PLEKHF2 gene and RNAi interference system and application thereof

Abstract

The invention discloses a PLEKHF2 gene as well as an RNAi (Ribonucleic Acid Interference) system and application thereof, and belongs to the technical field of biomedicine. The PLEKHF2 gene / protein disclosed by the invention is a prognosis judgment index and a treatment target spot of a glioma patient, is applied to preparation of a kit for prognosis judgment of the glioma patient or preparation of a medicine for treating glioma, provides a new strategy for treatment of the glioma patient, has good clinical application value, and has a good application prospect. Relates to the technical field of biomedicine. The invention also discloses a lentivirus RNAi interference system for constructing the PLEKHF2 gene, and results show that after the PLEKHF2 is knocked down, the proliferation activity and the clone formation ability of glioma cells are reduced, and the migration and invasion ability of the glioma cells are also reduced. The application provides a new gene target and a technical means for treatment and prognosis of glioma, and has good application value and prospect in clinical treatment and research.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the PLEKHF2 gene and its RNAi interference system and applications, specifically in the prognosis or therapeutic targets of gliomas. Background Technology

[0002] Gliomas are the most common primary malignant tumors of the central nervous system. Due to their pathological heterogeneity, local invasiveness, and high recurrence rate, treatment outcomes are poor and prognosis is bad, especially for glioblastoma patients, whose mortality rate in the first year after diagnosis is approximately 80%. In recent years, with the continuous development of multidisciplinary technologies such as pathological molecular subtyping, imaging, minimally invasive surgery, radiotherapy and chemotherapy, and targeted immunotherapy, the diagnosis and treatment of gliomas has entered a phase of precision, individualized, and multidisciplinary comprehensive treatment. However, the overall prognosis has not yet been significantly improved.

[0003] With the application of high-throughput technologies such as second- and third-generation sequencing, single-cell sequencing, proteomics, and metabolomics, identifying differentially expressed tumor genes or proteins closely related to clinical diagnosis and treatment has become a cutting-edge approach in clinical research. Despite a wealth of existing research on the mechanisms of glioma development and prognosis, including bioinformatics databases primarily based on transcriptome information, the search for convenient and effective diagnostic biomarkers and therapeutic targets remains a priority.

[0004] Based on the TCGA database, bioinformatics analysis was conducted to identify a prognostic biomarker for gliomas at the mRNA level, providing a basis for future research. The PLEKHF2 (Pleckstrin Homology and FYVE Domain Containing 2) gene (also known as EAPF or Phafin2) was identified through differential analysis. This gene regulates macropinocytosis; RAS gene-transformed cancer cells can selectively proliferate in dependence on PLEKHF2 macropinocytosis. Furthermore, PLEKHF2 accumulates in lysosomes and participates in regulating autophagy.

[0005] There is currently very little research on PLEKHF2 in malignant tumors, and its relationship with gliomas remains unclear. Summary of the Invention

[0006] In view of the above-mentioned problems existing in the prior art, the technical problems to be solved by the present invention are: 1. The PLEKHF2 gene / protein is a biomarker for predicting the prognosis of glioma patients and a potential target for treatment, and its application in the preparation of products for predicting the prognosis and treatment of glioma patients; 2. Providing the PLEKHF2 gene and its RNAi interference system; 3. The application of the PLEKHF2 gene and its RNAi interference system as a therapeutic target for glioma, for the preparation of drugs for treating glioma.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of the PLEKHF2 gene as a drug target in the preparation of drugs for the treatment of glioma.

[0009] In some embodiments, the treatment of glioma involves inhibiting the proliferative activity of glioma cells.

[0010] In some embodiments, the treatment of glioma involves inhibiting the migration ability of glioma cells.

[0011] In some embodiments, the treatment of glioma involves inhibiting the invasive ability of glioma cells.

[0012] A kit for the diagnosis, treatment, or prognosis of gliomas, containing a reagent for detecting the expression level of the PLEKHF2 gene.

[0013] In some embodiments, the reagent for detecting the expression level of the PLEKHF2 gene is specifically a fluorescently labeled antibody against the PLEKHF2 protein.

