Shrna, recombinant plasmid and lentivirus vector targeting silencing C15orf48 gene and application

Abstract

This invention discloses a targeted shRNA for silencing the C15ORF48 gene, a recombinant plasmid, a lentiviral vector, and its applications, relating to the field of biomedical technology. The shRNA comprises a nucleotide sequence as shown in SEQ ID No. 1 or SEQ ID No. 2. The targeted C15ORF48 shRNA of this invention significantly reduces C15ORF48 expression in TFE3 rearranged renal cell carcinoma cells and inhibits the proliferation, invasion, and migration of these cells. Therefore, the highly efficient and specific RNA interference fragment for TFE3 rearranged renal cell carcinoma of this invention has significant research value and application significance for advancing precision treatment of this disease, as it can efficiently and specifically silence the pathogenic gene and has the significant advantage of low toxicity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a targeted silencing shRNA, recombinant plasmid, and lentiviral vector for the C15ORF48 gene and its applications. Background Technology

[0002] Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. Genetic translocation renal cell carcinoma is a specific subtype of renal cell carcinoma, with TFE3 rearrangement renal cell carcinoma being the most prevalent. Compared to other renal cell carcinomas, TFE3 rearrangement renal cell carcinoma progresses and metastasizes earlier. Furthermore, TFE3 rearrangement renal cell carcinoma is highly malignant, and current first-line therapies for renal cell carcinoma are ineffective. Therefore, there is an urgent need to identify target genes to achieve therapeutic effects.

[0003] RNA interference (RNAi) is a highly conserved gene silencing mechanism in eukaryotes, induced by double-stranded RNA (dsRNA). Short hairpin RNA (shRNA) is an artificially designed shRNA sequence introduced into cells via a vector. After transcription, it forms a primary transcript with a stem-loop structure. This transcript is processed by the Dicer enzyme in the cytoplasm, cleaving it into potent double-stranded small interfering RNAs (siRNAs). These siRNAs bind to a complex called the RNA-induced silencing complex (RISC), using their antisense strand as a guide to precisely recognize and degrade complementary messenger RNA (mRNA), ultimately leading to the RISC's cleavage and degradation of the mRNA. This results in the specific downregulation of target gene expression at the translational level. Due to its ability to achieve stable and long-lasting gene silencing, shRNA technology has become an important tool in gene function research and gene therapy.

[0004] The rise of RNAi technology has greatly advanced functional genomics research in the post-genomic era and shown broad prospects in areas such as high-throughput screening of drug targets, gene therapy, and innovative drug development, providing novel treatment strategies for various intractable diseases, including cancer and hereditary diseases. In recent years, RNAi technology, as a precise gene regulation method, has been widely used in disease treatment research. Compared to traditional treatment methods, this technology can efficiently and specifically silence disease-causing genes and has the significant advantage of low toxicity and side effects.

[0005] In recent years, RNAi technology has developed rapidly in the field of cancer treatment. Among them, shRNA technology, with the widespread application of viral vector systems such as lentiviruses and adeno-associated viruses (AAVs), has seen a revolutionary expansion in delivery efficiency and application scope, achieving stable and heritable gene silencing. Due to its ability to achieve long-term and stable gene silencing, it has shown unique advantages in targeted cancer therapy. Taking pancreatic cancer as an example, KRAS gene mutations continuously activate signaling pathways such as MAPK and PI3K-AKT, driving abnormal proliferation and metastasis of cancer cells. Studies have shown that knocking out the KRAS gene using shRNA can not only inhibit the proliferation and invasiveness of pancreatic cancer cells but also enhance their sensitivity to chemotherapeutic drugs such as gemcitabine. In the treatment of glioblastoma, overexpression of the EGFR gene activates downstream STAT3 and NF-κB signaling pathways, promoting tumor angiogenesis and treatment resistance. Knocking out the EGFR gene using shRNA technology can significantly reduce tumor invasiveness, improve radiosensitivity, and significantly slow tumor growth in animal experiments. These studies provide important evidence for the development of novel targeted cancer therapies.

