CN111607616A - Research method for targeted silencing STAT3 on hypoxia induction effect of human brain glioma U251 cells and application of research method - Google Patents
Research method for targeted silencing STAT3 on hypoxia induction effect of human brain glioma U251 cells and application of research method Download PDFInfo
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Abstract
The invention discloses a research method for the targeted silent STAT3 on hypoxia induction influence of human brain glioma U251 cells and application thereof, wherein the method comprises the steps of constructing shRNA of a targeted STAT3 gene to transfect the U251 cells, observing infection efficiency by a microscope, and detecting the silent efficiency of STAT3 by real-time fluorescence quantitative PCR and Western blotting; the influence of STAT3 gene silencing on the angiogenesis mimicry formation and invasion capacity of U251 cells induced by hypoxia is detected by in vitro three-dimensional culture and a Transwell chamber invasion experiment; and detecting the expression level of VM/invasion related genes/proteins by real-time fluorescence quantitative PCR and Western blotting. Successfully constructs shRNA lentiviral plasmid targeting STAT3 gene and efficiently transfects U251 cells.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to a research method for influence of targeted silent STAT3 on human brain glioma U251 cell hypoxia induction and application thereof, in particular to a research method for influence of targeted silent STAT3 on human brain glioma U251 cell hypoxia induction angiogenesis mimicry and invasion and application thereof.
Background
Glioma is the most common primary tumor of the central nervous system of human and has the characteristics of high incidence rate, high recurrence rate, high death rate, low cure rate and the like. The effect is still not ideal though the comprehensive diagnosis and treatment such as operation, radiotherapy and chemotherapy. The research on the pathogenesis of the tumor is one of the problems to be solved at present by searching for a new therapeutic target. As a complex process of multifactorial involvement, polygenic changes, multistep evolution, glioma pathogenesis is closely related to the influence of the surrounding microenvironment. Studies have shown that hypoxia is the most prominent feature of the microenvironment around glioma cells. Under the condition of oxygen deficiency, tumor cells can form angiogenesis mimicry (VM) through a new tumor inner tubular network structure independent of original vascular endothelial cells, so that good blood perfusion is provided for malignant proliferation, invasion and metastasis of glioma cells. Signal transducers and transcription activators 3(Signal transducers and activators of transducers 3, STAT3) are important substrates of a cell Signal transduction pathway JAK-STAT pathway, and the abnormal expression and activation of the Signal transducers and the transcription activators play an important role in promoting the occurrence, development and malignant transformation of glioma, and are transcription factor oncogenes.
Like most solid tumors, human brain glioma is a malignant tumor that is extremely dependent on peripheral blood vessels, thereby achieving rapid proliferation and infiltration. The microenvironment and microecosystem reconstruction around the tumor and the activation state of the vascular endothelial cell colony play a crucial role in the process, and meanwhile, the expression of related matrix factors and the change of signal transduction pathways are accompanied.
VM is the blood supply mode which is found to be specific to tumors when Maniotis et al researches on uveal melanoma. Generally, more aggressive tumors are more likely to produce VM. In some highly invasive tumors including Ewing's sarcoma, malignant esophageal interstitial tumor, etc., tumor cells can form a specific microcirculation pipeline without the participation of vascular endothelial cells under the condition of hypoxia, so as to maintain the blood supply, proliferation and invasion abilities. With the research and development of tumor targeting therapy, VM is naturally and widely concerned by scholars at home and abroad as its unique blood supply mode.
STAT3 is a core transcription factor in a JAK-STAT signal transduction pathway mediated by a cytokine receptor, controls the transcriptional activation of a plurality of genes such as bcl-xl, bcl-2, c-myc, cyclinD1, survivin, mcl-1 and VEGF and the like, and plays an important role in regulating the growth, differentiation and apoptosis of cells. Abnormal STAT3 (abnormal expression and abnormal activation) is involved in malignant transformation and proliferation of various tumors including glioma, inhibits apoptosis of tumor cells by up-regulating expression of various oncogenes, promotes proliferation of tumor cells, finally causes occurrence and development of malignant tumors, and is determined to be an important transcription factor oncogene. Researches show that the targeting block of STAT3 signal channel can effectively reduce the expression level of various oncogenes in glioma cells and obviously inhibit the proliferation of tumor cells. STAT3 is a very effective molecular target in glioma gene therapy studies. Our previous studies indicate that the JAK-STAT signaling pathway plays an important role in the Crosstalk mechanism between glioma cells and human brain microvascular endothelial cells, but there are few reports on whether it can play a role by affecting glioma VM formation.