[0014] The application of the PLEKHF2 gene RNAi lentiviral interference system in the preparation of drugs for the treatment of gliomas, the sequence of which is shown below:

[0015] PLEKHF2-shRNA1: 5'-GGTCAACCTTTAACTATACCT-3', and / or

[0016] PLEKHF2-shRNA2: 5'-CCTTTAAATGATATGTCTGAT-3'.

[0017] In some embodiments, the RNAi lentiviral interference system of the PLEKHF2 gene targets the PLEKHF2 gene to inhibit the expression of PPP6R1 protein in glioma cells.

[0018] In some embodiments, the PLEKHF2 gene RNAi lentiviral interference system targets the PLEKHF2 gene to knock down the PLEKHF2 gene in glioma cells.

[0019] In some embodiments, the PLEKHF2 gene RNAi lentiviral interference system uses the PLEKHF2 gene as a target to silence the PLEKHF2 gene in glioma cells.

[0020] Compared with the prior art, the beneficial effects of this application are as follows:

[0021] This application, through bioinformatics analysis of public databases, clinical glioma tissue microarray analysis using multicolor immunofluorescence, in vitro glioma models, and molecular biology experiments, confirms that the expression level of the PLEKHF2 gene / protein in glioma tissue is significantly higher than that in benign brain tissue, and is associated with glioma tissue typing, molecular typing, and WHO classification. High expression of the PLEKHF2 gene / protein is an independent prognostic factor for poor prognosis in glioma patients. This invention knocks down / overexpresses the PLEKHF2 gene / protein in glioma cells, inhibiting or promoting tumor cell proliferation, colony formation, invasion, and migration. This application provides new gene targets and technical means for the treatment and prognosis of gliomas, and has good application value and prospects in clinical treatment and research. Attached Figure Description

[0022] Figure 1 shows the results of PLEKHF2 protein expression detection in glioma and benign brain tissue using multicolor immunofluorescence; where the expression level of PLEKHF2 protein (green fluorescence) in glioma tissue (A) is higher than that in benign brain tissue (B), and blue represents cell nucleus staining.

[0023] Figure 1C The graph shows the relationship between PLEKHF2 protein expression and prognosis in glioma patients. The Kaplan-Meier survival curve shows that the blue line represents low expression and the green line represents high expression.

[0024] Figure 2A Figure showing the different expression levels of PLEKHF2 protein in glioma cell lines (U87MG, T98G, Hs683, U251);

[0025] Figure 2B The graph shows the expression changes of PLEKHF2 protein after knockdown of the PLEKHF2 gene / protein in U87MG and U251 cells.

[0026] Figure 2C The graph shows the expression changes of PLEKHF2 protein after overexpression of PLEKHF2 gene / protein in T98G and Hs683 cells.

[0027] Figure 3 The results of the CCK-8 experiment are shown in the figure. Among them, A and B show that the proliferation ability of glioma cells decreased after knocking down the PLEKHF2 gene / protein in U251 and U87MG cells; C and D show that the proliferation ability of glioma cells increased after overexpressing the PLEKHF2 gene / protein in T98G and Hs683 cells.

[0028] Figure 4A The figure shows that knocking down the PLEKHF2 gene / protein in U251 and U87MG cells reduced the proliferation and cloning ability of glioma cells.

[0029] Figure 4B The graph shows the increase in glioma cell proliferation and cloning capacity after overexpression of the PLEKHF2 gene / protein in T98G and Hs683 cells.

[0030] Figure 5A The graph shows the reduced invasion and migration ability of glioma cells after knocking down the PLEKHF2 gene / protein in U87MG cells (invasion and migration assay results using the transwell assay).

[0031] Figure 5B The graph shows the reduced invasion and migration ability of glioma cells after knocking down the PLEKHF2 gene / protein in U251 cells (invasion and migration assay results using the transwell assay).

[0032] Figure 5C The graph shows the increase in glioma cell invasion and migration ability after overexpression of PLEKHF2 gene / protein in T98G3 cells (invasion and migration experiment results using the transwell assay).