[0006] Open reading frame 48 (C15ORF48) on chromosome 15 (also known as NMES1, Coxfa4l3, MISTRV, or MOCCI) is a small mitochondrial protein. Recent studies have shown that it primarily regulates mitochondrial metabolism and is significantly associated with pathways related to various cancer development and immunotherapy, such as cell death, the p53 pathway, and ferroptosis. It has also been shown to play a crucial role in promoting proliferation, migration, and apoptosis in thyroid, ovarian, and colorectal cancers. However, C15ORF48 is a human gene whose function is not yet fully understood. Therefore, targeted silencing of the C15ORf48 gene using shRNA is helpful in discovering its role and provides a foundation for further immunotherapy research. Current technologies require improvement and development; more effective inhibitors targeting C15ORf48 need to be developed to improve their specificity and selectivity for different tissues and cells. Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention provides an shRNA for targeted silencing of the C15ORF48 gene, a recombinant plasmid, a lentiviral vector, and its application. The technical solution is as follows:

[0008] A targeted shRNA for silencing the C15ORF48 gene, said shRNA comprising a nucleotide sequence as shown in SEQ ID No. 1 or a nucleotide sequence as shown in SEQ ID No. 2.

[0009] Optionally, the nucleotide sequence of the top strand of the shRNA is as shown in SEQ ID No. 3, and the nucleotide sequence of the bottom strand is as shown in SEQ ID No. 4; or

[0010] The nucleotide sequence of the top strand of the shRNA is shown in SEQ ID No. 5, and the nucleotide sequence of the bottom strand is shown in SEQ ID No. 6.

[0011] A recombinant plasmid comprising the shRNA and the vector;

[0012] And / or, the vector is a PCLenti plasmid;

[0013] And / or, the PCLenti plasmid is double-digested with AgeI and ECORI enzymes.

[0014] The method for constructing the recombinant plasmid includes the following steps:

[0015] 1) Synthesize the nucleotide sequences of the top and bottom strands of the shRNA, and anneal the nucleotide sequences of the top and bottom strands;

[0016] 2) The vector was double-digested with AgeI and ECORI enzymes;

[0017] 3) The enzyme-digested vector and the annealing product obtained in step 1) are ligated to obtain the recombinant plasmid.

[0018] A lentiviral vector containing shRNA, wherein the lentiviral vector comprises the shRNA.

[0019] The method for constructing the shRNA lentiviral vector includes the following steps:

[0020] (1) Thoroughly mix the recombinant plasmid, Pax2 plasmid and Vsvg plasmid;

[0021] (2) Add the transfection reagent and the mixed plasmid obtained in step (1) into the cells, culture them, collect the supernatant, and obtain the shRNA lentiviral vector.

[0022] A method for targeted silencing of the C15ORF48 gene, the method comprising: transfecting the shRNA lentiviral vector into target cells to obtain C15ORF48 silenced cells;

[0023] And / or, the target cells are TFE3 rearranged renal cell carcinoma cells.

[0024] The method described above resulted in C15ORF48 gene-silenced cells.

[0025] And / or, the target cells are TFE3 rearranged renal cell carcinoma cells.

[0026] The use of the shRNA, the recombinant plasmid, the shRNA lentiviral vector, and the C15ORF48 gene-silencing cells in the preparation of drugs, pharmaceutical compositions, or kits for treating TFE3 rearranged renal cell carcinoma.

[0027] A drug, pharmaceutical composition, or kit for treating TFE3 rearranged renal cell carcinoma, wherein the drug, pharmaceutical composition, or kit comprises: the shRNA, the recombinant plasmid, or the shRNA lentiviral vector.

[0028] The beneficial effects of the technical solutions provided by the embodiments of the present invention include at least the following:

[0029] This invention provides a targeted shRNA for silencing C15ORF48, a recombinant plasmid, and a lentiviral vector, as well as their applications. The targeted shRNA for silencing C15ORF48 of this invention can significantly reduce C15ORF48 expression in TFE3 rearranged renal cell carcinoma TFE301-1 cells and inhibit the proliferation, invasion, and migration of TFE3 rearranged renal cell carcinoma TFE301-1 cells.

