CN111996251B - Application of malignant glioma biomarker - Google Patents
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- CN111996251B CN111996251B CN202010568424.3A CN202010568424A CN111996251B CN 111996251 B CN111996251 B CN 111996251B CN 202010568424 A CN202010568424 A CN 202010568424A CN 111996251 B CN111996251 B CN 111996251B
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Abstract
The application of the glioblastoma biomarker is that RHOJ mRNA shown as SEQ ID No. 1 and RHOJ protein shown as SEQ ID No. 2 are used as detection targets in the preparation of a kit for diagnosing, treating or prognosis judging glioblastoma. We find that silencing RHOJ expression significantly inhibits malignant phenotypes such as glioma cell proliferation and invasion, RHOJ can be used as a molecular marker for diagnosis and prognosis judgment of malignant glioma, and therapeutic strategies such as antibodies or small molecule inhibitors targeting RHOJ are expected to improve malignant glioma treatment.
Description
Technical Field
The invention belongs to the field of biotechnology, pathological examination and oncology, and particularly relates to application of a malignant glioma biomarker.
Background
Glioblastoma (GBM) is the most common fatal primary intracranial malignancy in central nervous system tumors, the incidence rate of gliomas is 5/10 ten thousand-8/10 ten thousand, and the 5-year mortality rate is next to pancreatic cancer and lung cancer in systemic tumors, and is third in rank. The main characteristics are that the angiogenesis and diffuse infiltration growth are enriched, the pathological total excision is difficult, the recurrence is easy after the operation, and the composition is resistant to radiotherapy and chemotherapy. Gliomas can be classified according to the pathological malignancy of tumor cells: I-IV grade. Low Grade Gliomas (LGG) are mainly WHO grade II and III gliomas, while high grade Gliomas (GBM) are mainly WHO grade IV gliomas. With the development of molecular biology and molecular pathology, molecular typing of gliomas is becoming more and more important, such as IDH mutation, MGMT promoter methylation, 1p/19q co-deletion, TERT promoter mutation, EGFR amplification and egfrvlll rearrangement, ATRX mutation, and the like. Highly vascularized and invasive growth results in a poor prognosis for GBM, therefore, lock-mediated GBM angiogenesis and invasion key molecules are of clinical importance for the development of effective molecular markers and therapeutic targets.
Recently, GBM angiogenesis and endothelial barrier integrity have been found to be closely related to invasive metastasis, involving the regulation of various pathophysiological states such as cytoskeletal and intercellular junctions, in which Rho-gtpase plays a central role. Rho-gtpase is a key signaling element in GBM invasion regulation that mediates receptor initiation signals, can regulate dynamic changes in cell morphology and actin space structure, and stimulates extrusion of cells through the narrow extracellular space of neurons. Rho-gtpase is a family member of small G proteins, also part of the Ras superfamily, which is capable of modulating actin, involved in physiological processes such as cytoskeletal rearrangement, vesicle transport, and cell adhesion. It has now been found in mammals that 20 Rho-gtpases are divided into 8 subfamilies. Rho-gtpase plays a central role in the integrity of the vascular endothelial barrier, and Rho-gtpase and its modulators are important mediators of cytoskeletal and intercellular junctions. Rho-gtpase family molecules have a dual impact on the integrity of the vascular endothelial barrier. Rho-gtpase is involved in the inflammatory factor-induced loss of vascular endothelial stability on the one hand, and in maintaining vascular endothelial barrier stability on the other hand, and in inducing repair of vascular endothelial integrity after damage. The dual action of Rho-gtpase is closely related to its subcellular localization.
RHOJ is a homolog of CDC42 and is believed to be a key regulator of cell morphology, actin cytoskeleton, cell motility, and cell cycle. It is highly expressed in endothelial cells, and has important regulatory effects on migration and angiogenesis of endothelial cells. Previous studies have shown that RHOJ is highly expressed in melanoma and regulates tumor metastasis and progression through its downstream PAK-BAD signaling pathway, and that RHOJ also regulates melanoma resistance through a pathway that inhibits DNA damage. RHOJ as a small G protein rich in endothelial cells plays an important role in tumor angiogenesis, lung cancer metastasis and spontaneous breast cancer models. High RHOJ expression is an independent negative prognostic factor for gastric cancer survival and is associated with gastric cancer progression and metastasis. However, the expression of RHOJ in glioblastoma, and the use thereof in diagnosis, treatment and prognosis are not reported in the present day.
Disclosure of Invention
The technical problems to be solved are as follows: the invention provides an application of a malignant glioma biomarker, which confirms the difference of the action of RHOJ in regulating glioma cells, tumors and normal vascular endothelial cells and the regulating mechanism thereof; elucidating the role of RHOJ in regulating malignant phenotypes such as glioma proliferation, apoptosis, invasion and metastasis, angiogenesis and the like and molecular mechanisms thereof; how to target RHOJ or its binding key target molecule and elucidate its mechanism and signaling pathway that mediate GBM malignant phenotype. The specificity of the antibody and the kit for detecting RHOJ expression is prepared, the design and synthesis of a small molecular compound based on RHOJ protein and crystal structure combination computer prediction are realized, and the therapeutic antibody targeting RHOJ is screened by an antibody screening method such as phage display technology.
The technical scheme is as follows: the RHOJ mRNA shown in SEQ ID No. 1 is used as a detection target in the preparation of a kit for diagnosing, treating or prognosis judging glioblastoma.