[0033] Figure 5D This graph shows the increase in glioma cell invasion and migration ability after overexpression of the PLEKHF2 gene / protein in Hs683 cells (results of invasion and migration experiments using the transwell assay). Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0035] The main reagents, consumables, and animal models used in this application are:

[0036] A 7-color immunohistochemistry kit (Akoya Bioscience, USA) was used for multicolor immunofluorescence; anti-PLEKHF2 antibody (Novus, NBP2-19865) was used for Western blotting and multicolor immunofluorescence. Glioma cell lines (U87MG, T98G, Hs683) were purchased from the Cell Bank of the Chinese Academy of Sciences, and U251 was purchased from Nanjing Kebai Co., Ltd. RPMI-1640 medium (Gibco, USA) and fetal bovine serum (Lonsera, Uruguay) were used.

[0037] The instruments required for this application are: a multispectral pathological scanning system (Perkin Elmer, USA), an inverted fluorescence microscope (Zeiss, Germany), a gel imaging system (Tianneng, China), and a multifunctional microplate reader (Thermo Fisher Scientific, USA).

[0038] Example 1: Expression of PLEKHF2 in glioma tissue

[0039] Multicolor immunofluorescence histochemistry and statistical analysis of glioma tissue microarray

[0040] 1.1 Paraffin-embedded microarrays of glioma tissue from the Clinical Resource Translational Research Center of the Affiliated Hospital of Nantong University (surgeries performed between 2012 and 2017; patients had not received immunotherapy, chemotherapy, or radiotherapy prior to surgery; complete clinical case data). The study included 163 cases of glioma tissue and 21 cases of benign brain tissue. This research was approved by the hospital's ethics committee (approval number: 2018-K020).

[0041] 1.2 Multicolor immunofluorescence: (1) After baking the glioma tissue microarray, dewax it in xylene, then dehydrate it with graded alcohol, and rinse it with distilled water; (2) Place it on the slide rack in AR6 retrieval solution at pH 6.0 and perform high-temperature antigen retrieval in a microwave oven; (3) Cool it to room temperature naturally, then rinse it with PBS and block it with primary antibody blocking solution for 10 min; (4) Add 200 μL of anti-PLEKHF2 antibody (dilution ratio of 1:300) to the tissue microarray and incubate it at 4°C overnight; (5) Take out the tissue microarray, warm it for 0.5 h, and then rinse it with PBS; (6) Add 200 μL of secondary antibody working solution to the tissue microarray, incubate it at room temperature for 10 min, and then rinse it with PBS; (7) Add fluorescent dye to the tissue microarray, incubate it at room temperature in the dark for 10 min, and then rinse it with PBS; (8) After drying and clearing, mount it with DAPI.

[0042] 1.3 Result Judgment

[0043] Staining results were observed under a fluorescence microscope, with staining at corresponding cell sites considered positive. Fluorescence images of each sample were acquired using Vectra3 automated imaging software at 20x magnification. Images were analyzed and scored using inForm 4.3.0 (Perkin Elmer), with thresholds set for fluorescently positive or negative cells for each cell type. The percentage of cells in each region was calculated and scored (0-100). The cutoff point for PLEKHF2 protein expression score was determined using X-tile software based on survival time and 5-year survival status. All data were processed using SPSS V.22.0 statistical software. Chi-square test was used for intergroup comparisons, and Cox proportional hazards regression analysis was used to analyze prognostic factors. Univariate analysis of patient prognosis was performed using the Kaplan-Meier method and log-rank test. All test results were considered statistically significant with P < 0.05.