Disclosure of Invention
The invention aims to provide a research method for targeted silent STAT3 on hypoxia induction influence of human brain glioma U251 cells and application thereof, wherein the method is used for carrying out related research on a glioma cell line and further researching the effect of STAT3 on angiogenesis mimicry of glioma cells under the hypoxia condition, so that the method can be undoubtedly used as a new glioma treatment target, an experimental basis and a research basis of a new glioma treatment mechanism, and lays a firmer foundation for clinical application.
The technical scheme is as follows:
a research method for the targeted silencing STAT3 on the hypoxia induction effect of human brain glioma U251 cells comprises the following steps:
step 1, constructing and identifying sh-STAT3 lentiviral vector
Sequence information of human STAT3 gene (NM-003150) is searched from Genbank, and RNA interference sequence is designed according to the principle. Other non-specific suppressor sequences were excluded by BLAST homology analysis. And screening effective target sequences through an interference pre-experiment, determining a final construction sequence and synthesizing. Annealing to form double-stranded DNA, and performing enzyme digestion and connection to generate the lentiviral vector. And verifying the correctness by PCR identification and DNA sequencing. Whole virus particles were generated and titered in 293T cell packages.
Step 2, cell culture and detection of lentivirus infection efficiency
Human brain glioma cell U251 containing 10% fetal calf serum and 1 × 105DMEM culture solution of U/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO2Culturing and incubating under the condition. The cells are growing adherently. Cells were divided into 4 groups: blank control group (control), cobalt chloride (CoCl)2) A simulated hypoxia in vitro (hypoxia), an sh-NC infected cell simulated hypoxia group (hypoxia + sh-NC) and an sh-STAT3 infected cell simulated hypoxia group (hypoxia + sh-STAT 3).
The well-conditioned U251 cells were collected at 5 × 104Inoculating to 6-well plate at 37 deg.C and 5% CO2Culturing until the cell fusion degree reaches 30%, adding 0.9 mu L lentivirus according to MOI (maximum of identity) of 2.0, infecting by using a Normal + Polybrene mode, changing a culture medium after culturing for 24h, and observing the expression condition of a reporter gene GFP on the lentivirus after continuously infecting for 72 h.
After the sh-RNA infects U251 cells for 48h, the cells are collected, washed 3 times with PBS, and the total RNA of the cells is extracted according to the operation manual of the TRIzol kit, and GoScript is appliedTMReverse Transcription System for Reverse Transcription and PCR amplification. STAT3 gene primer sequence: the upstream primer is shown as SEQ ID NO 1, the downstream primer is shown as SEQ ID NO 2, and the amplification product size is 409 bp. GAPDH gene primer sequence: the upstream primer is shown as SEQ ID NO. 3, the downstream primer is shown as SEQ ID NO. 4, and the amplification fragment is 315 bp.
Step 4, Western blotting detection of expression level of each protein
Collecting each group of cells with 70-80% fusion degree, adding pre-cooled cell lysate to fully lyse the cells in an ice bath, extracting the whole protein, and analyzing and quantifying by a BCA method.
Dissolving BD matrix gel in ice water bath overnight, sucking 400 μ l Matrigel with precooling gun head, adding into 24-well plate horizontally placed on ice box, uniformly spreading, incubating in 37 deg.C incubator for 1h to solidify the gel, digesting each group of cells with trypsin, centrifuging, discarding supernatant, re-suspending with DMEM, and adjusting cell final concentration to 1.5 × 105And each/ml of the cells are inoculated into the 24-pore plate, 1ml of each pore is placed into an incubator and continuously cultured for 24 hours, then the culture medium is discarded, the cells are lightly washed by PBS for 2 times, a calcein solution of 1/10 culture medium system is added into each pore, the cells are incubated in the incubator at 37 ℃ for 30min, observed by a fluorescence microscope, 5 fields are randomly selected under a high power microscope to be photographed (200 ×), the number of tubes and the diameter of the tube cavity are respectively counted, and each group of experiments are repeated for 5 times.
Dissolving BD matrix gel in ice water bath overnight, diluting with serum-free culture medium to 10mg/ml (ratio of serum to BD matrix gel is 5: 1) in a clean bench, sucking 100 μ l per well, spreading gently into Transwell chamber of polycarbonate filter membrane with aperture of 8 μm, shaking and placingPolymerizing for 5h in an incubator at 37 ℃, digesting each group of cells by trypsin, centrifuging, discarding supernatant, then resuspending by serum-free DMEM, and adjusting the final concentration of the cells to be 5 × 105One/ml, 200. mu.l was added to the upper layer of the Transwell chamber. Adding 600 μ l complete culture medium into the lower layer, and standing at 37 deg.C with 5% CO2After fixing the cells for 15min with 4% paraformaldehyde, wiping off the upper cells of the chamber with a cotton swab, staining with 0.1% crystal violet for 30min, washing with PBS, naturally drying, taking pictures of the upper, lower, left, right and middle five visual fields under a microscope, observing and counting the cells (200 ×) passing through polycarbonate membrane of the Transwell chamber, and repeating the experiments for 5 times.
