CN112043837A - Application of MiR-205-5p in preparation of angiogenesis agent - Google Patents
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Abstract
The invention relates to the technical field of angiogenesis inhibition, and discloses an application of MiR-205-5p in preparation of a angiogenesis agent, which comprises the following steps: the expression of miR-205-5p and CD31 in human gastric cancer tissues and liver metastases is detected by adopting quantitative real-time qRT-PCR; the effects of miR-205-5p silencing and high expression are researched by detecting the formation of human umbilical vein endothelial cell capillaries in vitro and detecting a skin wrinkle chamber model on the back side of a human body; qRT-PCR and western blot are adopted to detect the influence of miR-205-5p on the expression of VEGFA and FGF1, and MiR-205-5p regulates the expression of VEGFA and FGF1 through ERK signaling pathway and direct targeting. According to the invention, MiR-205-5p is used for down-regulating the expression of VEGFA and FGF1 through an ERK signal pathway and direct targeting, so that gastric cancer angiogenesis is inhibited, especially important effects are played in angiogenesis, and miR-205-5p is used for reducing the formation of new blood vessels through negatively regulating VEGFA expression, so that miR-205-5p is a cancer inhibition factor for inhibiting angiogenesis in gastric cancer and plays an important role in the proliferation and invasion of different types of tumors.
Description
Technical Field
The invention relates to the technical field of angiogenesis inhibition, and in particular relates to application of MiR-205-5p in preparation of an angiogenesis agent.
Background
Gastric Cancer (GC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Almost two-thirds of gastric cancer cases occur in developing countries. With the introduction of postoperative adjuvant therapy, the prognosis of gastric cancer patients is greatly improved. However, median Overall Survival (OS) for metastatic and/or unresectable gastric cancer patients is less than 12 months [3], PD-1 and HER2 targeted therapies show good efficacy in gastric cancer therapy, but the proportion of patients who can benefit from targeted therapy is still very limited. Tumor angiogenesis plays an important role in the growth, invasion and metastasis and diffusion of gastric cancer, and anti-angiogenesis therapy is one of the most important and promising treatment methods in clinical oncology;
therefore, a signal molecule for regulating angiogenesis becomes a hot spot of current research, angiogenesis is not an active process per se and is controlled by some angiogenesis factors and some angiogenesis inhibitors, but the capability of miR-205-5p for regulating gastric cancer neovascularization is not clear, so that the technical personnel in the field provide an application of MiR-205-5p in preparing an angiogenesis agent to solve the problems in the background technology.
Disclosure of Invention
The invention aims to provide application of MiR-205-5p in preparation of an angiogenesis agent so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: an application of MiR-205-5p in preparing an angiogenesis agent comprises the following operations:
s1, detecting the expression of miR-205-5p and CD31 in human gastric cancer tissues and liver metastases by adopting quantitative real-time qRT-PCR;
s2, studying the silencing and high expression effects of miR-205-5p by detecting the formation of capillary vessels of endothelial cells of human umbilical veins in vitro and a skin wrinkle chamber model on the back side of a body;
s3, detecting the influence of miR-205-5p on VEGFA and FGF1 expression by adopting qRT-PCR and western blotting, and down-regulating the expression of VEGFA and FGF1 by MiR-205-5p through ERK signaling pathway and direct targeting.
As a further scheme of the invention: in order to further determine the influence of miR-205-5p expression on in vivo angiogenesis, different types of GC cell clones are adopted to establish a xenograft model, and an immunohistochemical method is adopted to detect the density of microvessels of liver metastases and xenograft tumors, wherein the xenograft model can adopt a small mouse model.
As a still further scheme of the invention: in the S3, a dual-luciferase report method is adopted to confirm the interaction between the 3'UTR of FGF1 and VEGFA, and the dual-luciferase report method shows that miR-205-5p can directly target the 3' UTR of VEGFA and FGF1 mRNA and regulate the expression of endogenous VEGFA and FGF1, so that VEGFA and FGF1 are direct targets of miR-205-5p, and miR-205-5p can regulate the expression of endogenous VEGFA and FGF1 through the direct interaction between a targeting ERK signaling pathway and miRNA-mRNA.