[0044] The results showed that PLEKHF2 protein was expressed to varying degrees in both glioma and benign brain tissues. The protein was cellularly localized in the cytoplasm (Figure 1, A and B). Using X-tile software, scoring cutoff values ​​were derived based on survival time and 5-year survival status: a score of 0-15 in glioma cells indicated low expression (92 cases), and ≥15-100 indicated high expression (71 cases). Statistical results showed that the high expression of PLEKHF2 protein in glioma cells (43.56%) was significantly greater than its high expression in brain glioma cells (4.76%), exhibiting a statistically significant difference (χ²). 2 =11.756, P<0.001); PLEKHF2 protein expression level in glioma cells and tumor histological classification (χ²) 2 =11.922, P=0.003), molecular typing (χ²) 2 =5.912, P=0.015), WHO classification (χ²) 2 =14.207, P<0.001) was statistically significant; and it was an independent prognostic factor for glioma patients (HR, 1.487, 95% CI: 1.020-2.168; P=0.039). Figure 1C ).

[0045] Example 2:

[0046] Differential expression of 1PLEKHF2 protein in glioma cell lines

[0047] 1.1 Glioma cell lines (U87MG, T98G, Hs683, U251) were all cultured in RPMI-1640 complete medium. The incubator was maintained at 37℃ and 5% CO2 saturated humidity. The cells were routinely passaged in the incubator, and cells in the logarithmic growth phase were selected for experiments.

[0048] 1.2 Extraction of total cell protein

[0049] (1) Collect glioma cells, discard the culture medium, and wash the cells twice with pre-cooled PBS; (3) Add different 1×SDS cell lysis buffers according to the size of the cell culture flask and the cell growth density, scrape the cells clean and transfer them to a clean EP tube; (4) Lyse the scraped cell proteins on ice for 30 min; (5) Centrifuge at 4℃ and retain the supernatant. Measure the concentration of cell proteins using the BCA method and a multi-functional microplate reader, and store in a -20℃ freezer for later use.

[0050] 1.3 Western blot assay

[0051] (1) Prepare polyacrylamide gel (5% stacking gel, 10% separating gel); (2) After loading the protein marker and the extracted protein sample, adjust the running voltage to 100V, and after the end, take out the gel for transfer (PVDF membrane); (3) Transfer the membrane at a constant current of 300mA for 1.5h in an ice box; (4) After the transfer, put the PVDF membrane into the blocking solution and block it at room temperature for 2h; (6) Prepare the anti-PLEKHF2 antibody dilution solution with the blocking solution (dilution ratio of 1:100), and evenly drop the diluted primary antibody onto the PVDF membrane, and incubate overnight at 4℃; (7) After washing the membrane, prepare the secondary antibody dilution solution with TBST, and evenly drop the diluted secondary antibody onto the PVDF membrane, and incubate at room temperature for 1.5h; (8) After washing the membrane, lay the PVDF membrane flat on the corresponding position of the developing instrument, dilute the ECL luminescent solution with TBST, and evenly drop it onto the membrane, and take pictures and save them using the gel imaging system.

[0052] The results showed that PLEKHF2 protein was expressed at relatively high levels in the U87MG and U251 cell lines, and at relatively low levels in the T98G and Hs683 cell lines. Figure 2A ).

[0053] 2. shRNA design and stable cell line selection

[0054] The gene sequence of the RNAi interference system specifically targeting the PLEKHF2 gene sequence is shown below. A lentivirus-mediated RNAi interference system was constructed.

[0055] PLEKHF2-shRNA1:5'-GGTCAACCTTTAACTATACCT-3';

[0056] PLEKHF2-shRNA2:5'-CCTTTAAATGATATGTCTGAT-3'.

[0057] (1) Select a suitable viral infection concentration, infect the target cells with lentivirus, and add a staining agent; after infection for 12-16 hours, change the medium and continue culturing, while observing whether there are any abnormalities in the cell state; after infection for 72-96 hours, observe the fluorescence under an inverted fluorescence microscope, and screen the infected cells for drugs to collect more successfully infected cells; (3) Preparation and growth of monoclonal cells: dilute the cells to 10 96-well plates using the limiting dilution method; observe the growth of monoclonal cells after one week, and transfer the grown monoclonal cells to 48-well plates for expansion culture after about two weeks; transfer the grown monoclonal cells to 24-well plates and 12-well plates for expansion culture in sequence; (4) When each monoclonal cell is expanded to two 12-well plates, take out the cells from one well, lyse them to extract protein, and use Western blot to detect the monoclonal cells with knockdown of the PLEKHF2 gene.