The RHOJ protein shown in SEQ ID No. 2 is used as a detection target in the preparation of a kit for diagnosing, treating or prognosis judging glioblastoma.
Use of a compound that inhibits the expression of the RHOJ protein in the preparation of a product for the treatment of glioblastoma.
The beneficial effects are that: according to the invention, through the second generation sequencing, gene chip, real-time fluorescent quantitative RT-PCR and immunohistochemical analysis on Rho-GTPase family molecular expression in malignant glioma tissues, RHOJ is found to be over-expressed in malignant glioma and related to poor prognosis of patients, and the important value of the RHOJ on diagnosis and prognosis judgment of malignant glioma is locked. Silencing RHOJ expression inhibits GBM cell proliferation and induces cell cycle G2/M phase arrest, stable knockdown of RHOJ significantly inhibits GBM cell invasion, migration and tumorigenicity, and affects cell morphology and EMT phenotype, suggesting that RHOJ plays an important role in GBM. The information or evidence of the malignant phenotype mediated by RHOJ regulatory difference target molecules is clarified, and novel molecular markers and intervention targets are provided for glioma molecular diagnosis and targeted treatment.
Drawings
FIG. 1 is a graph showing that RHOJ overexpression in high grade Glioma (GBM) is significantly correlated with patient recurrence and poor prognosis; wherein a is IHC staining expression difference of RHOJ in normal brain tissue and GBM tissue, b is Western blot analysis of RHOJ expression in umbilical cord blood endothelial cells (HUVEC) and human GBM cell lines, c-e is Kaplan-Meier analysis showing that high expression RHOJ in GBM is significantly related to prognosis differences of all GBM (c), primary GBM (d) and recurrent GBM (e) patients, f is relative expression difference of RHOJ in GBM and LGG, g is relative expression difference of recurrent GBM and primary GBM; FIG. 2 is a graph showing that silencing RHOJ expression inhibits GBM cell proliferation and induces G2 phase cell cycle arrest; wherein a-B is RT-qPCR or Western blot detection shows that siRNA inhibits RHOJ mRNA and protein expression, c-d and e-f are growth curves of GBM cell lines analyzed by cell counts (c-d) and Cell Counting Kit-8 (CCK-8 kit) (e-f) respectively after siRNA inhibits RHOJ expression, G is a cell growth curve obtained by analyzing stable knockdown and over-expression RHOJ in LN229 cells by Western blot, h-i is a cell growth curve obtained by analyzing stable knockdown and over-expression RHOJ in LN229 cells, cell counts (h) and CCK-8 analysis (i) after knocking down and over-expression RHOJ in LN229 cells, j-k is RHOJ-induced cell cycle arrest in G2 phase after knocking down RHOJ in LN229 cells, and l is Western blot shows that expression of Cyclin B1 is significantly reduced after stable knockdown in RHOJ in LN229 cells, all data are analyzed by t test as average value of three independent experiments.+ -. SEM. * P is less than 0.05; * P <0.01; p < 0.001;
FIG. 3 is a graph showing that stable knockdown RHOJ expression inhibits tumorigenicity and angiogenesis of mouse GBM cells; wherein a is RHOJ stable knockdown (shRHOJ) reducing the colony count of U251 and LN229 cells, b is an image of shRHOJ and RHOJ overexpression (RHOJ-oe) of U251 cells (500 cells/well) in a representative tumor sphere formation assay, and quantification of tumor sphere count after 9 days of culture; scale bar, 50 μm, c-d is the subcutaneous inoculation of 500 ten thousand U87 cells expressing shrhhoj or control-shRNA (shNC) into BALB/c nude mice, tumor size was measured every 4-5 days (n=6), mice and tumors were photographed and saved for IHC (immunohistochemistry) and western blotting 26 days after injection, E is H & E staining and expression of IHC assay proliferation marker Ki-67 in xenograft tumor tissue of control or RhoJ-shRNA, scale bar, 500 μm, f is RhoJ, p-Cofilin (Ser 3), western blot analysis of total Cofilin expression in xenograft tumor tissue. Data represent mean ± SEM of t-test analysis by three independent experiments, P <0.05, P <0.01;
FIG. 4 is a graph showing that silencing RHOJ expression inhibits the in vitro migration and invasion capacity of GBM cells; wherein a-b are representative images (a) and statistical plot (b) showing analysis of cell migration capacity after stable knockdown and overexpression of RHOJ of GBM cells, scale bar, 50 μm, c is representative images and relative migration areas of cell wound healing assays after stable knockdown and overexpression of RHOJ of GBM cells were counted, d is representative images and statistical number of invading cells measured after stable knockdown and overexpression of RHOJ of GBM cells, three independent in vitro assays were performed, including cell migration and invasiveness assays and wound healing assays, respectively, data expressed as mean value of three experiments.+ -. SEM,..times.P < 0.001;
FIG. 5 shows that glioblastoma cell RHOJ expression is related to GBM cell morphology and EMT phenotype; wherein a-c are the fluorescent intensity quantitative analysis of the cells of GBM showing RHOJ (green), F-actin (red), DAPI (blue) and Merge, and b-d are RHOJ and F-actin, scale bar: 200 μm, E is representative immunofluorescent staining of E-cadherin (green), vimentin (red) and DAPI (blue) in U251GBM cells, scale bar: 50. μm, f is a representative immunoblot for detection of RHOJ and EMT marker E-cadherin and vimentin expression, data expressed as mean ± SEM, t-test analyzed P values, P <0.05, P <0.01.
