behavior causing postoperative recurrence of colorectal cancer, and in colorectal cancer related research reports, miRNAs play a very important role in regulating the growth and proliferation processes of various colorectal cancer cells, and can induce cell growth and promote the progression of a cell cycle by directly or indirectly stimulating oncogenes, and can activate cell cycle arrest by stimulating oncogenes.
Studies such as ISLAM and the like find that in colorectal cancer cell line SW620 cells, the overexpression of miR-142-5p can increase the activity of colorectal cancer cells and promote migration of colorectal cancer cells. HUANG et al have found that in colorectal cancer cells HT-29, miRNA-19-5p can promote proliferation of colorectal cancer cells by targeted inhibition of TSPYL5 expression, and can reduce proliferation of colorectal cancer cells by targeted enhancement of TSPYL5 expression.
The main treatment for colorectal cancer is surgical excision, especially for early patients, with high 5-year survival rates after surgery, but most patients have already migrated lesions before surgery, which is clinically rare and difficult to perform surgical treatment, resulting in relatively high mortality rates for middle and late patients.
Tumor invasion and metastasis have progressive and continuous properties, and are a multi-factor and multi-link dynamic process. The basic process is as follows: firstly, the mass proliferation of tumor cells in the primary foci; secondly, tumor cells can fall off from the primary focus and attack the basal membrane part of the tumor cells, so that the tumor cells invade blood vessels-lymphatic vessels and even body cavities; thirdly, a few tumor cells can survive in the circulatory system to form cancer plugs, and the cancer plugs are migrated to another distant organ or part along with blood and lymph; and fourthly, tumor cells and target organs are adhered to the capillary vessel wall, pass through the vessel wall to form tiny metastasis focus, thereby forming secondary tumors of the same type as the primary tumors, and simultaneously, the tumor cells can be affected again and even metastasized. As can be seen from the above, invasion and metastasis of tumor cells is a complex process involving angiogenesis, tumor cell extravasation, organ-specific regrowth, and the like.
Current diagnostic means for colorectal cancer are mainly endoscopy. Endoscopy can cause some pain to the subject and is difficult for certain groups to tolerate, so there are limited groups that can be accepted and covered. There are also blood-based noninvasive detection means using blood septin9 methylation detection, but the sensitivity of the detection is not high; therefore, there is a need to develop a highly sensitive method for early detection of noninvasive colorectal cancer. Patent 201811126210.X discloses exosome miRNA markers for colorectal cancer diagnosis, said markers being selected from at least one of let-7b-3p, let-7b-5p, miR-150-3p, miR-145-3p, miR-139-5p, miR-15b-3p, miR-125a-5p, miR-140-5p, miR-342-3p, miR-132-5p and miR-92b-5p. Finding miRNA markers proves to be a potential solution to the problem of early detection of non-invasive colorectal cancer.
Disclosure of Invention
The invention aims to provide a colorectal cancer molecular marker miR-16-2-3P and application thereof.
A colorectal cancer molecular marker, which is miR-16-2-3P.
The molecular marker miR-16-2-3P is highly expressed in colorectal cancer cells and tissues.
A kit for diagnosing colorectal cancer, the kit comprising a colorectal cancer biomarker that is miR-16-2-3P.
The kit also includes enzymes and reagents for a PCR reaction.
Application of miR-16-2-3P in preparation of medicines for diagnosing colorectal cancer.
Application of miR-16-2-3P inhibitor in preparation of medicines for treating colorectal cancer is provided.
Application of miR-16-2-3P inhibitor in preparation of medicines for inhibiting colorectal cancer tumor angiogenesis.
The invention has the beneficial effects that: the miR-16-2-3P is found to be highly expressed in colorectal cancer cells and tissues for the first time, can be used as a molecular marker for diagnosing early colorectal cancer, is closely related to increase of tumor angiogenesis, and is expected to be developed into a therapeutic drug for colorectal cancer.
Drawings
FIG. 1 is the expression levels of miR-16-2-3P in different colorectal cancer cells.
FIG. 2 shows the change after transfection of human colorectal cancer cells HCT116 with miR-16-2-3P mimic and human colorectal cancer cells SW480 with miR-16-2-3P inhibitor.
FIG. 3 is a Western blot analysis of PTEN protein in human colorectal cancer cells HCT116 transfected with miR-16-2-3P mimics and human colorectal cancer cells SW480 transfected with miR-16-2-3P inhibitors.
FIG. 4 is a graph showing protein expression analysis of PTEN protein in human colorectal cancer cells HCT116 transfected with miR-16-2-3P mimics and human colorectal cancer cells SW480 transfected with miR-16-2-3P inhibitors.
FIG. 5 is closed tubule formation by Human Umbilical Vein Endothelial Cells (HUVECs) in conditioned medium from HCT116 cells with miR-16-2-3P overexpression.