[0030] Therefore, the highly efficient and specific RNA interference fragment of the present invention targeting TFE3 rearranged renal cell carcinoma has important research value and application significance for advancing the precision treatment of this disease. It can efficiently and specifically silence the pathogenic gene and has the significant advantage of low toxicity and side effects. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a schematic diagram of the PCLenti-C15ORF48-sh1 carrier provided in Embodiment 1 of the present invention;

[0033] Figure 2 This is a schematic diagram of the PCLenti-C15ORF48-sh2 carrier provided in Embodiment 1 of the present invention;

[0034] Figure 3This is a diagram showing the C15ORF48 knockdown efficiency in the TFE301-1 cell line provided in Example 3 of the present invention; where sh1 is shRNA1, sh2 is shRNA2, and shNC is the blank control group;

[0035] Figure 4 This is a diagram showing the results of a cell clonal proliferation experiment provided in Example 4 of the present invention; where sh1 is shRNA1, sh2 is shRNA2, and NC is the blank control group;

[0036] Figure 5 This is a statistical chart of the cell clonal proliferation experiment results provided in Example 4 of the present invention; where sh1 is shRNA1, sh2 is shRNA2, and nc is the blank control group;

[0037] Figure 6 This is a scratch test result diagram provided in Example 4 of the present invention; where sh1 is shRNA1, sh2 is shRNA2, and NC is the blank control group;

[0038] Figure 7 This is a statistical chart of scratch test results provided in Example 4 of the present invention; where sh1 is shRNA1, sh2 is shRNA2, and shNC is the blank control group; where sh1 is shRNA1, sh2 is shRNA2, and NC is the blank control group;

[0039] Figure 8 This is a diagram showing the results of the Transwell invasion and migration experiment provided in Example 5 of the present invention; where sh1 is shRNA1, sh2 is shRNA2, and shNC is the blank control group;

[0040] Figure 9 This is a statistical graph of the Transwell invasion and migration experiment results provided in Example 5 of the present invention, where sh1 is shRNA1, sh2 is shRNA2, and nc is the blank control group. Detailed Implementation

[0041] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0042] In embodiments of the present invention, the meaning of "and / or" can be both, or it can be either one of the two.

[0043] The present invention aims to overcome the above-mentioned deficiencies of the prior art and provide one or more shRNA molecules that can efficiently and specifically silence the human C15orf48 gene.

[0044] Specifically, the present invention provides a targeted silencing shRNA of C15ORF48 and its uses, a recombinant plasmid, and a lentiviral vector.

[0045] The technical solution is as follows:

[0046] A targeted shRNA that silences C15ORF48, with the following target sequence:

[0047] 5'-GAAGGAACTCATTCCCTTGGT-3' (SEQ ID No. 1); or

[0048] 5'-GCTTATAACAATCAACCAACA-3' (SEQ ID No. 2).

[0049] The shRNA comprises a nucleotide sequence as shown in SEQ ID No. 1 or a nucleotide sequence as shown in SEQ ID No. 2.

[0050] Preferably, the nucleotide sequence of the top strand of the shRNA containing the target site sequence is as shown in SEQ ID No. 3, and the nucleotide sequence of the bottom strand is as shown in SEQ ID No. 4; or

[0051] The nucleotide sequence of the top strand of the shRNA containing the target site sequence is shown in SEQ ID No. 5, and the nucleotide sequence of the bottom strand is shown in SEQ ID No. 6.

[0052] A recombinant plasmid comprising a vector and a target gene, wherein the target gene is the shRNA sequence of the targeted silencing C15ORF48. Preferably, the vector is a PCLenti plasmid. More preferably, the PCLenti plasmid is double-digested with AgeI and ECORI enzymes.

[0053] The method for constructing the recombinant plasmid includes the following steps:

[0054] 1) Synthesize the top strand sequence and bottom strand sequence of the shRNA, and anneal the top strand sequence and bottom strand sequence of the shRNA;

[0055] 2) The vector was double-digested with AgeI and ECORI enzymes;

[0056] 3) The digested vector and the annealing product obtained in step 1) are ligated together to obtain the recombinant plasmid.

[0057] A lentiviral vector containing shRNA, the shRNA lentiviral vector comprising the shRNA sequence shown in SEQ ID No. 3 or SEQ ID No. 5.

[0058] The method for constructing the shRNA lentiviral vector includes the following steps:

[0059] (1) The recombinant plasmid, Pax2 plasmid and Vsvg plasmid are thoroughly mixed;

[0060] (2) Add the transfection reagent and the mixed plasmid obtained in step (1) into the cells, culture them, collect the supernatant, and obtain the shRNA lentiviral vector.