As a still further scheme of the invention: the formation of the capillary vessels of the human umbilical vein endothelial cells in the S2 means that matrix gel is conveyed into a precooled 96-well plate by a pipeline, the human umbilical vein endothelial cells are cultured in a condition culture medium of 100 microliters, the condition culture medium is taken from different types of cells, and after the culture is carried out for 36 hours, the capillary vessels are photographed to form structures;
the dorsal skinfold chamber model refers to the formation of a dorsal air pocket in the body of the subject by subcutaneous injection of 5ml of air after complete anesthesia of the subject, the resulting different types of cells were injected into the diffusion chamber and placed subcutaneously, and 10 days later, these subjects were carefully peeled around the implanted chamber and the skin folds covering the room were photographed under visible light.
As a still further scheme of the invention: the number of the different types of cells is 1.0 × 106, and the subject may select an animal, preferably a mouse.
As a still further scheme of the invention: the qRT-PCR detection in S3 is to adopt Trizol reagent and miRNeasy-FFPE kit to respectively extract total RNA from cell lines and paraffin embedded tissue sections;
in order to detect miR-205-5p, RNA is reversely transcribed into a cDNATM miRNA first strand cDNA synthesis kit by All-in-One, and then the miRNA qRT-PCR detection kit is used for carrying out qRT-PCR on microRNAs;
for detection of mRNAs, the RNA was reverse transcribed into cDNA using the GoScript reverse transcription system, and
qRT-PCR-Master Mix carries out qRT-PCR on the mRNAs;
all qRT-PCR used ABI-Prism 7500 system with U6 or GAPDH as internal control for quantification of microRNAs and mRNAs, respectively.
As a still further scheme of the invention: the Western blot assay in S3 was performed by using FGF1, VEGFA and GAPDH antibodies for FGF1, VEGFA and GAPDH, respectively, and (p-) SAPK/JNK, (p-) ERK1/2 and (p-) p-38 antibodies for activation of MAPK signaling pathway;
wherein, VEGFA is 1: 1000 dilution, GAPDH 1: 2000 dilution, (p-) p-38 is 1: and (8) diluting.
Compared with the prior art, the invention has the beneficial effects that: in the practical application process, the expression of VEGFA and FGF1 is down-regulated by MiR-205-5p through an ERK signal pathway and direct targeting, so that gastric cancer angiogenesis is inhibited, the expression of miR-205-5p plays an important role in regulating and controlling gastric cancer canceration, particularly angiogenesis, and miR-205-5p can reduce the formation of new vessels through negatively regulating and controlling VEGFA expression, so that miR-205-5p is a cancer suppressor for inhibiting angiogenesis in gastric cancer, the expression in gastric cancer cells has important influence on gastric cancer cell proliferation and angiogenesis, and plays an important role in proliferation and invasion of different types of tumors.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the application experiment process, all patients obtain written consent, and 109 cases of paraffin-embedded gastric cancer, normal tissues and 7 cases of liver metastases are total;
cell culture and transfection human gastric carcinoma cell lines BGC823 and MGC803 were obtained from the institute of tumor, national institute of medical science, all cells were grown in RPMI1640 medium supplemented with 10% Fetal Bovine Serum (FBS), all cells were grown in monolayer medium at 37 deg.C in 5% CO2Storing in the humidified air;
cell transfection lentiviral vectors LV-hsa-miR-205-5p, LV-hsa-miR-205-5p inhibitor or LV-hsa-miR negative control, all from Sigma-Aldrich (MO, USA), using BGC-823 and MGC-803 cells: plating 2 × 105 cells in 12-well plates overnight, then diluting 10 μ Ι _ lentivirus (2 × 108IU/mL) in 1mL fresh RPMI1640 medium supplemented with 10% FBS, after 24 hours we replaced the medium with fresh RPMI1640 medium, after 3 days 1 μ Ι/mL puromycin (Sigma-Aldrich, MO, USA) was added to the medium, supplemented every 2 days for 2 consecutive weeks, selecting lentivirus transfected cells;
an application of MiR-205-5p in preparing an angiogenesis agent comprises the following operations:
the expression of miR-205-5p and CD31 in human gastric cancer tissues and liver metastases is detected by adopting quantitative real-time qRT-PCR;
the effects of miR-205-5p silencing and high expression are researched by detecting the formation of human umbilical vein endothelial cell capillaries in vitro and detecting a skin wrinkle chamber model on the back side of a human body;
in order to further determine the influence of miR-205-5p expression on in vivo angiogenesis, different types of GC cell clones are adopted to establish a xenograft model, and an immunohistochemical method is adopted to detect the density of microvessels of liver metastases and xenograft tumors, wherein the xenograft model can adopt a small mouse model;
formation of human umbilical vein endothelial cell capillaries
A total of 1.0 × 106 GC cells or their corresponding transfected clones were seeded and cultured in complete RPMI1640 in 6-well slides and allowed to grow for 24 hours, and then analyzed for capillary formation, briefly, matrix gel was piped into pre-cooled 96-well plates, human umbilical vein endothelial cells were cultured in 100 microliters of conditioned medium, taken from different types of cells, and after 36 hours of culture, capillary formation was photographed;
and in order to confirm the function of miR-205-5p in regulating the growth of cancer cells and neovascularization in vivo, the transfected GC cell clones are compared with control clones, the capacity of forming tumors in a subcutaneous model is observed, the size of the xenograft tumors with reduced MVD (tumor necrosis factor) is determined by the expression of miR-205-5p, immunohistochemistry shows that the VEGFA and FGF1 expression of the miR-205-5p group is also reduced, meanwhile, the miR-205-5p inhibitor promotes the growth of the xenograft tumors by increasing the MVD, and the miR-205-5p inhibitor can also increase the expression of VEGFA and FGF1, so that the miR-205-5p is a cancer suppressor factor for inhibiting angiogenesis in gastric cancer cells
Dorsal skin fold chamber model
After the subjects were completely anesthetized, a dorsal air sac was formed in the subject by injecting 5ml of air subcutaneously, the resulting different types of cells were injected into a diffusion chamber and placed subcutaneously, and after 10 days, these subjects were carefully peeled around the implanted chamber and photographed the skin fold covering the room under visible light, and the number of blood vessels was calculated, and the subject could select animals, preferably mice;
qRT-PCR and western blot are adopted to detect the influence of miR-205-5p on the expression of VEGFA and FGF1, and MiR-205-5p regulates the expression of VEGFA and FGF1 through ERK signal pathway and direct targeting;
miR-205-5p can regulate the expression of endogenous VEGFA and FGF1 by targeting the direct interaction of an ERK signaling pathway and miRNA-mRNA, and in order to confirm the role of miR-205-5p in regulating cancer cell growth and neovascularization in vivo, transfected GC cell clones are compared with control clones, and the ability of the clones to form tumors in a subcutaneous model is observed;
results show that miR-205-5p overexpression obviously reduces the size of the xenograft tumor with reduced MVD, immunohistochemistry shows that VEGFA and FGF1 expression of a miR-205-5p group is also reduced, meanwhile, miR-205-5p inhibitor promotes the growth of the xenograft tumor by increasing MVD, miR-205-5p inhibitor can also increase the expression of VEGFA and FGF1, and the results show that miR-205-5p is a cancer suppressor for inhibiting angiogenesis in gastric cancer;
the interaction of the 3'UTR of FGF1 and VEGFA is confirmed by a dual-luciferase report method, and the dual-luciferase report method shows that miR-205-5p can directly target the 3' UTR of VEGFA and FGF1 mRNA and regulate the expression of endogenous VEGFA and FGF1, so that VEGFA and FGF1 are direct targets of miR-205-5p, and miR-205-5p can regulate the expression of endogenous VEGFA and FGF1 by targeting the direct interaction of an ERK signaling pathway and miRNA-mRNA;
aiming at verifying whether VEGFA and FGF1 are direct targets of miR-205-5p, the method comprises the following steps:
human VEGFA and FGF 13 'UTR fragments containing wild-type or mutant miR-205-5P binding sequences were cloned downstream of the luciferase reporter gene in pGL3 vector, luciferase activity of the reporter gene containing wild-type 3' UTRs was significantly inhibited by miR-205-5 pmimic (P < 0.01) in HEK293T cells co-transfected with the reporter plasmid and miR-205-5 pmimic or negative control, whereas luciferase activity of the mutant reporter gene was unaffected, the results indicate that miR-205-5P may inhibit gene expression by the 3'UTR sequences binding VEGFA and FGF1, these findings indicate that at post-transcriptional level expression of VEGFA and FGF1 mrnas is also inhibited by miR-205-5P, and that the 3' UTRs of VEGFA and FGF1 mrnas comprise a site complementary to the miR-205-5P seed region, VEGFA and FGF1 can be proved to be direct targets of miR-205-5 p;
wherein, the construction of luciferase reporter plasmid: the 3'UTR fragment of VEGFA (1736-3660nt, Genbank accession number NM-001025366.3 was amplified using primers 5'-ACCACACCATCACCATCGAC-3'(forward) and 5'-CGTCTGACCTGGGGTAGAGA-3'(reverse), containing 1 putative miR-205-5p-binding sequence (1874-2280 nt), and the PCR product was cloned into firefly luciferase reporter vector pGL3(Promega, Wis., US), designated pGL3-VEGFA-3' UTR;
FGF 13 'UTR fragment, NM _000800.5 containing 1 putative mir-205-5p binding sequence (3438-3458nt) amplified using primers 5'-ATTTTCGGGCCAGCTCTGAA-3'(forward) and 5'-GGGTTCCTTTGCCTCTGACA-3'(reverse), PCR product was cloned into firefly luciferase reporter vector pGL3(Promega, WI, US) named pGL3-FGF1-3' UTR;
in addition, luciferase reporter genes pGL3-Mut VEGFA-3'UTR and pGL3-Mut FGF1-3' UTR, which have mutant sequences within the miR-205-5p binding site, were constructed using the mutanBEST Kit (Takara Bio, Shiga, JP);
HEK293T cells were seeded in 3 replicate 24 well plates, co-transfected with pGL3-VEGFA-3'UTR or pGL3-FGF1-3' UTR, miR-205-5p mimic or negative control miR mimic (Sigma-Aldrich, MO, USA) using FuGene HD transfection reagent (Promega, Wis., USA), pRL-TK (Promega, Wis., USA) also as normalization control; cells were harvested 24 hours after transfection and the firefly and renilla luciferase activities were measured using the BioTek Synergy H1 mixed with a multi-modal microplate reader, dual luciferase reporter gene system (Promega, WI, USA);
the qRT-PCR detection is that Trizol reagent and miRNeasy-FFPE kit are adopted to respectively extract total RNA from cell lines and paraffin embedded tissue sections;
in order to detect miR-205-5p, RNA is reversely transcribed into a cDNATM miRNA first strand cDNA synthesis kit by All-in-One, and then the miRNA qRT-PCR detection kit is used for carrying out qRT-PCR on microRNAs;
for detection of mRNAs, the RNA was reverse transcribed into cDNA using the GoScript reverse transcription system, and
qRT-PCR-Master Mix carries out qRT-PCR on the mRNAs;
all qRT-PCR adopts an ABI-Prism 7500 system, and U6 or GAPDH is used as an internal control for quantification of microRNAs and mRNAs respectively;
western blot assay was performed by using FGF1, VEGFA and GAPDH antibodies for detecting FGF1, VEGFA and GAPDH, respectively, and (p-) SAPK/JNK, (p-) ERK1/2 and (p-) p-38 antibodies for detecting activation of MAPK signaling pathway;
wherein, VEGFA is 1: 1000 dilution, GAPDH 1: 2000 dilution, (p-) p-38 is 1: 800, diluting;
GC and normal tissue were collected from 109 patients and their distribution is shown in table 1 below:
table 2: the expression of miR-205-5p in gastric cancer tissues is reduced, and the clinical characteristics of patients with gastric cancer are achieved;
as can be seen from the above table, in order to determine the expression level of miR-205-5P in gastric cancer, the expression level of miR-205-5P in a gastric cancer (gastric cancer) specimen and corresponding normal tissues of the gastric cancer (gastric cancer) specimen of 109 pairs is detected by adopting qRT-PCR, the miR-205-5P expression of most tumor tissues (86/109) is lower than that of the normal tissues, the relative level of miR-205-5P in a GC group is remarkably reduced compared with that of the normal tissues (paired student t test, P is less than 0.001), and further analysis shows that the miR-205-5P expression of GC tissues in a late TNM stage is lower than that in an early stage (P is less than 0.001), and miR-205-5P expression in 109 GC tissues is frequently reduced compared with that in the normal stomach tissues (P is less than 0.001);