FIG. 6 shows that RHOJ expression in GBM cells can be regulated by transcription factor c-Jun expression; wherein a is the predicted transcription factor c-Jun binding site between-1 kb and 0kb in the region upstream of RHOJ, WT comprises c-Jun binding site, mutations in the putative binding sequence are listed as Mut1, mut2 and Mut3, B is luciferase assay established using HEK-293T cells transfected with PGL3Basic or PGL3-RHOJ (PGL 3-plasmid containing the RHOJ DNA promoter [ -1000/0 ]), normalized luciferase activity to renilla luciferase activity, c is a dual luciferase assay, PGL3Basic, PGL3B-RHOJ or mutant plasmid Mut1, 2 or 3 are used to transduce c-Jun over-expression or empty vector control U251 cells, luciferase activity is normalized to renilla luciferase activity, d-e is Jun inhibitor SP 125 inhibits expression of oj mRNA (d) and protein (e) in U87 cells, and k shows a significant expression dependence on the expression of k in the cells, and k is a < p# 0.01, # 0.001, # p# is a test, and # 0 # p is confirmed by independent assay.
FIG. 7 shows RHOJ interaction with momin, an ERM family protein, driving its downstream Rac1-PAK-cofilin exchange, cell migration and proliferation. Wherein a is the interaction between RHOJ and moesin further verified by Western blotting, and then co-IP was performed, and b is a representative fluorescence image showing co-localization of RHOJ and moesin in U87 cells. Scale bar: 50 μm, c is Negative Control (NC), RHOJ shRNA and stable overexpressing cells were subjected to immunoblotting with antibodies recognizing downstream proteins, d is immunoblotting assay to prepare PAK2/4 activity in U251 cells expressing negative control or RHOJ-shRNA or shRHOJ and RHOJ overexpressing plasmids, rac1 and RHOJ expressed, tubulin was used as loading control, e is quantitative analysis of Western immunoblotting in d, f is transfection of U251 cells with RHOJ or Rac1siRNA for 72 hours, relative quantification of RHOJ and Rac1 in each group was shown by immunoblotting analysis of cell lysates, the experiment has been repeated 3 times independently, h is a summary of RHOJ upstream and downstream signals depicting potential to aid in GBM cell migration and proliferation. Data represent mean ± SEM of t-test analysis of 2 students by three independent experiments. * #p <0.05; * # p <0.01; * P <0.001.
Detailed Description
The following examples will provide those skilled in the art with a more complete understanding of the invention, but are not intended to limit the invention in any way.
Example 1
We performed immunohistochemical staining of the collected clinical tissue sections of glioblastoma patients with RHOJ antibody (Sigma, HPA 003050): placing the collected pathological section in xylene I for 5min, replacing xylene II, soaking for 5min, anhydrous ethanol for 3min,95% ethanol for 3min,80% ethanol for 3min,70% ethanol for 3min, dewaxing and hydrating tap water for 3min, placing in antigen retrieval liquid (citrate buffer solution, pH 6.0) and boiling at 95-98deg.C for 15min, naturally cooling to room temperature, and performing antigen retrieval, and washing with PBST buffer solution for 3 times each for 3min. And (5) dripping goat serum blocking solution for 30min at room temperature. The blocking solution was removed by shaking, and primary antibody (RHOJ, 1:400) was added overnight at 4 ℃. PBST buffer solution is washed for 3 times and 3min each time, secondary antibody is dripped, room temperature is 1-2h, PBST buffer solution is washed for 3 times and 3min each time, DAB color development (DAB peroxidase color development kit, biyun Tiansheng) is carried out, tap water is washed for 10min, hematoxylin (Soy Bao organism) is counterstained for about 20 seconds, tap water is rapidly used for washing, alcohol gradient dehydration (70%, 80%,95%,100%, dimethylbenzene I and dimethylbenzene II) and neutral resin sealing sheets are carried out. And photographing by an inverted microscope. The staining results show that RHOJ is highly expressed in patients with malignant glioma.
The clinical tissue sample of the malignant glioma patient is detected through second-generation sequencing and real-time fluorescent quantitative RT-PCR, the RHOJ is quantitatively measured through a dye method or a Taqman probe method, and the application prospect of the kit serving as a marker for molecular diagnosis or prognosis judgment and an intervention treatment target is explored by combining the relation between the expression level of the kit and glioma molecular typing, microvascular formation, patient survival and prognosis and the like through clinical analysis. RHOJ was found to be significantly higher in GBM than LGG, significantly higher in recurrent GBM than non-recurrent GBM, and high expression of RHOJ was significantly correlated with poor prognosis in both primary and recurrent GBM patients.
The human endothelial cells HUVEC and the glioblastoma cells LN229, U251, U87, T98G, KNS81 and LN18 total proteins are respectively extracted, the expression condition of RHOJ proteins is detected by western blot, and the expression of RHOJ in various glioblastoma cell strains is up-regulated, while the expression abundance in HUVEC cells of a control group cell strain is relatively low.
Example 2
siRNA that specifically inhibited RHOJ two sirnas were designed to prevent off-target effects based on the RHOJ sequence and on-line design website (https:// link.zhihu.com/. siRNA was synthesized and provided by Shanghai Ji Ma pharmaceutical technologies limited.