[0061] A method for targeted silencing of C15ORF48 includes transfecting target cells with the shRNA lentiviral vector to obtain C15ORF48-silenced cells.

[0062] The method described above produces C15ORF48 silenced cells.

[0063] The use of the shRNA, the recombinant plasmid, the shRNA lentiviral vector, and the C15ORF48 gene-silencing cells in the preparation of drugs, pharmaceutical compositions, or kits for treating TFE3 rearranged renal cell carcinoma.

[0064] A drug, pharmaceutical composition, or kit for treating TFE3 rearranged renal cell carcinoma, comprising: the shRNA, recombinant plasmid, or shRNA lentiviral vector.

[0065] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0066] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0067] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0068] In the following embodiments, the experimental methods such as Western blot detection of proteins are all conventional experimental methods well known to those skilled in the art. Where no specific conditions are specified in the experimental methods, they are usually operated according to conventional conditions.

[0069] The experimental materials are shown in Table 1:

[0070] Table 1. Experimental Materials and Source Companies

[0071]

[0072] Example 1

[0073] Two C15ORF48-shRNA knockdown sequences were constructed to target and silence C15ORF48. The target sequence is as follows:

[0074] 5'-GAAGGAACTCATTCCCTTGGT-3' (SEQ ID No. 1); or

[0075] 5'-GCTTATAACAATCAACCAACA-3' (SEQ ID No. 2).

[0076] The advantages of the target sequence constructed in this invention are as follows: the shRNA target sequence designed in this invention has good targeting specificity and is highly complementary to the C15ORF48 mRNA target region. This design can effectively inhibit malignant phenotypes such as cancer cell proliferation and migration, with low off-target effects, providing a reliable tool for in-depth exploration of the biological functions of C15ORF48 and the development of potential targeted therapy strategies.

[0077] Two C15ORF48-shRNA knockdown sequences were constructed, each containing a sense strand, an antisense strand, a sticky end, and a neck loop structure. The sequences are as follows:

[0078] The sequence of C15ORF48-sh1 is shown in Table 2:

[0079] Table 2

[0080]

[0081] Synthesized by Beijing Bomei Company.

[0082] The sequence of C15ORF48-sh2 is shown in Table 3:

[0083] Table 3

[0084]

[0085] Synthesized by Beijing Bomei Company.

[0086] The U6 promoter primer sequences are shown in Table 4:

[0087] Table 4. U6 promoter primer sequences

[0088]

[0089] The U6 promoter is located upstream of the shRNA sequence. Primers were designed for qPCR detection to verify whether the sequence and plasmid had successfully bound.

[0090] 2. Annealing reaction of the top and bottom strands of the shRNA targeting C15ORF48, with a total volume of 20 µl.

[0091] The reaction mixture consisted of 4 µl top strand, 4 µl bottom strand, 2 µl NEB buffer, and 10 µl double-distilled water. The reaction was performed in a thermal cycler according to the following procedure: incubation at 95 °C for 2 minutes, followed by slow cooling to 25 °C at a rate of 0.1 °C every 8 seconds, for a total of 700 cycles, and finally maintained at 4 °C.

[0092] 3. The PCLenti vector was subjected to double enzyme digestion, and the total reaction volume was 50 µl.

[0093] The reaction mixture contains: 1 µg PCLenti vector, 1 µl AgeI restriction enzyme, 1 µl EcoRI restriction enzyme, 5 µl special buffer, and double-distilled water to a final volume of 50 µl. The reaction mixture is incubated at 37°C for 3 hours for digestion.

[0094] 4. The enzyme digestion products were separated and purified using a 1% agarose gel electrophoresis. 1 g of agarose was dissolved in 100 ml of 1×TAE buffer, and the solution was microwaved until clear and transparent. After cooling, an appropriate amount of nucleic acid dye was added. The enzyme digestion products and the PCLenti empty vector plasmid (as a control) were simultaneously loaded onto the gel for electrophoresis analysis.

[0095] 5. Perform the ligation reaction in a total volume of 10 µl. This includes: 100 ng of enzyme-digested PCLenti vector, 1 µl of annealing product, 1 µl of T4 DNA ligase, and 1 µl of the corresponding buffer, to be made up to the required volume with double-distilled water. Initiate the reaction at room temperature for 30–60 minutes.

[0096] 6. The ligation product was transformed into DH5α competent cells (GENSTAR).