to determine correlation between miR-205-5P expression and microvascular density (MVD) in GC tissue, we also examined expression of vascular endothelial cell marker CD31 mRNA in GC tissue and normal tissues thereof by qRT-PCR, and miR-205-5P and CD31 expression were also negatively correlated in the collected GC tissue (R2 is 0.109, P is 0.005);
② test of miR-205-5p expression and gastric cancer patient clinical characteristics
Dividing gastric cancer patients into a miR-205-5p low expression group (the expression level of miR-205-5p in normal tissues is less than the average expression level of miR-205-5 p) and a miR-205-5p high expression group (the expression level of miR-205-5p in normal tissues is more than or equal to the average expression level of miR-205-5 p);
the low-expression miR-205-5P is closely related to gastric cancer patients with poor differentiation, stage + and distant metastasis (P <0.05, as shown in Table 1), however, the miR-205-5P expression has almost no significant correlation with age, sex and tumor position (P > 0.05), and in addition, the Kaplan Meier analysis shows that the miR-205-5P expression with higher total survival time of the patients performs far more than the low miR-205-5P expression of the patients (survival rate is more: chi)2＝4.603，P＝0.032)；
③ MiR-205-5p can inhibit the growth and angiogenesis of gastric cancer in vivo and in vitro
Stably transfecting MiR-205-5P and a negative control to a BGC-823 cell, selecting subclones which are respectively named as BGC + miR-205-5P and BGC-nc, wherein compared with the negative control cell, the miR-205-5P level in the BGC + miR-205-5P clone is remarkably increased (P is less than 0.001; FIG.2A), the miR-205-5P remarkably reduces the BGC cell growth speed (P is less than 0.001), on the contrary, the miR-205-5P inhibitor and the negative control stably transfect MGC-803 cells, the miR-205-5P level in the MGC + miR-205-5P inhibitor clone is remarkably reduced (P is less than 0.001; FIG.2A), and the miR-205-5P can stimulate the MGC cell growth speed (P is less than 0.001) through down regulation;
to understand the effect of miR-205-5P expression changes on the angiogenesis of the GC cell line, we cultured Human Umbilical Vein Endothelial Cells (HUVECs) in conditioned medium collected from the GC cell line, BGC + miR-205-5P supernatant induced HUVECs to differentiate to capillary-like structures to a lesser extent (P <0.05) than BGC-nc cells, whereas MGC cell supernatant transfected with miR-205-5P inhibitor induced differentiation of HUVECs to capillary-like structures was stronger than MGC-nc cells (P <0.05), BGC + miR-205-5P cells induced microvessels were significantly decreased (P <0.05), MGC + miR-205-5P cells were located in dorsal skin ruffled chambers, tumor-induced microvessels were significantly increased (P <0.05) in the back window model compared to negative control cells, these results clearly show that overexpression of miR-205-5p in gastric cancer inhibits angiogenesis in vitro and in vivo;
ERKs signaling pathways and expression of VEGFA and FGF1 are inhibited by miR-205-5p
In order to determine the influence of miR-205-5P expression change on VEGFA and FGF1, qRT-PCR and western blotting, the results show that VEGFA and FGF1 expression is negatively regulated by miR-205-5P expression, mRNA and protein levels of VEGFA and FGF1 in BGC + miR-205-5P cells are obviously lower than those of negative control cells (P <0.05), mRNA and protein levels of VEGFA and FGF1 in MGC + miR-205-5P inhibitor cells are obviously increased (P <0.05) compared with the negative control cells, the western blotting detects whether MAPK signaling pathway is regulated by miR-205-5P, SAPK/JNK, ERK1/2 and P38 levels, the results show that P-ERK1/2 expression level is obviously regulated by miR-205-5P expression change, and whether VEGFA and FGF1 expression is activated by ERK1/2 is studied, we carried out western blotting experiments using ERK1/2 inhibitor and showed that ERK1/2 inhibitor significantly inhibited the expression of VEGFA and FGF1 proteins.