Construction of shRNA for specifically inhibiting RHOJ and over-expression RHOJ plasmid. Specific shRNA sequences are obtained from sigma company, a functional network, and are inserted into PLKO.1 plasmid vectors to construct shRHOJ expression plasmids. Pubmed obtains the CDS region of RHOJ gene, designs primer, and the primer is synthesized by Kirschner Biotechnology Co., ltd, and PCR cloning to obtain the full length sequence of RHOJ, and inserts into eukaryotic expression vector plenti-CMV-GFP-puro. Transforming the shRHOJ plasmid and RHOJ expression plasmid into escherichia coli, culturing, selecting a monoclonal, culturing by shaking, extracting the plasmid by using a small-extraction medium-quantity kit of the biochemical endotoxin-free plasmid of the radix et rhizoma, sequencing by the engine biological company, and selecting and constructing the plasmid correctly for use. The RHOJ-flag label plasmid is obtained by designing a flag label primer based on the constructed RHOJ over-expression plasmid and carrying out cloning amplification.
siRNA, shRNA and RHOJ-oe expression plasmid transfection and stable transgenic cell line selection, RHOJ specific siRNA sequences and negative control (siNC) were purchased from Shanghai Ji Ma company. The siRNA used was an exact 2000 transfection reagent (Nanjinopran Bio Inc.). And (3) inoculating the cells into a 6-well plate, uniformly mixing 5 mu L of the Exfect 2000 with 125 mu L of serum-free DMEM after overnight, uniformly mixing 100pmol of siRHOJ/siNC with 125 mu L of serum-free DMEM, standing at room temperature for 5min, and mixing the incubated Exfect 2000 with siRNA. After 15min of standing, the mixture was added to a 6-well plate, and 1.75mL of serum-free medium was added to make up to 2mL, and after 6h, the mixture was replaced with DMEM complete medium. After 48h of transfection, subsequent experiments were performed. The ShRNA and the plenti expression plasmids are subjected to slow virus packaging by using 293T cells, the 293T cells are inoculated in a 6-well plate, target plasmids and virus packaging plasmids pRSV-Rev, pMDLg/pRRE and pMD2.G (purchased from addgene) are transfected for 48 hours to collect virus supernatant for infecting U87, LN229 and U251 cell strains, puromycin is added for stable transfection cell strain screening after 24 hours, and shRNA or RHOJ expression plasmid stable transfection cell strains are obtained after about one week.
The siRHOJ sequence is as follows:
#si-RHOJ-1:5′-AGAAACCUCUCACUUACGAG-3′；
#si-RHOJ-2:5′-CCACUGUGUUUGACCACUA-3′；
the shRHOJ sequence is as follows:
#sh-RHOJ-1:5'- CCGGCAACACUUGCUCGGACUGUAUCUCGAGAUACAGUCCGAGCAAGUGUUGUUUU UG-3'；
#sh-RHOJ-2:5'- CCGGGCCCGUUUGCUGUAUAUGAAACUCGAGUUUCAUAUACAGCAAACGGGCUUUU UG-3'；
the RHOJ expression plasmid primers were as follows:
an upstream primer of 5'-CGGAATTC-GCCACC-ATGAACTGCAAAGAGGGAAC-3';
a downstream primer 5'-CGGGATCC-TCAGATAATTGAACAGCAG C-3';
flag primer sequence:
an upstream primer 5'-GCTCTAGAGCCACCATGGGAGACTACAAGGACGATGATGACAAGA TGAACTGCAAAGAGGGAAC-3';
downstream, 5'-CGGAATTCGCCACCATGGGAGACTACAAGGACGATGATGACAAGATG AACTGCAAAGAGGGAAC-3';
cell growth curve experiment, U87 cells were inoculated in 6-well plates, 20 ten thousand per well, siNC (5'-UUCUCCGAACGUGUCACGUUU-3') and SiRHOJ (two siRNAs, siJ-1-5'-AGAAACCUCUCACUUACGAG-3' and siJ-2-5'-CCACUGUGUUUGACCACUA-3') in 3 multiple wells per group after siRNA transfection for 48h, cultured in complete medium, counted daily for 3d. The count results were plotted by Graphpad software.
Nude mice were subjected to subcutaneous oncology experiments, and for in vivo animal studies, U87 cells expressing shNC or shRHOJ which had been stably transformed were digested, counted, 500 ten thousand cells were resuspended in 100 μl of serum-free medium, mixed with 100 μl of Matrigel (Corning), and injected into 5 to 6 week old male BALB/C nude mice (purchased from the university of animal research center of the medical university of south kyo, china) subcutaneously, 6 animals per group. Tumor size was measured every 4-5 days after injection and calculated according to the following formula: (length. Times. Width) 2 )/2. Mice were sacrificed 26 days after injection. All animal experiments were performed under the approval of the national institutes of health.
Cell cycle assay (LN 229 cell line), 50 ten thousand cells were collected after 48h of LN229 cell transfection with siNC or siRHOJ (siJ-1), resuspended in 1.5mL of 75% ethanol and fixed overnight at-20 ℃. Centrifugation at 800rpm for 3min, washing with PBS 3 times, treating Propidium Iodide (PI) for 30min, then detecting with a flow cytometer (BD bioscience, USA), and analyzing the data using Modfit software, it was found that LN229 cell cycle was blocked in G2 phase after RHOJ expression as shown in FIGS. 2 j-k. After the RHOJ is knocked down through a cell growth curve experiment and a nude mouse subcutaneous tumor experiment, the growth of GBM cells and the growth of subcutaneous tumors are obviously inhibited, and the flow cytometry detection also shows that the cell cycle process is influenced.