FIG. 6 is closed tubule formation by Human Umbilical Vein Endothelial Cells (HUVECs) in conditioned medium from SW480 cells with miR-16-2-3P inhibition expression.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
Example 1 colorectal cancer-related exosome miRNA marker screening
30 cases of colorectal cancer specimens were collected from menstrual resections from month 07 to year 2021 of the department of Guangxi university affiliated oncology hospital 2020. Of these, 14 men and 16 women; ages 30-64. Wherein, 12 patients with early colorectal cancer, 18 patients with middle and late stage, all patients with selected patients are informed consent and approved by the ethical examination committee of the present department, the patients are not treated by radiotherapy and chemotherapy before operation, and the resected specimens are respectively stored in liquid nitrogen or neutral formaldehyde.
And selecting 12 cases of early colorectal cancer patients and controls, taking not less than 10ml of blood and separating plasma, separating exosomes in the plasma by using a classical ultracentrifugation method, extracting RNA by using qiagen miRNeasy mini kit, and carrying out miRNA library sequencing on the obtained RNA by using a NEB miRNA library kit. The obtained data are subjected to bioinformatics analysis, miRNAs which are differentially expressed in early colorectal cancer patients and controls are compared, and are subjected to correlation analysis with miRNA sequencing of tissues in TCGA, and the selected exosome miRNAs derived from tumors, including let-7b-5p, miR-150-3p, miR-145-3p, miR-139-5p, miR-140-5p, miR-16-2-3P, miR-132-5p and miR-92b-5p, are screened. These miRNA-level markers from exosomes can be used for early diagnosis of colorectal cancer. Wherein, let-7b-5P, miR-150-3P, miR-145-3P, miR-139-5P, miR-140-5P, miR-132-5P and miR-92b-5P have been disclosed by other documents, and the inventor selects miR-16-2-3P for subsequent experiments. The sequence of the miR-16-2-3P is as follows: CCAAUAUUACUGUGCUGCUUUA.
Example 2
Human colorectal cancer cells HCT116, HT29, SW480 were given by the affiliated synergetics of the university of china science and technology and the hospital. Cell culture: colorectal cancer cells HCT116, HT29 and SW480 were cultured in high-sugar DMEM complete medium, 37℃at 5% CO 2 Culturing, stably passaging for 2-4 generations, and taking cells in logarithmic growth phase for subsequent experiments.
Total RNA in colorectal cancer cells and normal colorectal epithelial Hertz membrane tissues is extracted according to the TRIzoI reagent instruction, and reverse transcription conditions are set according to the RNA reverse transcription kit instruction.
1 μg of total RNA was taken for each sample and subjected to reverse transcription as follows:
and (3) configuration of a reaction system: a5. Mu.L Reverse Transcription (RT) reaction system consisted of 1. Mu.L probe, 0.05. Mu.L dNTPs, 0.33. Mu.L RTase enzyme, 0.5. Mu.L reverse transcription buffer, 0.07. Mu.L LRNA enzyme inhibitor, 1.38. Mu.L LRNase-free water and 1.67. Mu.L RNA sample, which were gently shaken well.
Reverse transcription: the reaction was incubated at 16℃for 30min, at 42℃for 30min, at 85℃for 5min, and then maintained at 4 ℃.
Real-time quantitative PCR: real-time quantitative PCR reactions were performed using TaqMan PCR kit (Applied Biosystems Co., USA), and the specific steps were as follows:
1) Dilution of cDNA
To 5. Mu.L of cDNA product after the completion of the reverse transcription, 28.9. Mu.L of LRNase-free water was added, followed by shaking slightly and centrifugation.
2) Configuration of the reaction System
A reaction system is configured on an ice plate of an ultra-clean workbench: the 5. Mu.L PCR reaction system included 0.25. Mu.LPCR probe, 2.5. Mu.L LTaqMan universal PCR Master Mix, 2.25. Mu.L diluted cDNA, slightly shaken, and centrifuged.
3) Setting blank control
The 5. Mu.L blank included 0.25. Mu.L PCR probe, 2.5. Mu.L TaqMan Universal PCR Master Mix, 2.25. Mu.LRNase-free water.
4) Quantitative PCR
The settings were started, 2min at 50℃and 10min at 95℃and then 40 thermal cycles were performed: a blank was set at 95℃for 15s and 60℃for 1min for each group to detect contamination during the process.
And (3) expressing miR-16-2-3P by adopting an absolute quantitative method, establishing a standard curve and a regression equation according to a standard substance, and calculating the concentration of miR-16-2-3P. miRNAs were normalized to quantification and the results were expressed as miRNA in total RNA/. Mu.g, content (fmol/. Mu.g total RNA). RNA extracted from cells is taken as an internal reference, the relative expression quantity of each group of miR-16-2-3P is calculated, the experiment is repeated 3 times, and the average value is calculated.
Statistical analysis using SPSS19.0 software for metering data resultsThe data is normally tested by a Kolmogorov-Smirnov test method, and for data conforming to normal distribution, the mean difference between two groups is compared by a t test to obtain P<0.05 is the difference with statisticsMeaning.