Step 7, statistical processing
Statistical analysis was performed using SPSS 18.0 software, and the measurements were expressed as means. + -. standard deviation. The comparison between groups adopts one-factor variance analysis, and the comparison between two groups adopts LSD-t test. P <0.05 is statistically significant for the differences.
Further, in step 3, the PCR reaction conditions are pre-denaturation at 95 ℃ for 30s, then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 6min after 35 cycles. The relative expression of STAT3mRNA is expressed as a 2-. DELTA.Ct value (Ct represents the cycle threshold). Each experiment was repeated 5 times.
Further, in step 4, each group of cells with a degree of fusion of 75% was collected.
Further, in step 4, 10. mu.g of total protein sample was subjected to SDS-PAGE using 4% -12% TrisGlycine gel and transferred to PVDF membrane, which was then blocked with milk prepared from TBST for 1 hour, and added with primary antibody (volume dilution ratio 1: 3000) and incubated overnight at 4 ℃. Then washing the membrane with TBST for 5min × 5 times, incubating with fluorescent secondary antibody (volume dilution ratio of 1: 5000) in the dark at room temperature for 1h, washing the membrane with TBST for 5min × 5 times, and exposing with Odyssey (ODYSSEY CLx). Scanning gray scale and calculating the corresponding expression amount of the protein.
Further, SDS-PAGE was performed using 8% TrisGlycine gel.
The method of the invention is applied to the preparation process of the medicine for treating human brain glioma.
The invention has the beneficial effects that:
the invention successfully constructs shRNA lentiviral plasmid targeting STAT3 gene and efficiently transfects the shRNA lentiviral plasmid to U251 cells, and compared with the infected sh-NC lentiviral cells, the level of STAT3mRNA of the shRNA lentiviral plasmid is obviously reduced, which indicates that the expression of the glioma cell STAT3 is silenced by RNA interference technology. Compared with a control group and an sh-NC infected group, the cell hypoxia-induced VM formation capacity of the sh-STAT3 infected group is remarkably reduced, and the targeted silent STAT3 can inhibit the U251 hypoxia-induced VM.
Drawings
FIG. 1 shows the sh-STAT3 lentivirus vector structure and sequence, wherein, a is vector map and information; b, carrying out PCR electrophoresis on the vector; c, vector sequencing result.
FIG. 2 shows the infection efficiency and targeted silencing effect of sh-STAT3 lentivirus on U251 cells, wherein a is the infection efficiency of sh-RNA observed under a microscope (A1: optical microscope, sh-NC; A2: fluorescence microscope, sh-NC; B1: optical microscope, sh-STAT 3; B2: fluorescence microscope, sh-STAT 3; 100 ×), B is the expression level of STAT3mRNA detected by real-time fluorescence quantitative PCR,**comparison with control group P<0.01; and c, detecting the expression level of STAT3 protein by Western blotting.
FIG. 3 is a three-dimensional in vitro culture experiment to examine the effect of silent STAT3 on hypoxia-induced U251 cell angiogenic mimicry (VM); wherein, A, a fluorescence microscope observes the VM formation condition of a control group; b: observing the formation condition of VM in the anoxic group by using a fluorescence microscope; c: observing the formation condition of VM when the sh-NC is used for infection under the condition of oxygen deficiency by using a fluorescence microscope; d: observing the formation condition of VM when the sh-STAT3 is used for infection under the condition of oxygen deficiency by using a fluorescence microscope; e: VM is quantitatively counted under a high-power visual field of a fluorescence microscope,﹟﹟comparison with control group P<0.01，**Comparison with hypoxic group P<0.01。
FIG. 4 is a Transwell cell invasion assay to examine the effect of silent STAT3 on hypoxia-induced U251 cell invasion capacity; wherein, A, the invasion capacity of the U251 cells of the control group is observed by an optical microscope; b: observing the invasion capacity of the anoxic group U251 cells by using an optical microscope; c: observing the invasion capacity of U251 cells infected by sh-NC under the condition of hypoxia by using an optical microscope; d: observing the invasion capacity of U251 cells infected by sh-STAT3 under the condition of hypoxia by using an optical microscope;
FIG. 5 shows the results of quantitative analysis of U251 cell invasive potential,﹟﹟comparison with control group P<0.01，**Comparison with hypoxic group P<0.01。
FIG. 6 shows the expression of target protein after STAT3 gene silencing by Western blotting; wherein, a, Westernblotting detects protein expression conditions of MMP9, MMP14, EphA2, Laminin5 gamma 2 and the like after STAT3 gene silencing; b: the result of the statistical analysis of the Western blotting test,﹟comparison with control group P<0.05，**Comparison with hypoxic group P<0.01。
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific embodiments.