[0097] 7. Preparation of antibiotic-containing solid culture medium: Add 6 µl of Amp+ antibiotic to 6 ml of melted LB medium, mix well, pour into a 6 cm sterile petri dish, and allow it to solidify. Spread the transformation product evenly on the surface of the plate, let it stand at room temperature for 10-30 minutes, then invert the petri dish and incubate at 37°C for 16 hours.

[0098] 8. Select morphologically regular single colonies and resuspend them in 9 µl of double-distilled water. Take 1-2 µl of bacterial suspension as a template, add 0.3 µl of U6 promoter forward primer, 0.3 µl of U6 promoter reverse primer, and 10 µl of Taq PCR StarMix, and bring the volume to 20 µl with double-distilled water. The PCR reaction program is as follows: 95℃ pre-denaturation for 5 minutes; followed by 30 cycles of 94℃ for 30 seconds, 55℃ for 30 seconds, and 72℃ for 60 seconds; finally, extend at 72℃ for 5 minutes and store at 4℃. Analyze the PCR products by agarose gel electrophoresis to verify the recombinant plasmid construction results.

[0099] Example 2: Lentiviral packaging of C15ORF48 lentivirus interference vector:

[0100] 24 hours before cell transfection, 293T cells were seeded in 100mm culture dishes, with 10ml of LDM complete medium added to each dish, and pre-cultured at 37°C for 12-24 hours. When the cell confluence reached 40%-50%, the transfection system was prepared: each transfection sample required a plasmid mixture containing 5μg of packaging plasmids (Pax2 and Vsvg, purchased from Sigma) and 5μg of PCLenti-puro lentiviral recombinant plasmid (i.e., the product constructed in Example 1), which was thoroughly mixed before use. Simultaneously, 20μL of JetPrime transfection reagent and 500μL of dedicated buffer were prepared for each sample. The shNC viral vector containing a non-target gene sequence was used as a negative control. After gently mixing the transfection reagent and plasmid mixture separately, the mixtures were incubated at room temperature for 10 minutes. The transfection complex (1 mL total volume) was then slowly added dropwise to a 100 mm culture dish, and the mixture was incubated at 37°C for 8-12 hours before being replaced with fresh complete culture medium. To obtain a high concentration of virus, the culture medium volume was adjusted to 8 mL during the medium change. Forty-eight hours after transfection, the cell supernatant was collected, and an equal volume of fresh complete culture medium was added for further incubation. The collected supernatant was centrifuged at 3500 rpm for 10 minutes; the resulting supernatant was the primary virus solution, which was aliquoted into sterile 1.5 mL EP tubes at 700 μL each. The above collection steps were repeated 72 hours after transfection, and the virus supernatant was aliquoted again. All virus samples were stored at -80°C for later use.

[0101] Example 3

[0102] 1. Cell Culture: TFE301-1 cells in good growth condition were cultured in F12 complete medium containing 5% FBS, centrifuged and counted, and cultured at 6×10⁻⁶ cells / mL. 5 Inoculate each well in a 60mm dish and incubate at 37°C with 5% CO2 for 24 hours.

[0103] 2. shC15ORF48 lentivirus infecting TFE301-1:

[0104] TFE301-1 cells were planted at a density of 1.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μL in 6-well plates and cultured for 12-24 hours before lentiviral transduction. The experimental group was treated with a mixed infection system consisting of 500 μL F12 medium, 500 μL shC15ORF48 lentiviral solution, and 0.5 μL polybrene (final concentration 10 μg / μL). A control group was established, treating TFE301-1 cells with shNC lentivirus targeting non-target gene sequences under the same conditions to ensure the reliability of the experimental results.

[0105] 3. 72 h after cell infection with the virus, add sterile blastidin S (Sterile) culture medium with a final concentration of 2 μg / μL and continue culturing the cells until a cell line stably infected with sh C15ORF48 lentivirus is selected.

[0106] 4. Protein sample and protein quantification:

[0107] Discard the cell culture medium and wash the cells twice with pre-chilled PBS. Add 200 μL of pre-chilled RIPA lysis buffer (containing inhibitor) to the cell culture flask and incubate on ice for 30 minutes, shaking occasionally. Scrape off cell debris and lysis buffer with a cell scraper and collect the residue into a pre-chilled 1.5 mL EP tube. Centrifuge at 12,000–14,000 g for 15 minutes at 4°C. Carefully aspirate the supernatant (total protein solution) into a new pre-chilled EP tube, incubate on ice, and discard the precipitate.