Example 3
Transwell migration experiments, cells were grown overnight in serum-free medium and then trypsinized, cells were suspended in serum-free DMEM medium, and the concentration was adjusted to 2X 10 after counting 4 /mL; 600. Mu.L of DMEM complete medium containing 10% serum was added to the bottom of the 24-well plate, 200. Mu.L of the cell suspension was added to the upper chamber, and then placed in an incubator for cultivation; after 24h, carefully taking out the chamber with forceps, sucking out the liquid in the upper chamber, adding about 600. Mu.L of 4% paraformaldehyde, and fixing for 30min at room temperature; sucking out 4% paraformaldehyde in the upper chamber, adding about 600 μl of crystal violet, and dyeing at room temperature for 30min; after the PBS is gently washed for a plurality of times, the small chamber is taken out, the liquid in the upper chamber is sucked off, and the small chamber is carefully rubbed by a wet cotton swabRemoving cells from the surface of the membrane at the bottom of the upper chamber; randomly selecting 5 visual fields under a 40-fold optical lens for observation, counting and carrying out statistical result analysis to find that the knockdown RHOJ inhibits cell migration.
Transwell invasion assay approximately 30. Mu.L Matrigel dilution gel, i.e., serum free medium, was spread in a Transwell chamber: matrigel gum = 3:1, coagulated in an incubator at 37 ℃ for 1-2h; cells were grown overnight in serum-free medium and then trypsinized, suspended in serum-free DMEM medium, and the concentration was adjusted to 2×10 after counting 4 /mL; 600. Mu.L of DMEM complete medium containing 10% serum was added to the bottom of the 24-well plate, 200. Mu.L of the cell suspension was added to the upper chamber, and then placed in an incubator for cultivation; after 24h, carefully taking out the chamber with forceps, sucking out the liquid in the upper chamber, adding about 600. Mu.L of 4% paraformaldehyde, and fixing for 30min at room temperature; sucking out 4% paraformaldehyde in the upper chamber, adding about 600 μl of crystal violet, and dyeing at room temperature for 30min; after the PBS is gently washed for a plurality of times, the small chamber is taken out, the liquid in the upper chamber is sucked, and the cells on the surface of the membrane at the bottom of the upper chamber are carefully wiped off by a wet cotton swab; randomly selecting 5 visual fields under a 40-fold optical lens for observation, counting and carrying out statistical result analysis to find that RHOJ knockdown inhibits cell invasion.
Scratch healing experiments, for healing analysis, a marker pen was used to evenly draw a transverse line with a ruler behind a 6-well plate, and 3 lines were drawn per well; after digestion of the cells, the cells were incubated with serum-free DMEM medium at 2 mL/well, 5X 10 5 The individual cells/wells were plated on 6-well plates overnight and cultured until the cells were substantially confluent; vertically scratching 10 mu L of a trace gun head in a 6-hole plate for the next time, washing cells for 3 times by using PBS, removing the scratched cells, and adding a serum-free culture medium; adding 5% CO 2 Is cultured in an incubator at 37 ℃. The distances of the scratch voids were compared by observing in 6 100X fields of view under an inverted microscope at 0h, 24h, 48h, respectively. The knockdown RHOJ is found to inhibit cell migration, and in vitro functional experiments find that the knockdown RHOJ expression inhibits malignant glioma cell migration and invasion.
Example 4
Cell immunofluorescence experiments U251 and LN229 cells stably expressing shrheoj were seeded into 6 well plates with coverslips 2.0 x 105 cells per well. After 72h, the culture was aspirated, the cells were washed 3 times with PBS, then 1mL of 4% paraformaldehyde was added to each well for 30min, the paraformaldehyde was aspirated, the cells were washed twice with PBS, 1mL of blocking solution containing 0.5% Triton X-100 was added to each well for 1h at room temperature, and the cells were washed twice with PBS. The cells were washed 3 times with PBS and 5min each time after incubation at 4℃overnight. Adding corresponding fluorescent secondary antibodies, incubating for 2 hours at room temperature, washing 3 times by PBS, adding rhodomine-conjugated phalloidin (bright holy organisms) to dye the cytoskeleton for 30min, washing 3 times by PBS, adding DAPI dye solution, washing the cells 3 times by PBS after 10min, sealing with 90% glycerol, and observing under a fluorescent microscope. Phalidin immunofluorescence staining shows that cells become larger after RHOJ is knocked down, and the fluorescence density of Phalidin-labeled cytoskeletal protein F-actin is reduced.
The dual-luciferase reporter gene experiment, JASPAR and ALGGEN-PROMO databases predict on line that the upstream region of the RHOJ promoter has 3 binding sites for transcription factors c-Jun, and then the dual-luciferase reporter gene experiment is used for verification, and the method is as follows: 1) Constructing pGL3-RHOJ reporter gene plasmid: plasmid was constructed by NCBI query RHOJ sequence full length, wild type sequence (WT) and 3 c-Jun binding site mutant sequences (Mut 1, mut2 and Mut 3) were synthesized, inserted into pGL3-Basic reporter plasmids, respectively, and PGL3-WT and pGL3-Mut1/Mut2/Mut3 plasmids were constructed. The C-jun expression plasmid was purchased from Sigma. 2) Cell transfection: 293T cells or U251 cells were inoculated into 96-well ELISA plates at a certain density one day before transfection, 6 multiplex wells were set per group, the transfection method was the same as siRNA, the amount of vector plasmid transferred was 0.25. Mu.g, and cells were collected after 24h of transfection. 3) Luciferase activity assay: the medium was discarded, gently washed 2 times with PBS, 20. Mu.L of cell lysate (1 XPLB) was added to each well, and the mixture was shaken on a shaker for 30min to ensure sufficient cell lysis; adding 100 mu L Luciferase Assay Reagent II, mixing, and immediately placing into an instrument for reading; 100. Mu.L of pre-mixed Stop & Glo Reagent was added, mixed well, put into an instrument for reading, and the values of Firefly-Luc and Renilla-Luc were recorded for comparative analysis of fluorescence activity. Double Luciferase report experiments show that c-Jun can enhance the activity of Luciferase and promote the expression of RHOJ.