By taking normal colorectal epithelial hertz (NT) as a control, the expression level of miR-16-2-3P in 3 human colorectal cancer cells is significantly higher than that of the normal control, and the difference is statistically significant (P < 0.05). Wherein HCT116 cell miR-16-2-3P is expressed lowest, and SW480 cell miR-16-2-3P is expressed highest (figure 1).
Example 3
The PTEN gene is an oncogene with dual specific phosphatase activity found subsequent to p53, having 9 exons and 8 introns, with the 5' -terminal ATG and CGG repeats of the cDNA forming the structural basis for methylation, and its lipid phosphatase activity depending on the structure homologous to the protein serine/threonine enzyme and protein tyrosine phosphatase catalytic centers. Many factors responsible for cancer are reducing PTEN gene expression, so the aim of this experiment was to verify whether miR-16-2-3P overexpression affected PTEN gene expression.
Establishing a cell model for high expression and inhibition of miR-16-2-3P expression: human colorectal cancer cells HCT116 and SW480 in logarithmic growth phase were transiently transfected. Cells were seeded in 6-well plates and cultured until the confluence reached about 60%. The experimental components are respectively added with 50nM miR-16-2-3P mic and 100nM miR-16-2-3P inhibitor and 5 mu L transfection reagent; the control groups were each added with a corresponding Negative Control (NC). The final volume of the l×Opti-MEM medium was adjusted to 2mL and the culture was continued for 6 hours with medium replacement. Cells from each group were collected 48h after transfection for RNA extraction, and cell proteins and serum-free culture supernatants (conditioned medium) were collected 72h after transfection.
The procedure of RNA extraction from transfected cells, reverse transcription and real-time quantitative PCR is described in example 2.
Western blot: extracting cell total protein by RAPA, and quantifying the extracted protein by a BCA protein quantification method; preparing 5% SDSPAGE concentrated gel and 15% SDS-PAGE separating gel, and loading 100ug of total protein; carrying out protein running electrophoresis by 80V 20min and 150V 1h; transferring the film for 2 hours by adopting 100V film transferring; sealing 5% of skimmed milk for 1h; goat anti-rabbit secondary antibody (1:4000) was added, incubated at room temperature for 1h, and TBST rinsed 6 times for 5 min/time. Dripping ECL developer, imaging by using a chemiluminescent imager, measuring the gray value of a target band, analyzing protein expression, and calculating the relative expression quantity of PTEN protein by taking GAPDH as an internal reference.
The measurement results are shown in FIGS. 2 to 4: transfection of miR-16-2-3P mic leads miR-16-2-3P in human colorectal cancer cells HCT116 to be obviously increased, and the grey value of PTEN protein strips is reduced; transfection of miR-16-2-3P inhibitor significantly reduces miR-16-2-3P expression level in human colorectal cancer cells SW480, and the grey value of PTEN protein strips is increased. The results suggested that the expression level of miR-16-2-3P was inversely related to PTEN protein expression.
Example 4
Tubule formation experiments: 50 mu L Matrigel was spread on each well of 96-well plate and inoculated with 1X 10 4 HUVEC (human umbilical vein endothelial cells) were cultured for 24h, and the experimental group and control group were respectively added with 100. Mu.L of HCT116 and SW480 cell conditioned medium corresponding to the collected transfected miR-16-2-3P mic and inhibitor, at 37deg.C, 5% CO 2 After 6h incubation in the environment, HUVEC growth was observed under a microscope and counted as 1 nascent tubule forming a closed loop tubule-like structure. Each group had 3 complex wells, and each well had 3 fields of view averaged.
Experimental results show that, HCT116 cells which are used for up-regulating miR-16-2-3P expression by using transfected miR-16-2-3P mic act for 6 hours on a conditioned medium for miR-16-2-3P expression, and the number of HUVECs forming complete closed tubules is obviously higher than that of a control group (t=5.586, P <0.05, figure 5); in contrast, SW480 cells transfected with miR-16-2-3P inhibitor for inhibiting miR-16-2-3P expression are used, the conditioned medium for miR-16-2-3P expression acts for 6 hours, and the number of closed tubules formed by HUVEC is obviously lower than that of a control group (t=)
-6.232, p <0.05, fig. 6).
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (7)
1. A colorectal cancer molecular marker, which is characterized by being miR-16-2-3P.
2. The molecular marker of colorectal cancer according to claim 1, wherein the molecular marker miR-16-2-3P is highly expressed in colorectal cancer cells and tissues.
3. A kit for diagnosing colorectal cancer, characterized in that the kit comprises a colorectal cancer biomarker that is miR-16-2-3P.
4. A kit for diagnosing colorectal cancer according to claim 3, wherein the kit further comprises enzymes and reagents for a PCR reaction.
Application of miR-16-2-3P in preparation of medicines for diagnosing colorectal cancer.
Application of miR-16-2-3P inhibitor in preparation of medicines for treating colorectal cancer.
Application of miR-16-2-3P inhibitor in preparation of medicines for inhibiting colorectal cancer tumor angiogenesis.
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