1 materials and methods
1.1 Primary reagent Material
Human brain glioma cell line U251 was purchased from Shanghai cell resource center of Chinese academy of sciences, and is a glioblastoma monoclonal adult line with WHO classification standard of grade IV. DMEM culture medium, trypsin, was purchased from Gibco, USA, and fetal bovine serum was purchased from Hyclone, USA. Rabbit anti-human STAT3, STAT3Phospho (pY705) and MMP9, MMP14, EphA2, Laminin5 gamma 2 monoclonal antibodies were purchased from Cell Signal Technology, Inc., Rabbit anti-human GAPDH polyclonal antibody (AB-P-R001) was purchased from Hangzhou xian till Biotech, Inc., and fluorescent secondary antibody Goat anti-Rabbit 926-. Western blotting-related reagents were purchased from Katy Biotech development Ltd, Nanjing. BD matrix gel was purchased from Sigma, USA, and Transwell cell from Corning, USA. Calcein was purchased from AAt bioquest.
Lentiviral vector systems (including pGCSIL-GFP vector plasmids, helper packaging vector plasmids pHelper 1.0, pHelper2.0), restriction enzymes Age I, EcoR I, ligase T4 DNA ligase, Taq DNA polymerase, 293T cells, etc. were purchased from Kjekay GeneTech, Shanghai. The PCR kit was purchased from Promega corporation, and the primers were synthesized by Kjeka Gene technology, Shanghai. The plasmid small extraction kit and the gel recovery kit are products of QIAGEN company, and the DH5 alpha competent cells are stored in a key laboratory of Ningxia craniocerebral diseases.
1.2 construction and identification of sh-STAT3 Lentiviral vector
Sequence information of human STAT3 gene (NM-003150) is searched from Genbank, and RNA interference sequence is designed according to the principle. Other non-specific suppressor sequences were excluded by BLAST homology analysis. And screening effective target sequences through an interference pre-experiment, determining a final construction sequence and synthesizing. Annealing to form double-stranded DNA, and performing enzyme digestion and connection to generate the lentiviral vector. And verifying the correctness by PCR identification and DNA sequencing. Whole virus particles were generated and titered in 293T cell packages.
1.3 cell culture and detection of lentivirus infection efficiency
Human brain glioma cell U251 containing 10% fetal calf serum and 1 × 105DMEM culture solution of U/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO2Culturing and incubating under the condition. The cells are growing adherently. Cells were divided into 4 groups: blank control group (control), cobalt chloride (CoCl)2) A simulated hypoxia in vitro (hypoxia), an sh-NC infected cell simulated hypoxia group (hypoxia + sh-NC) and an sh-STAT3 infected cell simulated hypoxia group (hypoxia + sh-STAT 3).
The well-conditioned U251 cells were collected at 5 × 104Inoculating to 6-well plate at 37 deg.C and 5% CO2Culturing until the cell fusion degree reaches 30%, adding 0.9 mu L lentivirus according to MOI (maximum of identity) of 2.0, infecting by using a Normal + Polybrene mode, changing a culture medium after culturing for 24h, and observing the expression condition of a reporter gene GFP on the lentivirus after continuously infecting for 72 h.
1.4 real-time fluorescent quantitative PCR detection of mRNA expression level of each gene in U251 cells
After the sh-RNA infects U251 cells for 48h, the cells are collected, washed 3 times with PBS, and the total RNA of the cells is extracted according to the operation manual of the TRIzol kit, and GoScript is appliedTMReverse Transcription System for Reverse Transcription and PCR amplification. STAT3 gene primer sequence: the upstream primer is 5'-GTCAGA TGCCAAATGC-3', the downstream primer is 5'-CCTGGAGGCTTAGTGC-3', and the size of the amplification product is 409 bp. GAPDH gene primer sequence: 5'-CATCTTCTTTTGCGTCGCCA-3' upstream and 5'-TCGCCCCACTTGATTTTGG-3' downstream, and 315 amplified fragmentsAnd bp is adopted. The PCR reaction conditions are pre-denaturation at 95 ℃ for 30s, then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 6min after 35 cycles. The relative expression of STAT3mRNA is expressed as a 2-. DELTA.Ct value (Ct represents the cycle threshold). Each experiment was repeated 5 times.
1.5 Western blotting detection of expression level of each protein
Collecting cells of each group with the fusion degree of 70-80%, wherein the range of the values has a preferable value point: 75 percent. Adding pre-cooled cell lysate, fully cracking cells in an ice bath, extracting holoprotein, and analyzing and quantifying by using a BCA method. A10 mu g total protein sample is taken, SDS-PAGE electrophoresis is carried out by using 4% -12% TrisGlycine gel, the sample is transferred to a PVDF membrane, the PVDF membrane is sealed by using milk prepared by TBST for 1h, and primary antibody (the volume dilution ratio is 1: 3000) is added for incubation at 4 ℃ overnight. Then washing the membrane with TBST for 5min × 5 times, incubating with fluorescent secondary antibody (volume dilution ratio of 1: 5000) in the dark at room temperature for 1h, washing the membrane with TBST for 5min × 5 times, and exposing with Odyssai (ODYSSEYCLx). Scanning gray scale and calculating the corresponding expression amount of the protein.