Further administration of the c-jun small molecule inhibitor SP600125 (available from MedChemExpress Co.) cells were seeded at moderate density in 6 well plates, the following day cells were treated with SP600125 at a concentration gradient of 0,5, 10, 20. Mu.M, and after 48h the cells were harvested to extract total RNA and protein, respectively. Row qPCR and western blot validation found that SP600125 significantly inhibited mRNA and protein levels of RHOJ and was significantly dose dependent (fig. 6d and e). JNK belongs to the MAPK pathway and is an upstream regulator of c-Jun, and expression of JNK is inhibited by constructing shRNA targeting JNK, and similarly, expression of RHOJ is found to be effectively inhibited (fig. 6 f).
The RHOJ qPCR primers were as follows:
upstream primer 5'-CCTGAGTGACAGAGAAAGAACC-3'
Downstream primer 5'-GGAGTGTGTGCGTATGAAAGA-3'
The Jun qPCR primers were as follows:
upstream primer 5'-AAGAACTCGGACCTCCTCA-3'
Downstream primer 5'-CCGTTGCTGGACTGGATTAT-3'
Example 5:
immunoprecipitation (Co-IP) experiments, blocking the beads with 1% BSA for 30min (4 ℃) and washing twice with PBS, adding 1. Mu.L of Flag anti body (Sigma) or mouse IgG (abclon), mixing the beads with antibody in 700. Mu.L of IP buffer, coupling for 1-2h at 4 ℃, discarding the buffer, collecting cells, washing twice with PBS, adding about 800. Mu.L of IP buffer, placing on a shaker for lysis for 10min, centrifuging for 10min at 14000rpm, taking the supernatant, adding protein supernatant into the coupled beads with antibody respectively, incubating at 4 ℃ overnight, washing with IP buffer for 5 times, discarding the buffer, adding SDS loading buffer, and boiling at 95 ℃ for 10min for western blot detection. Co-IP experiments found an interaction between RHOJ and moesin, and further confirmed the existence of Co-localization between the two by immunofluorescence experiments. Western blot detection shows that PAK channels are down-regulated after RHOJ knockdown, the phosphorylation level of skeleton-related proteins Rac1 and cofilin is reduced, and the CDH1 of the EMT-related protein is up-regulated and VIM is down-regulated.
To sum up, the applicant obtains clinical evidence of the relationship between the expression level of RHOJ in clinical glioma patient tissues and blood samples and glioma patient survival and prognosis; elucidating the role of RHOJ in regulating malignant phenotypes such as glioma proliferation, apoptosis, invasion and metastasis, angiogenesis and the like and molecular mechanisms thereof. The information or evidence of the malignant phenotype mediated by RHOJ regulatory difference target molecules is clarified, and novel molecular markers and intervention targets are provided for glioma molecular diagnosis and targeted treatment. It is drawn that "transcription factor c-Jun can regulate the expression of RHOJ, RHOJ and interact with moesin to activate PAK-Rac1 pathway to promote cytoskeletal dynamics and proliferation and migration of glioma cells, RHOJ has also been demonstrated to play a key role in the epithelial-mesenchymal transition process. "mechanical drawing. The important roles of RHOJ in diagnosis, treatment and prognosis judgment of malignant glioma are proved for the first time, specific antibodies and kits for detecting RHOJ expression are developed, immunohistochemical or enzyme-linked immunosorbent assay (ELISA) technology and application thereof are developed, and the research and development of pharmaceutical compositions for treating malignant glioma, including nucleic acid molecules for inhibiting or silencing human RHOJ expression, RHOJ small molecule inhibitors or therapeutic antibodies and the like and application thereof are all included in the scope of the invention.