VEGFA is a multifunctional cytokine that stimulates blood vessel growth, vascular repair and regeneration, increased VEGFA expression is associated with poor gastric cancer prognosis, FGF1 plays an important role in the regulation of angiogenesis, cell survival, cell differentiation and cell migration, unlike vegf, FGFs are pleiotropic molecules that can act on cells of various cell types, including endodermal, mesenchymal and neuroectodermal origin. This raises the possibility that FGF1 function coordinates angiogenesis by regulating various cell-cell interactions, and, to sum up, secretion of VEGFA and FGF1 in the extracellular microenvironment may be important mediators of miR-205-5p regulation of angiogenesis, however, the signaling pathways involved in miR-205-5p regulated GC cell neovascularization are not clear;
the names in the invention are abbreviated as follows:
3' -UTR: a 3' untranslated region;
ERK: extracellular signal-regulated kinase;
EMT: epithelial to mesenchymal transition;
FGF 1: fibroblast growth factor 1;
VEGFA: vascular endothelial growth factor a;
and (3) IHC: immunohistochemistry;
MicroRNA: micro;
MAPK: mitogen-activated protein kinase;
HER 2: human epidermal growth factor receptor 2;
HUVECs: human umbilical vein endothelial cells;
recombinant human purlin;
rhFGF 1: recombinant human FGF 1;
ICT 1: immature colon cancer transcript-1;
MVD: microvascular density;
HMGB 1: a high mobility group box 1;
MTS：
one aqueous cell proliferation assay;
NC: negative control;
GC: gastric cancer;
operating the system: overall survival rate;
qRT-PCR: quantitative reverse transcription polymerase chain reaction;
CD 31: platelet endothelial cell adhesion molecule-1 (PECAM-1, or CD 31);
PD-1: programmed cell death protein 1;
ZEB 1: the zinc finger electronic cassette binds to homeobox 1.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. An application of MiR-205-5p in preparing an angiogenesis agent is characterized by comprising the following operations:
s1, detecting the expression of miR-205-5p and CD31 in human gastric cancer tissues and liver metastases by adopting quantitative real-time qRT-PCR;
s2, studying the silencing and high expression effects of miR-205-5p by detecting the formation of capillary vessels of endothelial cells of human umbilical veins in vitro and a skin wrinkle chamber model on the back side of a body;
s3, detecting the influence of miR-205-5p on VEGFA and FGF1 expression by adopting qRT-PCR and western blotting, and down-regulating the expression of VEGFA and FGF1 by MiR-205-5p through ERK signaling pathway and direct targeting.
2. The use of MiR-205-5p for the preparation of an angiogenesis agent according to claim 1, wherein to further determine the effect of MiR-205-5p expression on angiogenesis in vivo, a xenograft model was established using different types of GC cell clones, and the microvascular density of liver metastases and xenografts was determined using an immunohistochemical method, wherein the xenograft model used was a mouse model.