1.6 Effect of lentivirus infection of the angiogenic mimicry of U251 cells
Dissolving BD matrix gel in ice water bath overnight, sucking 400 μ l Matrigel with precooling gun head, adding into 24-well plate horizontally placed on ice box, uniformly spreading, incubating in 37 deg.C incubator for 1h to solidify the gel, digesting each group of cells with trypsin, centrifuging, discarding supernatant, re-suspending with DMEM, and adjusting cell final concentration to 1.5 × 105And each/ml of the cells are inoculated into the 24-pore plate, 1ml of each pore is placed into an incubator and continuously cultured for 24 hours, then the culture medium is discarded, the cells are lightly washed by PBS for 2 times, a calcein solution of 1/10 culture medium system is added into each pore, the cells are incubated in the incubator at 37 ℃ for 30min, observed by a fluorescence microscope, 5 fields are randomly selected under a high power microscope to be photographed (200 ×), the number of tubes and the diameter of the tube cavity are respectively counted, and each group of experiments are repeated for 5 times.
1.7 Effect of lentivirus infection on the invasiveness of U251 cells
The BD matrigel was dissolved overnight in an ice-water bath and diluted to a concentration of 10mg/ml (5: 1 ratio of serum to BD matrigel) in serum-free medium in a clean benchSucking 100 μ l of the cell, spreading into a Transwell cell of 8 μm polycarbonate filter membrane, shaking, polymerizing in an incubator at 37 deg.C for 5h, digesting each group of cells with trypsin, centrifuging, discarding supernatant, re-suspending with serum-free DMEM, and adjusting the final cell concentration to 5 × 105One/ml, 200. mu.l was added to the upper layer of the Transwell chamber. Adding 600 μ l complete culture medium into the lower layer, and standing at 37 deg.C with 5% CO2After fixing the cells for 15min with 4% paraformaldehyde, wiping off the upper cells of the chamber with a cotton swab, staining with 0.1% crystal violet for 30min, washing with PBS, naturally drying, taking pictures of the upper, lower, left, right and middle five visual fields under a microscope, observing and counting the cells (200 ×) passing through polycarbonate membrane of the Transwell chamber, and repeating the experiments for 5 times.
1.8 statistical treatment
Statistical analysis was performed using SPSS 18.0 software, and the measurements were expressed as means. + -. standard deviation. The comparison between groups adopts one-factor variance analysis, and the comparison between two groups adopts LSD-t test. P <0.05 is statistically significant for the differences.
2 results
2.1 identification of STAT3shRNA Lentiviral vectors
According to the sequence information of human STAT3mRNA (NM-003150) published by Genbank, 3 interference target sequences are designed according to the RNA interference sequence design principle (Table 1), and are compared with a human genome database to eliminate homology with other coding sequences, plasmids are respectively constructed, a 293T cell is transfected, PSC-2 is determined to be an effective target sequence according to the inhibition rate of the plasmid to STAT3, and finally the DNA oligonucleotide sequence for constructing STAT3shRNA is determined to be F: 5'-CCGG AGAAGG ACA TCAGCG GTA AGA CTCGAG TCT TAC CGC TGA TGT CCT TCT TTTTTG-3' and R: 5'-AATTCAAAAA AGAAGG ACA TCA GCG GTA AGA CTCGAG TCT TAC CGC TGA TGT CCT TCT-3', the DNA oligonucleotide sequence is synthesized by Shanghai Kjek gene technology company, is annealed to form double-stranded DNA, and is connected with pGCSIL-GFP vector after double enzyme digestion by Age I and EcoR I endonuclease to generate shRNA lentiviral vector, PCR identification and DNA sequencing confirm that the oligonucleotide chain of the synthesized lentiviral vector containing STAT3shRNA is inserted correctly (figure 1 a-figure 1c), the complete viral particles are generated by packaging the 293T cell, and the titer is determined to be 3 × 108TU/mL。
TABLE 1 STAT3 RNA interference designed target sequences
Table 1 Interfering sequence specified for STAT3RNA
| Sequence name | Position of | Target sequence | GC content/%) |
| PSC-1 | 672-692bp | CGG AAG AGA GTG CAG GAT CTA | 52.38 |
| PSC-2 | 2092-2112bp | AGA AGG ACA TCA GCG GTA AGA | 47.62 |
| PSC-3 | 2119-2139bp | TCC AGT CCG TGG AAC CAT ACA | 52.38 |
2.2 infection efficiency of lentivirus-infected U251 cells and Targeted silencing of STAT3 expression
To determine the transfection efficiency of sh-RNA into U251 cells, fluorescence microscopy was used for detection. The results after 96h infection showed that significant green fluorescent protein expression was observed in more than 90% of cells regardless of sh-NC or sh-STAT3 lentivirus, indicating that the infection efficiency could reach 90% (FIG. 2 a).