Sequence listing
<110> Anhui nan medical college first affiliated Hospital (Anhui nan medical college Yi Angeles mountain Hospital)
<120> application of malignant glioma biomarker
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3556
<212> RNA
<213> Artificial sequence (Artificial Sequence)
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aacucacacu gagagcggaa aggggcagac ccuuuucaua acucccucaa guguguguua 240
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ucaugugcuu uggaaaaagc aggagaagca auagcagcag gaguccccag cagcuggagc 420
cgcaagaaug aacugcaaag agggaacuga cagcagcugc ggcugcaggg gcaacgacga 480
gaagaagaug uugaagugug uggugguggg ggacggugcc guggggaaaa ccugccugcu 540
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cugcuucucu gucguaaacc cugccucuua ccacaauguc caggaggaau ggguccccga 780
gcucaaggac ugcaugccuc acgugccuua uguccucaua gggacccaga uugaucuccg 840
ugaugaccca aaaaccuugg cccguuugcu guauaugaaa gagaaaccuc ucacuuacga 900
gcauggugug aagcucgcaa aagcgaucgg agcacagugc uacuuggaau guucagcucu 960
gacucagaaa ggucucaaag cgguuuuuga ugaagcaauc cucaccauuu uccaccccaa 1020
gaaaaagaag aaacgcuguu cugaggguca cagcugcugu ucaauuaucu gagguugucu 1080
gggaccugcc uccaccccau ccagggauga gaauggcagc caaucucugu ggccaagcuc 1140
cagccaaaaa ggagggcacg accagaaagg aacucccuuu gcacggaggc uugccccauc 1200
acccucugag cccucccaac acagcacacu agucagccca cugccacgac cucccugcca 1260
gccagaagca uccguacugc acgcugucug agaaugcugg gccuggauug cagacagugc 1320
cgcugcugau cgcaucaaaa acaaagucaa aggccaucuc acauuuuaca aauccccagc 1380
ucaugaacgu gaagcugaua ggaaaucacc ccagggaacc cgaaaaagaa acuugauucc 1440
ucuauugcug gccuuacuug augucuuuua uaaaacuugg gacuacaaua cuaaccuuuu 1500
uuucugaauc ugcuguucua cccauguguc ucacauucau uuguauuauu ucaagaaaug 1560
uacuaauuuc caguucacuc aggccuuacu aauccauacc aaauuagccu aaagacaagg 1620
cauuuuauau ucauuucuau uuucagcaug uuucuaccaa agcuauuaga accaacacgu 1680
accucugaau gcccgauuau aagaagacau gagaagacuu uaaaaguuuu ggaaauuuac 1740
agagccauga uuuuugaacc uaauugaaag aaaaccaucu gaauuguugc agguccacau 1800
uuuugccaaa gauacacucu auagaugcuu aguaguggcc ugauuuuuuu ccauguauug 1860
ccacgacaaa cuaaaaauga acuguguuua agaauguagu auuucuguuu uucauccaag 1920
uugauugggg gaagaauaug gcaggaucca ucuuuuacag uauuuuguau ucaguaaagu 1980
ggacauuccu gcuccucccu ucccccauug caugcccucu uccucccuug auuucacuuu 2040
cucucaugcc cggauccuuu uauucucccc aguuauaacc caguuauaaa agaaagaucu 2100
gagcauaaag auacguguuu aaaaauaacu aaaaguaaag gaaagugccu uaauuuuucu 2160
auuugcuuca acugaaagug cuucucagcu cgccccaugu aaguucucau uccauguaaa 2220
ugacauuuuc caguuacaac ugguacugag auuuugccuc ucucuuuccu uacucauccu 2280
cccaaauguc uuugugggag ccauaucagu ggauaccaag cucuguaucc auuugucccc 2340
ugcccuccac aaugugugac auagaacagg gacuuuggcc cugggaaagc aaaagcuccc 2400
aguaaggaau ccugugccca augauguaaa acaauuccaa acauccagga auuuuuguau 2460
cauagagcga auuacuuccu aucuuuucau uagaggcuau gaggacuucu aauuagucuu 2520
aguugcuuau aagugcccug gaaucaccca gguaggcacu uaauuuuuuu uucaguugca 2580
ugagcaaagu gcuucuuagu agugugaaau uacaacaacu uuaagacuuu ccagauucaa 2640
gcucccacug uuggaaaaag ccagccuuuc uaaucucuuc ugcuacugga auaagcacuu 2700
aagaauugcg ugauagccag gcaccguggc ucaugccugu aaucccaaca cuuagggagg 2760
cugagguggg ugggccgcuu gagcucagga guucaagacc agccugggua auauagugag 2820
auccuguguc ucuauaaaaa aauuaaaaau uagucaguug uagugacaca uaccuguagu 2880
cccagcuacu caggaggcug agguggaagg aucacuugag cccagaaggu aaggcugcag 2940
ugagcuguga cugugccacu acacuccagc cugagugaca gagaaagaac cugucaaaaa 3000
aaaaaaaaaa acaaccuaca uuucaaguac uauuucccuu cucucccauc uaauugcuaa 3060
agauuuucuu ucauacgcac acacuccagu gacuggaaaa acgggaguuu ucagucaaag 3120
cuugacauuu agagaaaaca aggacuuucu gccuuuauaa auggaaauca acuguguaug 3180
aacuauaacu cugcagaggu uaugaauuca uccuuuacaa acaauaauga acuuuuaguc 3240
cuguaauaaa ugaaauguua uuaggcagcu uuguugcaug auugcauagu uauaucuugc 3300
uaacgggcca cucauuucuc acugaugugg augaaaaaau gagagcagua uguuuccagg 3360
ugugugcacu caacaggcaa auagcucccg aggucaccac uucccuaaug ggccacagga 3420
aguaaguuga ucuugauggg gagaucacgu cacccagaac cagcaacugg auagagacug 3480
uuguuagugu cuggguagag cacaggcucc caggggucuu aagagcuaau uacugaauaa 3540
aacaaucuag aacaaa 3556
<210> 2
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Asn Cys Lys Glu Gly Thr Asp Ser Ser Cys Gly Cys Arg Gly Asn
1 5 10 15
Asp Glu Lys Lys Met Leu Lys Cys Val Val Val Gly Asp Gly Ala Val
20 25 30
Gly Lys Thr Cys Leu Leu Met Ser Tyr Ala Asn Asp Ala Phe Pro Glu
35 40 45
Glu Tyr Val Pro Thr Val Phe Asp His Tyr Ala Val Thr Val Thr Val
50 55 60
Gly Gly Lys Gln His Leu Leu Gly Leu Tyr Asp Thr Ala Gly Gln Glu
65 70 75 80
Asp Tyr Asn Gln Leu Arg Pro Leu Ser Tyr Pro Asn Thr Asp Val Phe