[0108] Follow the BCA kit instructions. Plot a standard curve and dilute the samples 10-fold appropriately before measurement. Measure the protein concentration of each sample, ensuring that all sample concentrations are within the linear range of the standard curve. Calculate the concentration of each sample after adding the loading buffer.

[0109] Add 1 / 4 volume of 5× Loading Buffer to the protein sample and mix well. Heat in a metal bath at 95-100℃ for 5-10 minutes to denature the protein. After brief centrifugation, place on ice for later use or store at -20℃.

[0110] 5. Western blot analysis of protein expression levels:

[0111] After protein sample preparation and quantification, an equal volume of 20 µg of protein sample was used for SDS-PAGE analysis under constant voltage of 80 V. Subsequently, the protein was transferred to a PVDF membrane using a wet transfer method at a constant current of 120 mA for 1.5 hours. After transfer, the membrane was blocked in 5% skim milk solution at room temperature for 1 hour, followed by washing twice with TBST buffer. Then, primary antibody working solution diluted 1:1000 with 3% skim milk was added, and the membrane was incubated overnight at 4°C with gentle shaking. The next day, the membrane was washed three times with TBST buffer for 5 minutes each time, then the corresponding secondary antibody diluted 1:5000 with 3% skim milk was added, and the membrane was incubated at room temperature for 1 hour. Finally, chemiluminescence staining was performed.

[0112] Experimental results are as follows Figure 3 As shown in the figure. sh1 is shRNA1, sh2 is shRNA2, and shNC is the blank control group. From... Figure 3 It can be seen that the expression level of C15ORF48 protein in the knockdown group was significantly lower than that in the control group.

[0113] Example 4

[0114] 1. shC15ORF48 can inhibit the proliferation of TFE301-1.

[0115] In the cell colony formation experiment, single-cell suspensions of C15ORF48-sh1, C15ORF48-sh2, and shNC-TFE301-1 cells obtained in Example 3 were first revived and allowed to adhere to the culture plate, followed by viable cell counting. 1000 cells were precisely seeded into each well of a six-well plate, and 2 ml of F12 complete culture medium (purchased from Prologis) was slowly added to ensure uniform cell distribution. The culture plate was placed in a 37°C, 5% CO2 incubator for static incubation, with the medium replaced with fresh complete culture medium every 3 days. After 10-14 days of continuous incubation, when clear cell colony formation was visible to the naked eye, the culture medium was discarded, and the cells were gently washed twice with PBS. The cells were fixed with 4% paraformaldehyde solution for 15 minutes, followed by staining with 0.1% crystal violet solution for 30 minutes. Finally, excess staining was removed by slow rinsing with water, and the cells were allowed to air dry at room temperature before colony counting and statistical analysis.

[0116] Results analysis:

[0117] Experimental results are as follows Figure 4 and Figure 5 As shown, the area occupied by the clonal clusters in the C15ORF48-sh1 and C15ORF48-sh2 groups was significantly smaller than that in the control group, indicating that knockdown of the C15ORF48 gene significantly inhibited the proliferation of TFE301-1 cells.

[0118] 2. shC15ORF48 inhibits TFE301-1 migration.

[0119] Scratch test: Inoculate with 1×10 5 SH1, SH2, and SHNC-TFE301-1 cells were cultured in 6-well plates and allowed to adhere for 24 hours. A vertical line was then drawn in the center using a 200µl pipette tip, and the degree of closure of the scratch was observed at 0h and 24h.

[0120] Results analysis:

[0121] Experimental results are as follows Figure 6 and Figure 7 As shown, the scratch closure ratio of the C15ORF48-sh1 and C15ORF48-sh2 groups was significantly lower than that of the control group at 24 hours, indicating that knockdown of the C15ORF48 gene significantly inhibited the migration of TFE301-1 cells.