In order to determine the expression effect of the sh-RNA targeted silent U251 cell STAT3mRNA and protein, real-time fluorescence quantitative PCR and Western blotting are used for detection. The PCR results showed that the expression level of STAT3mRNA was significantly lower in cells of sh-STAT 3-infected group (P <0.01) compared to control and sh-NC-infected group, indicating that it was able to effectively reduce the expression level of STAT3 in U251 cells at the transcriptional level (fig. 2 b). Western blotting results showed that, compared with the control group and sh-NC, the expression of STAT3 protein and P-STAT3 protein in the phosphorylation activation state thereof in sh-STAT 3-infected U251 cells was significantly lower, indicating that the expression and activation of STAT3 in U251 cells could be effectively reduced at the translation level (FIG. 2 c).
2.3 Effect of Targeted silencing STAT3 Gene expression on hypoxia-induced angiogenesis mimicry of U251 cells
To determine the effect of targeted silent STAT3 expression on hypoxia-induced angiogenesis mimicry of U251 cells, in vitro three-dimensional culture experiments were used to test. The observation under a fluorescence microscope after Calcein staining shows that the growth sequence of the U251 cells is disordered under the normoxic condition of a control group, no obvious rule exists, and only a few network structures can be found (figure 3A); and the hypoxic cells are connected with each other and fused to form a network structure formed by connecting single or multiple rings (figure 3B), and the angiogenesis mimicry is remarkably increased (P < 0.01). After targeted silencing of STAT3 gene expression, U251 cells again exhibited growth disorders, reduced tubular structure (fig. 3D); the control sh-RNA-infected group performed similarly to the hypoxic group (FIG. 3C). Randomly selecting 5 high-power visual fields, respectively counting the tube forming number and the tube cavity diameter, and calculating the length of the tube forming number and the tube cavity diameter. Statistical results of multiple independent experiments show (fig. 3E) that targeted silencing of STAT3 expression can significantly inhibit the formation of angiogenesis mimicry (P <0.01) of U251 cells induced by hypoxia.
2.4 Effect of Targeted silencing STAT3 Gene expression on hypoxia-induced U251 cell invasiveness
To determine the effect of targeted silencing STAT3 gene expression on hypoxia-induced U251 cell invasiveness, a Transwell cell invasiveness assay was used for the detection. Microscopic observation after crystal violet staining showed that the number of U251 cells crossing Matrigel was significantly greater (P <0.05) under hypoxic conditions (fig. 4B) than under normoxic conditions (fig. 4A) in the control group. Upon targeted silencing of STAT3 expression (fig. 4D), the number of U251 penetrating Matrigel cells was again significantly reduced compared to the hypoxic group; whereas the sh-NC-infected group performed similarly to the hypoxic group (FIG. 4C). Randomly selecting 5 high power fields, and counting the number of the cells penetrating the Matrigel. Statistical results of multiple independent experiments show (fig. 5) that targeted silencing of STAT3 expression can significantly inhibit U251 cell invasion capacity induced by hypoxia (P < 0.05).
2.5 Effect of Targeted silencing STAT3 expression on hypoxia-induced expression of the mimotope-related protein in U251 cells
To further determine the effect of targeted silent STAT3 expression on hypoxia-induced angiogenesis mimicry of U251 cells and its molecular mechanism, Western blotting was used to detect the expression levels of VM-associated proteins. The results show (fig. 6 a-fig. 6b) that compared with the control group, the MMP9 and MMP14 protein expression of the hypoxia group was significantly reduced (P < 0.05); compared with the hypoxia group and the sh-NC infection group, the expression levels of MMP9, MMP14, EphA2 and Lamin 5 gamma 2 proteins of the U251 cells of the sh-STAT3 infection group are remarkably reduced (P <0.01), which shows that the expression of targeted silent STAT3 can remarkably inhibit the increase of protein expression levels of MMP9, MMP14, EphA2, Lamin 5 gamma 2 and the like of the glioma U251 cells caused by hypoxia, and further proves that the expression of the silent STAT3 can remarkably inhibit the VM formation and cell invasion capacity of the U251 cells induced by hypoxia.