3. The use of MiR-205-5p in the preparation of an angiogenesis agent according to claim 1, wherein in S3, the dual-luciferase reporting method is used to confirm the interaction of the 3'UTR of FGF1 and VEGFA, and the dual-luciferase reporting method shows that MiR-205-5p directly targets the 3' UTR of VEGFA and FGF1 mRNA, regulating the expression of endogenous VEGFA and FGF1, so VEGFA and FGF1 are direct targets of MiR-205-5p, and MiR-205-5p regulates the expression of endogenous VEGFA and FGF1 by targeting the ERK signaling pathway and the miRNA-mRNA direct interaction.
4. The use of MiR-205-5p in the preparation of an angiogenesis agent according to claim 1, wherein the formation of capillaries in human umbilical vein endothelial cells in S2 is performed by piping the matrix gel into a pre-cooled 96-well plate, culturing the human umbilical vein endothelial cells in 100 microliters of conditioned medium, wherein the conditioned medium is obtained from different types of cells, and after culturing for 36 hours, photographing the capillary formation structure;
the dorsal skinfold chamber model refers to the formation of a dorsal air pocket in the body of the subject by subcutaneous injection of 5ml of air after complete anesthesia of the subject, the resulting different types of cells were injected into the diffusion chamber and placed subcutaneously, and 10 days later, these subjects were carefully peeled around the implanted chamber and the skin folds covering the room were photographed under visible light.
5. The use of MiR-205-5p for the preparation of an angiogenesis agent as claimed in claim 4, wherein the number of said different cell types is 1.0 x 106, and the subject is selected from the group consisting of animals.
6. The use of MiR-205-5p for the preparation of an angiogenesis agent as claimed in claim 1, wherein qRT-PCR assay in S3 is performed by using Trizol reagent and miRNeasy-FFPE kit to extract total RNA from cell lines and paraffin embedded tissue sections, respectively;
for detection of miR-205-5p, RNA is reverse transcribed into cDNA using All-in-OneTMSynthesizing a miRNA first strand cDNA (complementary deoxyribonucleic acid) kit, and then carrying out qRT-PCR (quantitative reverse transcription-polymerase chain reaction) on the microRNAs by using the miRNA qRT-PCR detection kit;
for detection of mRNAs, the RNA was reverse transcribed into cDNA using the GoScript reverse transcription system, and
qRT-PCR-Master Mix carries out qRT-PCR on the mRNAs;
all qRT-PCR used ABI-Prism 7500 system with U6 or GAPDH as internal control for quantification of microRNAs and mRNAs, respectively.
7. The use of MiR-205-5p for the preparation of an angiogenesis agent according to claim 1, wherein the Western blot assay in S3 is performed by using FGF1, VEGFA and GAPDH antibodies for the detection of FGF1, VEGFA and GAPDH, respectively, and (p-) SAPK/JNK, (p-) ERK1/2 and (p-) p-38 antibodies for the detection of activation of MAPK signaling pathway;
wherein, VEGFA is 1: 1000 dilution, GAPDH 1: 2000 dilution, (p-) p-38 is 1: and (8) diluting.
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| WO2023177275A1 (en) * | 2022-03-18 | 2023-09-21 | (주)에스엔이바이오 | Biomarker composition for diagnosing hemostatic disorders, comprising mirnas in extracellular vesicles |
| WO2024047914A1 (en) * | 2022-08-31 | 2024-03-07 | Kabushiki Kaisha Toshiba | Analysis method, kit, and detection device for cancer diagnosis by means of microrna expression |
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| WO2023177275A1 (en) * | 2022-03-18 | 2023-09-21 | (주)에스엔이바이오 | Biomarker composition for diagnosing hemostatic disorders, comprising mirnas in extracellular vesicles |
| WO2024047914A1 (en) * | 2022-08-31 | 2024-03-07 | Kabushiki Kaisha Toshiba | Analysis method, kit, and detection device for cancer diagnosis by means of microrna expression |
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