[0058] The results of quantitative analysis showed that ( Figure 2B Compared with the control group, the expression level of PLEKHF2 protein in cells treated with the RNAi interference system was significantly reduced, indicating that the expression level of PLEKHF2 gene / protein in U87MG and U251 cell lines was effectively suppressed.

[0059] 3. Construction of overexpression system and screening of stable strains

[0060] (1) The open reading frame region of PLEKHF2 mRNA was amplified using first and last primers, and the PLEKHF2 expression fragment was recombined into the pCDH-CMV-MCS-EF1-puro vector using gene recombination technology.

[0061] (2) After packaging into a virus, select an appropriate viral infection concentration and infect the target cells with lentivirus, and add a staining agent; after 12-16 hours of infection, change the medium and continue culturing; after 72-96 hours of infection, observe the fluorescence under an inverted fluorescence microscope, and screen the infected cells for drugs in order to collect more successfully infected cells.

[0062] (3) Preparation and growth of single clones: Cells were diluted into 10 96-well plates using the limiting dilution method; the growth of single clones was observed after one week, and the grown single clones were transferred to 48-well plates for expansion culture after about two weeks; the grown single clones were then transferred to 24-well plates and 12-well plates for expansion culture in sequence.

[0063] (4) When each single clone is expanded to two 12-well cells, cells from one well are removed, lysed and extracted for protein extraction. Western blot is used to detect single clones overexpressing PLEKHF2 protein.

[0064] Quantitative analysis results show that ( Figure 2C Compared with the control group, the expression level of PLEKHF2 protein in the cells after expression treatment was significantly increased, indicating that the expression of PLEKHF2 gene / protein in T98G and Hs683 cell lines was effectively upregulated.

[0065] 4. Cell proliferation assay (CCK-8 assay)

[0066] (1) Digest and collect cells from each group 48 h after transfection, and centrifuge them for later use;

[0067] (2) Resuspend the cells in complete culture medium and adjust the cell density to 30,000 cells / mL;

[0068] (3) Add 100 μL of cell suspension to each well of the 96-well plate, and set 5 replicates per group. Gently tap the plate to distribute the cells evenly.

[0069] (4) After the cells adhere to the plate (about 6-8h), add CCK-8 reagent (10μL per well) at 0, 24, 48, 72 and 96h respectively, gently tap the 96-well plate, put it in the incubator for 2h and then take it out. Detect the absorbance value at 450nm on the microplate reader. Note the linear range of the microplate reader.

[0070] (5) Use Graphpad Prism to statistically process the measured data and draw a line graph.

[0071] The results showed that knockdown of the PLEKHF2 gene / protein using the RNAi interference system reduced the proliferation of U87MG and U251 cells. Figure 3 In the middle (A, B), T98G and Hs683 cells overexpressing PLEKHF2 gene / protein showed significantly increased proliferation activity. Figure 3 (C, D)

[0072] 5. Cell clone formation experiment

[0073] (1) Digest and collect cells from each group 48 h after transfection, and centrifuge them for later use;

[0074] (2) Resuspend the cells in complete culture medium and adjust the cell density to 30,000 cells / mL;

[0075] (3) Add 250 μL of cell suspension to each well of a 6-well plate, and set up 3 replicates per group. Gently tap the plate to distribute the cells evenly.

[0076] (4) Remove the cells after 2 weeks in a cell culture incubator.

[0077] (5) Fix cells in the well plate with 4% paraformaldehyde for 15 min, wash twice with 1×PBS, and then stain with crystal violet for 20 min.

[0078] The results showed that knocking down the PLEKHF2 gene / protein using the RNAi interference system inhibited the proliferation of U87MG and U251 cells, and the number of colony units was significantly lower than that in the control group. Figure 4A ); while T98G and Hs683 cells overexpressing the PLEKHF2 gene / protein showed promoted cell proliferation and a significantly higher number of colony units than the control group. Figure 4B ).