The invention constructs shRNA of a target STAT3 gene to transfect U251 cells, observes infection efficiency by a microscope, and detects the silencing efficiency of STAT3 by real-time fluorescence quantitative PCR and Western blotting; the method comprises the following steps of (1) detecting the influence of STAT3 gene silencing on the formation and invasion capacity of angiogenesis mimicry (VM) of U251 cells induced by hypoxia of the cells through in-vitro three-dimensional culture and a Transwell chamber invasion experiment; and detecting the expression level of VM/invasion related genes/proteins by real-time fluorescence quantitative PCR and Western blotting. Successfully constructing shRNA lentiviral plasmid targeting STAT3 gene and efficiently transfecting U251 cells; compared with the sh-NC infected group, the mRNA and protein expression level of STAT3 in the sh-STAT3 infected group is remarkably reduced (P is less than 0.05); compared with a blank control group and an sh-NC infected group, the cell hypoxia-induced VM formation and invasion capacity of the sh-STAT3 infected group is remarkably reduced (P is less than 0.05); compared with a blank control group and an sh-NC infected group, the expression levels of proteins such as MMP9, MMP14, EphA2, Laminin5 gamma 2 and the like of the U251 cells of the sh-STAT3 infected group are obviously reduced (P is less than 0.01). The targeted silent STAT3 expression can inhibit angiogenesis mimicry and invasion induced by U251 cell hypoxia, and the targeted silent STAT3 expression can be a multi-action target point of human glioma gene therapy.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
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Claims (6)
1. A research method for the targeted silencing STAT3 on the hypoxia induction effect of human brain glioma U251 cells is characterized by comprising the following steps:
step 1, constructing and identifying sh-STAT3 lentiviral vector
Searching sequence information of human STAT3 gene NM-003150 from Genbank, and designing RNA interference sequence according to principle; the possibility of other nonspecific suppressor sequences was excluded by BLAST homology analysis; screening effective target sequences through an interference pre-experiment, determining and synthesizing final construction sequences; annealing to form double-stranded DNA, and performing enzyme digestion connection to generate a lentiviral vector; verifying the correctness by PCR identification and DNA sequencing; packaging 293T cells to generate complete virus particles and measuring the titer of the complete virus particles;
step 2, cell culture and detection of lentivirus infection efficiency
Human brain glioma cell U251 containing 10% fetal calf serum and 1 × 105DMEM culture solution of U/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO2Culturing and incubating under the condition; the cells are grown adherent; cells were divided into 4 groups: blank control group, cobalt chloride CoCl2Simulating the hypoxia of an in-vitro hypoxia group, the sh-NC infected cell simulated hypoxia group hypoxia + sh-NC and the sh-STAT3 infected cell simulated hypoxia group hypoxia + sh-STAT 3;
the well-conditioned U251 cells were collected at 5 × 104Inoculating to 6-well plate at 37 deg.C and 5% CO2Culturing until the cell fusion degree reaches 30%, adding 0.9 mu L lentivirus according to MOI (maximum of identity) of 2.0, infecting in a Normal + Polybrene mode, changing a culture medium after culturing for 24h, and observing the expression condition of a reporter gene GFP (green fluorescent protein) on the lentivirus after continuously infecting for 72 h;
step 3, detecting the mRNA expression level of each gene in the U251 cells by real-time fluorescence quantitative PCR
After sh-RNA infects U251 cells for 48h, the cells are collected and washed 3 times by PBS,extracting total RNA from cells according to the manual of TRIzol kit, and applying GoScriptTMReverse Transcription and PCR amplification are carried out in a Reverse Transcription System; STAT3 gene primer sequence: the upstream primer is shown as SEQ ID NO 1, the downstream primer is shown as SEQ ID NO 2, and the amplification product size is 409 bp; GAPDH gene primer sequence: the upstream primer is shown as SEQ ID NO. 3, the downstream primer is shown as SEQ ID NO. 4, and the amplification fragment is 315 bp;
step 4, Western blotting detection of expression level of each protein
Collecting each group of cells with the fusion degree of 70-80%, adding precooled cell lysate to fully lyse the cells in an ice bath, extracting the whole protein, and analyzing and quantifying by a BCA method;
step 5, Effect of lentivirus infection of U251 cell angiogenic mimicry