85 90 95
Leu Ile Cys Phe Ser Val Val Asn Pro Ala Ser Tyr His Asn Val Gln
100 105 110
Glu Glu Trp Val Pro Glu Leu Lys Asp Cys Met Pro His Val Pro Tyr
115 120 125
Val Leu Ile Gly Thr Gln Ile Asp Leu Arg Asp Asp Pro Lys Thr Leu
130 135 140
Ala Arg Leu Leu Tyr Met Lys Glu Lys Pro Leu Thr Tyr Glu His Gly
145 150 155 160
Val Lys Leu Ala Lys Ala Ile Gly Ala Gln Cys Tyr Leu Glu Cys Ser
165 170 175
Ala Leu Thr Gln Lys Gly Leu Lys Ala Val Phe Asp Glu Ala Ile Leu
180 185 190
Thr Ile Phe His Pro Lys Lys Lys Lys Lys Arg Cys Ser Glu Gly His
195 200 205
Ser Cys Cys Ser Ile Ile
210
<210> 3
<211> 20
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
agaaaccucu cacuuacgag 20
<210> 4
<211> 19
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ccacuguguu ugaccacua 19
<210> 5
<211> 58
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ccggcaacac uugcucggac uguaucucga gauacagucc gagcaagugu uguuuuug 58
<210> 6
<211> 58
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ccgggcccgu uugcuguaua ugaaacucga guuucauaua cagcaaacgg gcuuuuug 58
<210> 7
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
cggaattcgc caccatgaac tgcaaagagg gaac 34
<210> 8
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
cgggatcctc agataattga acagcagc 28
<210> 9
<211> 64
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gctctagagc caccatggga gactacaagg acgatgatga caagatgaac tgcaaagagg 60
gaac 64
<210> 10
<211> 64
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
cggaattcgc caccatggga gactacaagg acgatgatga caagatgaac tgcaaagagg 60
gaac 64
<210> 11
<211> 21
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
uucuccgaac gugucacguu u 21
<210> 12
<211> 20
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
agaaaccucu cacuuacgag 20
<210> 13
<211> 19
<212> RNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ccacuguguu ugaccacua 19
<210> 14
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
cctgagtgac agagaaagaa cc 22
<210> 15
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ggagtgtgtg cgtatgaaag a 21
<210> 16
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
aagaactcgg acctcctca 19
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ccgttgctgg actggattat 20
Claims (1)
1. The siRNA of the RHOJ mRNA which is shown as SEQ ID No. 1 is knocked out and applied to the preparation of products for reducing proliferation and migration of human high-grade glioma cell lines in vitro.
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| CN202010568424.3A CN111996251B (en) | 2020-06-19 | 2020-06-19 | Application of malignant glioma biomarker |
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| CN111996251B true CN111996251B (en) | 2024-03-26 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111032662A (en) * | 2017-06-21 | 2020-04-17 | 尚医治疗有限责任公司 | Compounds interacting with the RAS superfamily for the treatment of cancer, inflammatory diseases, RAS proteinopathies and fibrotic diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005017161A2 (en) * | 2003-08-13 | 2005-02-24 | Children's Hospital Medical Center | Chimeric peptides for the regulation of gtpases |
| WO2015015306A2 (en) * | 2013-06-17 | 2015-02-05 | Korea Advanced Institute Of Science And Technology (Kaist) | Method for targeting vascular rhoj for inhibiting tumor angiogenesis |
| US10456470B2 (en) * | 2013-08-30 | 2019-10-29 | Genentech, Inc. | Diagnostic methods and compositions for treatment of glioblastoma |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111032662A (en) * | 2017-06-21 | 2020-04-17 | 尚医治疗有限责任公司 | Compounds interacting with the RAS superfamily for the treatment of cancer, inflammatory diseases, RAS proteinopathies and fibrotic diseases |
Non-Patent Citations (6)
| Title |
|---|
| Bhaduri et al..Outer Radial Glia-like Cancer Stem Cells Contribute to Heterogeneity of Glioblastoma.《 Cell Stem Cell》.2020,第48–63页. * |
| Chan Kim et al..Vascular RhoJ Is an Effective and Selective Target for Tumor Angiogenesis and Vascular Disruption.《Cancer Cell》.2014,第102-117页. * |
| Clarke K et al..High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer.《PLoS Genet》.2015,第11卷(第7期),第3页倒数第二段-第5页第1段. * |
| Mei Wang et al..Rhoj Is a Novel Target for Progression and Invasion of Glioblastoma by Impairing Cytoskeleton Dynamics.《Neurotherapeutics》.2020,第2028-2040页. * |
| NCBI Reference Sequence: NM_020663.5.Homo sapiens ras homolog family member J (RHOJ), mRNA.《GenBank》.2020,第1-4页. * |
| 王梅.小G蛋白RhoJ在人脑胶质瘤中的生物学功能及其分子机制研究.《中国博士学位论文全文数据库医药卫生科技辑》.2022,第E070-15页. * |
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