[0079] 6. Cell invasion and migration assay (transwell assay)

[0080] (1) For invasion experiments, first prepare hydrogel (50 μL hydrogel mixed in 350 μL diluent, then add 50 μL of basal culture medium and mix), add 100 μL to the upper chamber of the Transwell chamber, and avoid generating air bubbles; without hydrogel, it is used for migration experiments.

[0081] (2) Digest and collect cells from each group 48 h after transfection, and centrifuge them for later use;

[0082] (3) Resuspend the cells in RPMI-1640 medium and adjust the cell density to 5×10⁴ / mL;

[0083] (4) Add 800 μL of complete culture medium to a 24-well plate, place it in a small chamber, fully infiltrate it, and add 100 μL of cell suspension to the upper chamber of the transwell chamber.

[0084] (5) After culturing for 24-48 hours, remove the product, wash twice with 1×PBS, fix with 4% paraformaldehyde for 20 min, and wash twice with 1×PBS.

[0085] (6) Add 500 μL of crystal violet staining solution to a 24-well plate, place the chamber in the well, remove it after 10 min, wash twice with 1×PBS, invert the chamber, and gently wipe away any cells that have not passed through the upper chamber with a cotton swab.

[0086] Observation using an inverted microscope showed that after knocking down the PLEKHF2 gene / protein using the RNAi interference system, the invasion and migration abilities of U87MG and U251 cells decreased. Figure 5A , Figure 5B Overexpression of the PLEKHF2 gene / protein increased the invasion and migration abilities of T98G and Hs683 cells. Figure 5C , Figure 5D ).

[0087] Based on the above embodiments, this application has confirmed through clinical glioma tissue microarrays, multicolor immunofluorescence, cell models, and molecular biology experiments that PLEKHF2 protein expression in glioma tissues is significantly higher than its expression in benign brain tissues, and is associated with glioma tissue typing, molecular typing, and WHO grading. High expression of PLEKHF2 protein in glioma tissues is an independent factor for poor patient prognosis. This invention knocks down / overexpresses the PLEKHF2 gene / protein in glioma cells, inhibiting or promoting the proliferation, colony formation, invasion, and migration of cancer cells.

[0088] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of PLEKHF2 gene as a drug target in the preparation of drugs for the treatment of glioma.

2. The application according to claim 1, characterized in that, The treatment for glioma involves inhibiting the proliferation of glioma cells.

3. The application according to claim 1, characterized in that, The treatment for glioma involves inhibiting the migration ability of glioma cells.

4. The application according to claim 1, characterized in that, The treatment for glioma involves inhibiting the invasive ability of glioma cells.

5. A kit for the diagnosis, treatment, or prognosis of gliomas, characterized in that, A reagent containing the detection level of the PLEKHF2 gene.

6. The kit for the diagnosis, treatment, or prognosis of glioma according to claim 5, characterized in that, The specific reagent for detecting PLEKHF2 gene expression is a fluorescently labeled antibody against the PLEKHF2 protein.

7. The application of the PLEKHF2 gene RNAi lentiviral interference system in the preparation of drugs for treating gliomas, characterized in that, The sequence of the RNAi lentiviral interference system is shown below: PLEKHF2-shRNA1: 5'-GGTCAACCTTTAACTATACCT-3', and / or PLEKHF2-shRNA2: 5'-CCTTTAAATGATATGTCTGAT-3'.

8. The application according to claim 7, characterized in that, The aforementioned RNAi lentiviral interference system for the PLEKHF2 gene targets the PLEKHF2 gene to inhibit the expression of PPP6R1 protein in glioma cells.

9. The application according to claim 7, characterized in that, The aforementioned RNAi lentiviral interference system for the PLEKHF2 gene targets and knocks down the PLEKHF2 gene in glioma cells.

10. The application according to claim 7, characterized in that, The aforementioned RNAi lentiviral interference system for the PLEKHF2 gene targets and silences the PLEKHF2 gene in glioma cells.

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