Dissolving BD Matrigel in ice water bath overnight, sucking 400 μ l Matrigel with precooling gun head, adding into 24-well plate horizontally placed on ice box, uniformly spreading, incubating in 37 deg.C incubator for 1h to solidify gel, digesting each group of cells with trypsin, centrifuging, discarding supernatant, re-suspending with DMEM, and adjusting cell final concentration to 1.5 × 105Inoculating the seed/ml into the 24-pore plate, wherein each pore is 1ml, and placing the seed/ml in an incubator for continuous culture for 24 hours; then removing the culture medium, lightly washing with PBS for 2 times, adding a calcein solution of 1/10 culture medium system into each hole, and incubating for 30min in an incubator at 37 ℃; observing by a fluorescence microscope, randomly selecting 5 visual fields under a high power microscope for photographing, and respectively counting the number of tubes and the diameter of the tube cavity; each set of experiments was repeated 5 times;
step 6, influence of invasion capacity of lentivirus infected U251 cells
Dissolving BD matrix gel in ice water bath overnight, diluting with serum-free culture medium to 10mg/ml in a superclean bench at a ratio of 5: 1, sucking 100 μ l per well, spreading gently into Transwell chamber with polycarbonate filter membrane having pore diameter of 8 μm, shaking, polymerizing at 37 deg.C for 5 hr, digesting each group of cells with trypsin, centrifuging, discarding supernatant, re-suspending with serum-free DMEM, and adjusting final cell concentration to 5 × 105One/ml, 200. mu.l was added to the upper layer of the Transwell chamber; lower layerAdding 600 μ l complete medium, standing at 37 deg.C and 5% CO2Culturing for 24 hours in an incubator; after fixing the cells for 15min by 4% paraformaldehyde, wiping the upper cells of the chamber by a cotton swab, dyeing for 30min by 0.1% crystal violet, washing by PBS, naturally drying, taking five visual fields of upper, lower, left, right and middle under a microscope, photographing, observing and counting the cells passing through a polycarbonate membrane of the Transwell chamber; each set of experiments was repeated 5 times;
step 7, statistical processing
Statistical analysis is carried out by using SPSS 18.0 software, and the measurement data are expressed by mean +/-standard deviation; the comparison among groups adopts single-factor variance analysis, and the comparison between every two groups adopts LSD-t test; p <0.05 is statistically significant for the differences.
2. The method for researching the hypoxia-induced influence of targeted silent STAT3 on human brain glioma U251 cells according to claim 1, wherein in the step 3, the PCR reaction condition is pre-denaturation at 95 ℃ for 30s, then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ is carried out for 6min after 35 cycles; relative expression of STAT3mRNA is expressed as a 2-. DELTA.Ct value; ct represents the threshold cycle and each set of experiments was repeated 5 times.
3. The method for studying the hypoxia-induced influence of targeted silent STAT3 on human brain glioma U251 cells as claimed in claim 1, wherein in step 4, groups of cells with a confluency of 75% are collected.
4. The method for studying the hypoxia-induced influence of targeted silent STAT3 on human brain glioma U251 cells according to claim 1, wherein in step 4, a 10 μ g total protein sample is taken, subjected to SDS-PAGE electrophoresis by using 4% -12% TrisGlycine gel, transferred onto a PVDF membrane, sealed for 1h by using milk prepared from TBST, and added with a volume dilution ratio of 1: 3000 primary antibody 4 ℃ overnight; the membrane was then washed 5min × 5 times with TBST, diluted with a volume ratio of 1: incubating 5000 fluorescent secondary antibody at room temperature in dark condition for 1h, washing membrane with TBST for 5min × 5 times, and exposing with ODYSSEY CLx; scanning gray scale and calculating the corresponding expression amount of the protein.
5. The method for studying the hypoxia-induced influence of targeted silent STAT3 on human brain glioma U251 cells according to claim 4, wherein 8% TrisGlycine gel is used for SDS-PAGE electrophoresis.
6. Use of the method of claim 1 in the manufacture of a medicament for the treatment of human brain glioma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010448903.1A CN111607616A (en) | 2020-05-25 | 2020-05-25 | Research method for targeted silencing STAT3 on hypoxia induction effect of human brain glioma U251 cells and application of research method |
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| CN102433336A (en) * | 2010-09-29 | 2012-05-02 | 上海生博生物医药科技有限公司 | Small interfering RNA for inhibiting glioma proliferation and promoting glioma apoptosis and preparation method and application thereof |
| CN103882060A (en) * | 2012-12-24 | 2014-06-25 | 苏州中同生物科技有限公司 | Construction and identification of STAT3-targeting lentivirus interference vector |
| CN111154723A (en) * | 2020-01-15 | 2020-05-15 | 宁夏医科大学 | Research method for siRNA targeted inhibition of Zwilch and application thereof |
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| CN102433336A (en) * | 2010-09-29 | 2012-05-02 | 上海生博生物医药科技有限公司 | Small interfering RNA for inhibiting glioma proliferation and promoting glioma apoptosis and preparation method and application thereof |
| CN103882060A (en) * | 2012-12-24 | 2014-06-25 | 苏州中同生物科技有限公司 | Construction and identification of STAT3-targeting lentivirus interference vector |
| CN111154723A (en) * | 2020-01-15 | 2020-05-15 | 宁夏医科大学 | Research method for siRNA targeted inhibition of Zwilch and application thereof |
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