[0122] Example 5: Transwell experiment:

[0123] Migration assay: sh1, sh2, and shNC-TFE301-1 cells were resuspended in serum-free F12 medium and precisely seeded at 5 × 10⁶ cells / day in the upper chamber of a Transwell. 4 Cells were cultured in a chamber with 600 μL of serum-containing complete culture medium as a chemokine. After 24 hours of culture, the chamber was removed, washed with PBS, fixed with 4% paraformaldehyde for 10 minutes, stained with crystal violet solution for 5 minutes, gently rinsed with running water, dried at room temperature, and photographed under a microscope.

[0124] Invasion assay: 40 μL of Matrigel was diluted with F12 complete medium at an appropriate ratio and uniformly coated onto the membrane surface of each Transwell upper chamber. The coated membrane was cured at 37°C to form a biomimetic matrix barrier. Subsequently, sh1, sh2, and shNC-TFE301-1 cells were resuspended in serum-free F12 medium and seeded at 5 × 10⁶ cells / year in the Matrigel-coated upper chamber. 4 Cells were cultured in a chamber with 600 μL of complete culture medium. After 48 h of culture, the upper chamber was removed, and the cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and then washed with PBS again. The cells were then stained with crystal violet for 5 min, rinsed thoroughly with running water, air-dried, and photographed under a microscope.

[0125] Experimental results:

[0126] Experimental results are as follows Figure 8 and Figure 9 As shown. From Figure 8 and Figure 9As can be seen, the number of cells passing through the C15ORF48-sh1 and C15ORF48-sh2 groups was significantly lower than that of the control group in both invasion and migration experiments, indicating that knocking down C15ORF48 significantly inhibits the invasion and migration of TFE301-1 clear cell renal cell carcinoma cells.

[0127] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A shRNA for targeted silencing of the C15ORF48 gene, characterized in that, The shRNA comprises a nucleotide sequence as shown in SEQ ID No. 1 or a nucleotide sequence as shown in SEQ ID No.

2.

2. The shRNA according to claim 1, characterized in that, The nucleotide sequence of the top strand of the shRNA is shown in SEQ ID No. 3, and the nucleotide sequence of the bottom strand is shown in SEQ ID No. 4; or The nucleotide sequence of the top strand of the shRNA is shown in SEQ ID No. 5, and the nucleotide sequence of the bottom strand is shown in SEQ ID No.

6.

3. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the shRNA and vector according to any one of claims 1-2; And / or, the vector is a PCLenti plasmid; And / or, the PCLenti plasmid is double-digested with AgeI and ECORI enzymes.

4. The method for constructing recombinant plasmids according to claim 3, characterized in that, Includes the following steps: 1) Synthesize the nucleotide sequences of the top and bottom strands of the shRNA, and anneal the nucleotide sequences of the top and bottom strands; 2) The vector was double-digested with AgeI and ECORI enzymes; 3) The enzyme-digested vector and the annealing product obtained in step 1) are ligated to obtain the recombinant plasmid.

5. A shRNA lentiviral vector, characterized in that, The shRNA lentiviral vector comprises shRNA according to any one of claims 1-2.

6. The method for constructing the shRNA lentiviral vector according to claim 5, characterized in that, Includes the following steps: (1) Thoroughly mix the recombinant plasmid according to claim 3, as well as the Pax2 plasmid and the Vsvg plasmid; (2) Add the transfection reagent and the mixed plasmid obtained in step (1) into the cells, culture them, collect the supernatant, and obtain the shRNA lentiviral vector.

7. A method for targeted silencing of the C15ORF48 gene, characterized in that, The method includes: transfecting the shRNA lentiviral vector according to claim 5 into target cells to obtain C15ORF48 silenced cells; And / or, the target cells are TFE3 rearranged renal cell carcinoma cells.

8. C15ORF48 gene-silenced cells constructed according to the method described in claim 7; And / or, the target cells are TFE3 rearranged renal cell carcinoma cells.

9. The use of shRNA according to any one of claims 1-2, recombinant plasmid according to claim 3, shRNA lentiviral vector according to claim 5, and C15ORF48 gene-silencing cells according to claim 8 in the preparation of drugs, pharmaceutical compositions, or kits for treating TFE3 rearranged renal cell carcinoma.

10. A medicament, pharmaceutical composition, or kit for treating TFE3 rearranged renal cell carcinoma, characterized in that, The drug, drug composition, or kit comprises: shRNA according to any one of claims 1-2, recombinant plasmid according to claim 3, or shRNA lentiviral vector according to claim 5.

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